**Real-Time Monitoring of Nitric Oxide Dynamics in the Myocardium: Biomedical Application of Nitric Oxide Sensor**

Gi-Ja Lee, Young Ju Lee and Hun-Kuk Park

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/67255

#### **Abstract**

Nitric oxide (NO) is an important physiological mediator that regulates a wide range of cellular processes in many tissues. Therefore, the accurate and reliable measurement of physiological NO concentration is essential to the understanding of NO signaling and its biological role. Most methods used for NO detection are indirect including spectroscopic approaches such as the Griess assay for nitrite and detection of methemoglobin after NO reaction with oxyhemoglobin. These methods cannot accurately reflect the changes in NO concentration in vivo and in real time. Therefore, direct methods are necessary for investigating biological process and diseases related to NO in biological conditions. There is a growing interest in the development of electrochemically based sensors for direct, in vivo, and real-time monitoring of NO. Electrochemical methods offer simplicity, good sensitivity, high selectivity, fast response times, and long-term calibration stability compared to other techniques including electron paramagnetic resonance, chemiluminescence, and fluorescence. In this article, we present real-time NO dynamics in the myocardium during myocardial ischemia-reperfusion (IR) utilizing electrochemical NO microsensor. And applications of electrochemical NO sensor for the evaluation of cardioprotective effects of therapeutic treatments such as drug administration and ischemic preconditioning are reviewed.

**Keywords:** nitric oxide, real-time detection, myocardial ischemia-reperfusion, electrochemical sensor, therapeutic treatments

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

#### **1. Introduction**

Nitric oxide (NO) is one of gaseous cellular-signaling molecules, which regulates a wide range of physiological and cellular processes in various tissues. In particular, it plays a vital role in a variety of biological processes including immune defense, neurotransmission, regulation of cell death (apoptosis), and cell motility [1–4]. NO has some key features that make this molecule suited to its cellular-signaling functions. NO is a lipophilic diatomic gas under atmospheric conditions. As it has a relatively small Stokes radius and neutral charge, it can rapidly diffuse the cell membrane. The presence of an unpaired electron in NO supports its high reactivity with oxygen (O2 ), superoxide (O2 − ), transition metals, and thiols [5, 6]. The removal of the unpaired electron in NO generates the nitrosonium cation NO+ , while the addition of an electron forms the nitroxyl anion (NO<sup>−</sup> ). These different forms of NO represent distinct chemical reactivities [6, 7]. And NO reacts with O2 − to form peroxynitrite (OONO<sup>−</sup> ), a particularly destructive molecule within biological systems [8].

NO is known to play a major role in vascular biology and heart failure. NO is a doubleedged sword; NO inhibits ischemia-reperfusion (IR) injury, represses inflammation, and prevents left ventricular remodeling, whereas excess NO and coexistence of reactive oxygen species (ROS) with NO are injurious [6]. In that, low concentration of NO has beneficial effects on heart function, while high concentration of NO has opposite effects. Recently, it was reported that the final action of NO is not only regulated by its concentration and cellular confinement but also strongly depends on the level of oxidative stress in the myocardium [4]. But, the cardioprotective mechanism of NO is not yet clear, and it is not known whether NO effectively acts during ischemia or during reperfusion. Therefore, the accurate and quantitative detection of physiological NO concentration is crucial to the understanding of NO signaling and its biological role. This review focuses on the role of NO in myocardial IR injury. In addition, we will summarize the studies from our laboratory, which evaluates the cardioprotective effects of therapeutic treatments such as drug administration and ischemic preconditioning in the myocardium during myocardial IR utilizing electrochemical NO sensor.

#### **2. Production of nitric oxide**

In general, NO is produced from the conversion of L-arginine to L-citrulline, a reaction catalyzed by a family of enzymes called NO synthases (NOSs). Endothelial NOS (eNOS, also known as NOS3) and neuronal NOS (nNOS, also known as NOS1) are constitutive and low-output enzymes, whereas the macrophage-type NOS isoform, known as inducible NOS (iNOS, also known as NOS2), is an inducible and high-output enzyme [9]. NOS is a homodimeric oxidoreductase containing iron protoporphyrin IX (heme), flavin adenine dinucleotide, flavin mononucleotide, and tetrahydrobiopterin (BH<sup>4</sup> ), which is a cofactor essential for the catalytic activity of all three NOS isoforms [6, 10, 11]. NO biosynthesis by the three NOS isoforms can be suppressed by several small-molecule inhibitors: NGmethyl-L-arginine (L-NMA) inhibits all NOS isoforms, and L-NG-nitroarginine methyl ester (L-NAME) has some selectivity for the constitutive NOS isoforms (i.e., nNOS and eNOS), whereas other inhibitors such as aminoguanidine, 1400 W, and many others show selectivity for iNOS [9].

Although NOS had been generally considered to be the primary source of NO in biological systems, nonenzymatic NO synthesis also occurs. NO can be produced in tissues by either direct disproportionation or reduction of nitrite to NO under the acidic and highly reduced conditions which occur in disease states, such as ischemia [12]. Tissue acidosis occurring during ischemia increases NO production independent from eNOS [13], and even at normal pH, xanthine oxidase in the presence of low *p*O2 and high nicotinamide adenine dinucleotide (NADH) concentration is capable of producing NO from nitrite [14]. Besides, in the isolated rat heart [15] and in rabbit hindlimb muscle [16], the NO concentration is still increased during ischemia after complete NOS inhibition by Nw-nitro-L-arginine (L-NNA).

#### **3. Nitric oxide measurements**

**1. Introduction**

nium cation NO+

systems [8].

NO sensor.

**2. Production of nitric oxide**

to form peroxynitrite (OONO<sup>−</sup>

74 Nitric Oxide Synthase - Simple Enzyme-Complex Roles

O2 −

Nitric oxide (NO) is one of gaseous cellular-signaling molecules, which regulates a wide range of physiological and cellular processes in various tissues. In particular, it plays a vital role in a variety of biological processes including immune defense, neurotransmission, regulation of cell death (apoptosis), and cell motility [1–4]. NO has some key features that make this molecule suited to its cellular-signaling functions. NO is a lipophilic diatomic gas under atmospheric conditions. As it has a relatively small Stokes radius and neutral charge, it can rapidly diffuse the cell membrane. The presence of an unpaired

metals, and thiols [5, 6]. The removal of the unpaired electron in NO generates the nitroso-

different forms of NO represent distinct chemical reactivities [6, 7]. And NO reacts with

NO is known to play a major role in vascular biology and heart failure. NO is a doubleedged sword; NO inhibits ischemia-reperfusion (IR) injury, represses inflammation, and prevents left ventricular remodeling, whereas excess NO and coexistence of reactive oxygen species (ROS) with NO are injurious [6]. In that, low concentration of NO has beneficial effects on heart function, while high concentration of NO has opposite effects. Recently, it was reported that the final action of NO is not only regulated by its concentration and cellular confinement but also strongly depends on the level of oxidative stress in the myocardium [4]. But, the cardioprotective mechanism of NO is not yet clear, and it is not known whether NO effectively acts during ischemia or during reperfusion. Therefore, the accurate and quantitative detection of physiological NO concentration is crucial to the understanding of NO signaling and its biological role. This review focuses on the role of NO in myocardial IR injury. In addition, we will summarize the studies from our laboratory, which evaluates the cardioprotective effects of therapeutic treatments such as drug administration and ischemic preconditioning in the myocardium during myocardial IR utilizing electrochemical

In general, NO is produced from the conversion of L-arginine to L-citrulline, a reaction catalyzed by a family of enzymes called NO synthases (NOSs). Endothelial NOS (eNOS, also known as NOS3) and neuronal NOS (nNOS, also known as NOS1) are constitutive and low-output enzymes, whereas the macrophage-type NOS isoform, known as inducible NOS (iNOS, also known as NOS2), is an inducible and high-output enzyme [9]. NOS is a homodimeric oxidoreductase containing iron protoporphyrin IX (heme), flavin adenine

essential for the catalytic activity of all three NOS isoforms [6, 10, 11]. NO biosynthesis

dinucleotide, flavin mononucleotide, and tetrahydrobiopterin (BH<sup>4</sup>

, while the addition of an electron forms the nitroxyl anion (NO<sup>−</sup>

), superoxide (O2

), a particularly destructive molecule within biological

−

), which is a cofactor

), transition

). These

electron in NO supports its high reactivity with oxygen (O2

It is difficult to directly measure NO concentration in vivo because NO is present at nanomolar concentrations in the body and highly reactive with numerous endogenous species including free radicals, oxygen, peroxides, transition metals, and metalloproteins. Indeed, the half-life of NO in biological milieu is <10 s [17].

#### **3.1. Indirect method (Griess assay)**

Indirect methods measure the stable metabolites of NO such as nitrites (NO<sup>2</sup> − ) and nitrates (NO3 − ). The most widely used method for NO detection is based on Griess assay reagents, which can react with nitrite to form a purple azo dye. This method requires that nitrate first be reduced to nitrite and then nitrite determined by the Griess reaction [18]. Briefly, the Griess reaction is a two-step diazotization reaction. First, the NO-derived nitrosating agent, dinitrogen trioxide (N2 O3 ), generated from the acid-catalyzed formation of nitrous acid from nitrite (or autoxidation of NO) reacts with sulfanilamide to produce a diazonium ion. And then it is coupled to N-(1-napthyl)ethylenediamine to form a chromophoric azo product that strongly absorbs at 540 nm [19]. This method has some disadvantages including its sensitivity and its ability to detect nitrate. Therefore, nitrate must be converted to nitrite before the total nitrite is detected. The reduction of nitrate to nitrite can be achieved by using bacterial nitrate reductase or reducing metal such as cadmium [20]. However, these methods often fail to accurately reflect the spatial and temporal distributions of NO in biological environments. In addition, the concentration of nitrate and nitrite metabolite may not show the accurate amount of NO in the specific site because other parts of the body can also produce these compounds. Therefore, direct measurement strategies are necessary for investigating the physiological origin and action of endogenously produced NO.

#### **3.2. Direct methods (nitric oxide sensor)**

Several methods exist for directly measuring NO including electron paramagnetic resonance spectroscopy [21], chemiluminescence [22], fluorescence [23, 24], and electrochemical sensing [17, 25, 26]. Of these approaches, miniaturized electrochemical (e.g., amperometric and voltammetric) sensors represent the most promising means for determining the spatial and temporal distributions of NO near its physiological source [17]. Electrochemical methods provide simplicity, fast response times, good sensitivity, high selectivity, and long-term calibration stability [27, 28]. The most simple detection scheme to date involves the electrochemical oxidation of NO at a metal (e.g., platinum and gold) or carbon electrode [27, 29]. However, it is necessary to use a relatively high working potential (+0.7 to +0.9 V vs. Ag/AgCl) for the direct electrooxidation of NO. In this condition, several interferences from other readily oxidizable biological species such as nitrite, ascorbic acid, uric acid, and acetaminophen often disturb selective detection of NO [25, 27]. Therefore, further surface modification with permselective membranes is required to achieve the desired selectivity for NO via size exclusion or electrostatic repulsion [4]. Indeed, several polymeric materials have been evaluated as gaspermeable or permselective membranes including nafion, collodion, polycarbazole, o- and m-phenylenediamine, poly(tetrafluoroethylene), cellulose acetate, and multilayer hybrids of these polymers [27, 30–36]. In particular, Shin et al. reported that sol-gel-derived electrochemical sensors showed good sensitivity and selectivity for NO detection [17, 27].

#### **4. Nitric oxide in myocardial ischemia-reperfusion injury**

Myocardial infarction (MI) is one of the major causes of morbidity and mortality in industrialized countries, despite the improvement in clinical management of the disease. MI is caused by sudden stoppage of blood supply to the heart that leads to tissue necrosis. The normal myocardium produces more than 90% of its adenosine triphosphate (ATP) by oxidative metabolism and less than 10% by anaerobic glycolysis [4, 37]. After the induction of ischemia, the myocardium can be completely recovered to its normal state if blood supply is adequately restored. But cellular necrosis eventually occurs if ischemia persists [4]. During severe cardiac ischemia, cardiac myocytes must drastically reduce ATP demand or utilization to meet the needs for survival and thus balance the reduced ATP supply with reduced demand during severe ischemia [4, 38].

NO has been extensively studied in the setting of myocardial IR injury. Previous studies demonstrate that the deficiency of eNOS deteriorates myocardial IR injury [39], whereas the overexpression of eNOS [40], the administration of NO donors [41], and inhaled NO gas therapy [42] significantly protect the myocardium. NO possesses several physiological properties that make it a potent cardioprotective-signaling molecule, as follows [43]: First, NO is a potent vasodilator in the ischemic myocardium which enables an essential perfusion of injured tissue. Second, NO reversibly inhibits mitochondrial respiration during early reperfusion. It leads to a decrease in mitochondrial-driven injury by extending the zone of adequate tissue cellular oxygenation away from vessels [43–45]. It is known that restoration of oxygen at reperfusion **3.2. Direct methods (nitric oxide sensor)**

76 Nitric Oxide Synthase - Simple Enzyme-Complex Roles

demand during severe ischemia [4, 38].

Several methods exist for directly measuring NO including electron paramagnetic resonance spectroscopy [21], chemiluminescence [22], fluorescence [23, 24], and electrochemical sensing [17, 25, 26]. Of these approaches, miniaturized electrochemical (e.g., amperometric and voltammetric) sensors represent the most promising means for determining the spatial and temporal distributions of NO near its physiological source [17]. Electrochemical methods provide simplicity, fast response times, good sensitivity, high selectivity, and long-term calibration stability [27, 28]. The most simple detection scheme to date involves the electrochemical oxidation of NO at a metal (e.g., platinum and gold) or carbon electrode [27, 29]. However, it is necessary to use a relatively high working potential (+0.7 to +0.9 V vs. Ag/AgCl) for the direct electrooxidation of NO. In this condition, several interferences from other readily oxidizable biological species such as nitrite, ascorbic acid, uric acid, and acetaminophen often disturb selective detection of NO [25, 27]. Therefore, further surface modification with permselective membranes is required to achieve the desired selectivity for NO via size exclusion or electrostatic repulsion [4]. Indeed, several polymeric materials have been evaluated as gaspermeable or permselective membranes including nafion, collodion, polycarbazole, o- and m-phenylenediamine, poly(tetrafluoroethylene), cellulose acetate, and multilayer hybrids of these polymers [27, 30–36]. In particular, Shin et al. reported that sol-gel-derived electrochem-

ical sensors showed good sensitivity and selectivity for NO detection [17, 27].

Myocardial infarction (MI) is one of the major causes of morbidity and mortality in industrialized countries, despite the improvement in clinical management of the disease. MI is caused by sudden stoppage of blood supply to the heart that leads to tissue necrosis. The normal myocardium produces more than 90% of its adenosine triphosphate (ATP) by oxidative metabolism and less than 10% by anaerobic glycolysis [4, 37]. After the induction of ischemia, the myocardium can be completely recovered to its normal state if blood supply is adequately restored. But cellular necrosis eventually occurs if ischemia persists [4]. During severe cardiac ischemia, cardiac myocytes must drastically reduce ATP demand or utilization to meet the needs for survival and thus balance the reduced ATP supply with reduced

NO has been extensively studied in the setting of myocardial IR injury. Previous studies demonstrate that the deficiency of eNOS deteriorates myocardial IR injury [39], whereas the overexpression of eNOS [40], the administration of NO donors [41], and inhaled NO gas therapy [42] significantly protect the myocardium. NO possesses several physiological properties that make it a potent cardioprotective-signaling molecule, as follows [43]: First, NO is a potent vasodilator in the ischemic myocardium which enables an essential perfusion of injured tissue. Second, NO reversibly inhibits mitochondrial respiration during early reperfusion. It leads to a decrease in mitochondrial-driven injury by extending the zone of adequate tissue cellular oxygenation away from vessels [43–45]. It is known that restoration of oxygen at reperfusion

**4. Nitric oxide in myocardial ischemia-reperfusion injury**

leads to a lethal burst of reactive oxygen species (ROS) generation. An important source of ROS is the mitochondria. In mitochondria, electrons from intermediary metabolism move down the electron transport chain (ETC) and transferred to oxygen at complex IV [46]. When oxygenation is normal, complex I activity is high because a cysteine residue on its ND3 subunit is protected from modification. During ischemia (without oxygen), electrons accumulate along the ETC [46]. Reperfusion leads to a burst of ROS production from multiple sites which can attack proteins, lipids, and DNA, as well as lethal activation of the mitochondrial permeability transition pore [46]. NO inhibits mitochondrial complex I by S-nitrosation (or S-nitrosylation) of cysteines, which subsequently prevents damage during IR injury [47]. Reversible S-nitrosation of complex I slows the reactivation of mitochondria during the crucial first minutes of the reperfusion, thereby decreasing ROS production, oxidative damage, and tissue necrosis [48]. Third, NO is a potent inhibitor of neutrophil adherence to the vascular endothelium which is a significant event initiating further leukocyte activation and superoxide radical production [43, 49, 50]. Fourth, NO prevents platelet aggregation [51], and this effect attenuates capillary plugging together with the anti-neutrophil actions of NO [52]. Finally, NO inhibits apoptosis either directly or indirectly by inhibiting caspase-3-like activation via a cGMP-dependent mechanism [43, 53] and by direct inhibition of caspase-3-like activity through protein S-nitrosylation [43, 54]. In summary, the release of low concentrations of NO by constitutive NOS played a role in the regulation of coronary blood flow, inhibition of platelet aggregation, adherence to the endothelium, and possibly modulation of myocardial oxygen consumption.

But, excessive generation of NO is detrimental to cardiovascular function as exemplified in septic shock where burst generation of iNOS-derived NO causes hypotension, cardiodepression, and vascular hyporeactivity [55]. The detrimental effect of excess NO is attributed to the action on mitochondria. NO inhibits the mitochondrial respiratory chain, resulting in inhibition of ATP production, increased oxidant production, and increased susceptibility to cell death [56]. Inhibition of mitochondrial respiration by NO and its derivatives stimulates production of reactive oxygen and nitrogen species by mitochondria [56], which contribute to cell death in excess.

In conclusion, NO can preserve blood flow in the ischemic tissues and reduce platelet aggregation and neutrophil-endothelium interaction following IR. Besides, low concentrations of NO improve cardiomyocyte function. On the contrary, higher NO concentrations diminish cardiomyocyte function, mediate inflammatory processes following IR, worsen mitochondrial respiration, and even induce cardiomyocyte death. Therefore, it seems that NO can mediate both protective and detrimental myocardial effects which are crucially dependent upon the experimental conditions. Consensus is being reached in the debate regarding a NO protective effect, with most studies reporting its protective effects. However, the role of their product, NO, in the process of IR is still not well defined mainly because of the difficulty in measuring NO concentration in the body tissue.

In the next section, we summarize real-time NO dynamics in the myocardium during myocardial IR. And applications of electrochemical NO sensor for the evaluation of cardioprotective effects of therapeutic treatments such as hypothermia, drug administration, and ischemic preconditioning are summarized.

### **5. Real-time monitoring of nitric oxide dynamics in the myocardium during myocardial ischemia-reperfusion**

#### **5.1. Hypothermia**

In general, hypothermia is thought to reduce the metabolic needs of cells, specifically perhaps by reducing the oxygen demand in the hypothermic tissues [57]. Besides, in isolated heart perfusion system, hearts were placed in ice-cold buffer as quickly as possible to avoid any detrimental effects of hypoxia. Therefore, myocardial hypothermia might be a useful technique to limit ischemic damage during infarction or as adjunctive therapy during minimally invasive cardiac surgery [58]. Lee et al. reported changes in NO dynamics during myocardial IR utilizing a sol-gel-modified electrochemical NO sensor and isolated heart perfusion system [4]. They attempted to clarify the role of endogenous NO release by comparing intact and cardioprotected hearts, in which cardioprotection was conferred by hypothermic treatment of the hearts. For the hypothermic group, hearts were immediately immersed in ice-cold perfusion buffer for 3 min after harvest. In the ischemic myocardium, NO showed a time-dependent change during the 40 min ischemic episode. After myocardial ischemia and early reperfusion, the restoration level of NO was decreased below the pre-ischemic level (**Figure 1**). However, the myocardium with hypothermic treatment (151 ± 37 nM) generated more NO during the ischemic period than that without any treatment (59 ± 15 nM). Besides, the restoration level of NO in the hypothermic group (−57 ± 26 nM) was significantly higher than that of the intact group (−170 ± 50 nM, p < 0.05) [4]. As a result, they inferred that hypothermic treatment of the heart would promote endogenous NO production in the ischemic myocardium. It might be a helpful therapeutic strategy for protecting the myocardium from IR injury [4].

#### **5.2. Myocardial oxygen dynamics**

Because oxygen plays a critical role in the pathophysiology of myocardial injury during subsequent reperfusion, as well as ischemia, the accurate measurement of myocardial oxygen tension is crucial for the assessment of myocardial viability by IR injury. Lee et al. reported a sol-gel-derived electrochemical oxygen microsensor to monitor changes in oxygen tension (*p*O2 ) during myocardial IR [59]. And they analyzed differences in oxygen tension recovery in the post-ischemic myocardium depending on ischemic time to investigate the correlation between recovery parameters for oxygen tension and the severity of IR injury. **Figure 2** shows the nine parameters for *p*O2 dynamics during myocardial IR and the maximum and restoration values of *p*O2 at different ischemic times. As a result, they observed that if ischemia was stopped within 20 min, the *p*O2 in the myocardium after the onset of reperfusion restored to pre-ischemic levels. However, the *p*O2 in the myocardium did not recover to its pre-ischemic state, if the ischemic time was >30 min [59]. These results show that the maximum and restoration values of *p*O2 in the post-ischemic myocardium were closely related to the infarct size [59]. In summary, they demonstrated that the degree of reoxygenation in the post-ischemic myocardium was an important index of IR injury and myocardial viability, utilizing a sol-gelderived electrochemical oxygen microsensor and recovery parameters.

**5. Real-time monitoring of nitric oxide dynamics in the myocardium** 

In general, hypothermia is thought to reduce the metabolic needs of cells, specifically perhaps by reducing the oxygen demand in the hypothermic tissues [57]. Besides, in isolated heart perfusion system, hearts were placed in ice-cold buffer as quickly as possible to avoid any detrimental effects of hypoxia. Therefore, myocardial hypothermia might be a useful technique to limit ischemic damage during infarction or as adjunctive therapy during minimally invasive cardiac surgery [58]. Lee et al. reported changes in NO dynamics during myocardial IR utilizing a sol-gel-modified electrochemical NO sensor and isolated heart perfusion system [4]. They attempted to clarify the role of endogenous NO release by comparing intact and cardioprotected hearts, in which cardioprotection was conferred by hypothermic treatment of the hearts. For the hypothermic group, hearts were immediately immersed in ice-cold perfusion buffer for 3 min after harvest. In the ischemic myocardium, NO showed a time-dependent change during the 40 min ischemic episode. After myocardial ischemia and early reperfusion, the restoration level of NO was decreased below the pre-ischemic level (**Figure 1**). However, the myocardium with hypothermic treatment (151 ± 37 nM) generated more NO during the ischemic period than that without any treatment (59 ± 15 nM). Besides, the restoration level of NO in the hypothermic group (−57 ± 26 nM) was significantly higher than that of the intact group (−170 ± 50 nM, p < 0.05) [4]. As a result, they inferred that hypothermic treatment of the heart would promote endogenous NO production in the ischemic myocardium. It might be a helpful therapeutic strategy for protecting the myocardium from

Because oxygen plays a critical role in the pathophysiology of myocardial injury during subsequent reperfusion, as well as ischemia, the accurate measurement of myocardial oxygen tension is crucial for the assessment of myocardial viability by IR injury. Lee et al. reported a sol-gel-derived electrochemical oxygen microsensor to monitor changes in oxygen tension

) during myocardial IR [59]. And they analyzed differences in oxygen tension recovery in the post-ischemic myocardium depending on ischemic time to investigate the correlation between recovery parameters for oxygen tension and the severity of IR injury. **Figure 2** shows

state, if the ischemic time was >30 min [59]. These results show that the maximum and resto-

[59]. In summary, they demonstrated that the degree of reoxygenation in the post-ischemic myocardium was an important index of IR injury and myocardial viability, utilizing a sol-gel-

derived electrochemical oxygen microsensor and recovery parameters.

dynamics during myocardial IR and the maximum and restora-

in the myocardium after the onset of reperfusion restored to

in the myocardium did not recover to its pre-ischemic

at different ischemic times. As a result, they observed that if ischemia was

in the post-ischemic myocardium were closely related to the infarct size

**during myocardial ischemia-reperfusion**

78 Nitric Oxide Synthase - Simple Enzyme-Complex Roles

**5.1. Hypothermia**

IR injury [4].

(*p*O2

**5.2. Myocardial oxygen dynamics**

the nine parameters for *p*O2

stopped within 20 min, the *p*O2

pre-ischemic levels. However, the *p*O2

tion values of *p*O2

ration values of *p*O2

**Figure 1.** Representative real-time measurement of NO in intact (A) and hypothermic (B) groups during myocardial ischemia-reperfusion of Langendorff-perfused rat hearts. Reproduced with permission from Lee et al. [4]. © 2011 Elsevier B.V.

**Figure 2.** (A) Definition of analysis parameters proposed for changes in oxygen tension throughout the experimental protocol and box and whisker graph depicting maximum (B) and restoration levels (C) of oxygen tension during the reperfusion period as a percentage of pre-ischemic levels at different ischemic times (n = 3 per group). Reproduced from Lee et al. [59]. © The Royal Society of Chemistry 2012.

#### **5.3. Remote ischemic preconditioning**

Ischemic preconditioning is an adaptive response of briefly ischemic tissues that serves to protect against subsequent prolonged ischemic insults and reperfusion injury [60]. In particular, remote ischemic preconditioning (RIPC) is a novel method where ischemia followed by reperfusion of one organ is believed to protect remote organs either by the release of biochemical messengers into circulation or by the activation of nerve pathways, resulting in the release of messengers that have a protective effect [60–62]. This preserves the target tissue without trauma to major vessels or direct stress to the target organ [63]. Although some studies have demonstrated that endothelial NO is one of the major contributors to the candidate mechanism of RIPC [60, 64], the mechanism of RIPC-induced cardioprotection has not yet been fully elucidated. Lee et al. simultaneously measured NO and O2 dynamics in the myocardium during myocardial IR utilizing sol-gel-modified electrochemical NO and O2 microsensors [65]. By comparing control and RIPC-treated hearts, we attempted to clarify the correlation between NO release in the ischemic period and O<sup>2</sup> restoration in the myocardium after reperfusion. **Figure 3** represents the schematic diagrams of experimental design and experimental setup of an isolated heart perfusion system and a real-time monitoring system for NO and oxygen tension dynamics during myocardial IR of the rat.

Real-Time Monitoring of Nitric Oxide Dynamics in the Myocardium: Biomedical Application of Nitric Oxide Sensor http://dx.doi.org/10.5772/67255 81

**Figure 3.** Schematic diagrams of (A) experimental design and (B) experimental setup of an isolated heart perfusion system and a real-time monitoring system for nitric oxide and oxygen tension dynamics during myocardial ischemiareperfusion of the rat. Reproduced from Kang et al. [65]. © 2013 Elsevier B.V.

**5.3. Remote ischemic preconditioning**

80 Nitric Oxide Synthase - Simple Enzyme-Complex Roles

Lee et al. [59]. © The Royal Society of Chemistry 2012.

sured NO and O2

electrochemical NO and O2

Ischemic preconditioning is an adaptive response of briefly ischemic tissues that serves to protect against subsequent prolonged ischemic insults and reperfusion injury [60]. In particular, remote ischemic preconditioning (RIPC) is a novel method where ischemia followed by reperfusion of one organ is believed to protect remote organs either by the release of biochemical messengers into circulation or by the activation of nerve pathways, resulting in the release of messengers that have a protective effect [60–62]. This preserves the target tissue without trauma to major vessels or direct stress to the target organ [63]. Although some studies have demonstrated that endothelial NO is one of the major contributors to the candidate mechanism of RIPC [60, 64], the mechanism of RIPC-induced cardioprotection has not yet been fully elucidated. Lee et al. simultaneously mea-

**Figure 2.** (A) Definition of analysis parameters proposed for changes in oxygen tension throughout the experimental protocol and box and whisker graph depicting maximum (B) and restoration levels (C) of oxygen tension during the reperfusion period as a percentage of pre-ischemic levels at different ischemic times (n = 3 per group). Reproduced from

attempted to clarify the correlation between NO release in the ischemic period and O<sup>2</sup>

system for NO and oxygen tension dynamics during myocardial IR of the rat.

in the myocardium after reperfusion. **Figure 3** represents the schematic diagrams of experimental design and experimental setup of an isolated heart perfusion system and a real-time monitoring

dynamics in the myocardium during myocardial IR utilizing sol-gel-modified

microsensors [65]. By comparing control and RIPC-treated hearts, we

restoration

As a result, the concentration change of NO in the RIPC group was different from those in the control group during the ischemic period. In the control group, the NO level initially declined but then gradually inclined during the ischemic episode. In contrast, the NO level in the RIPC group rapidly increased after the onset of ischemia and continued to rise throughout the entire ischemic period [65]. When reperfusion was initiated, the pattern of both NO level and *p*O2 in the RIPC group was different from that of the control group. As a result, the NO level and the *p*O2 of the myocardium in the RIPC group were restored to pre-ischemic levels, unlike those in the control group that did not recover to their pre-ischemic state (**Figures 4** and **5**). In summary, the endogenous production of NO during the ischemic period appears to be correlated with the restoration of NO and *p*O2 in the post-ischemic myocardium after early reperfusion. Additionally, RIPC would promote endogenous NO release against ischemic stimuli and subsequently facilitate reoxygenation in post-ischemic myocardia after reperfusion [65].

**Figure 4.** Representative real-time and simultaneous measurement of nitric oxide and oxygen tension in (A) control and (B) remote ischemic preconditioning (RIPC) groups during myocardial ischemia-reperfusion in Langendorff-perfused rat hearts. Reproduced from Kang et al. [65]. © 2013 Elsevier B.V.

#### **5.4. Effect of prostaglandin E1**

Prostaglandin E1 (Alprostadil®, PGE1), which is an important member of the prostaglandin (PG) family, is a product of arachidonic acid metabolism by cyclooxygenase [66, 67]. Similar to NO, PGE1 has cardioprotective effects during IR [67, 68], as well as vasodilator effects on the systemic and pulmonary circulation [69]. Fang et al. reported that pretreatment of human umbilical vein endothelial cells with PGE1 significantly protected those cells from H<sup>2</sup> O2 induced cell death [66]. And this effect might depend, at least in part, on the upregulation of NO expression [66]. On the other hand, Huk et al. reported that PGE1 prevents the excessive generation of NO, superoxide, and ONOO<sup>−</sup> which trigger a cascade of events leading to IR injury [70]. Though it is known that PGE1 has cardioprotective effects against IR injury, its mechanism and the correlation between NO and PGE1 are not yet clear. Lee et al. monitored the changes in NO and O2 levels in the myocardium during myocardial IR that were induced by PGE1 pretreatment, utilizing sol-gel-modified amperometric NO and O<sup>2</sup> microsensors [67]. They investigated the correlation between endogenous NO and PGE1 in the ischemic episode, as well as oxygen recovery in the post-ischemic myocardium [67]. For statistical and quantitative analysis, they utilized analytical parameters such as %NO and %*p*O2 , which are defined as the percentage of normalized NO (Eq. (1)) and *p*O2 (Eq. (2)), respectively:

Real-Time Monitoring of Nitric Oxide Dynamics in the Myocardium: Biomedical Application of Nitric Oxide Sensor http://dx.doi.org/10.5772/67255 83

restored to pre-ischemic levels, unlike those in the control group that did not recover to their pre-ischemic state (**Figures 4** and **5**). In summary, the endogenous production of NO during the ischemic period appears to be correlated with the restoration of NO and *p*O2

the post-ischemic myocardium after early reperfusion. Additionally, RIPC would promote endogenous NO release against ischemic stimuli and subsequently facilitate reoxygen-

Prostaglandin E1 (Alprostadil®, PGE1), which is an important member of the prostaglandin (PG) family, is a product of arachidonic acid metabolism by cyclooxygenase [66, 67]. Similar to NO, PGE1 has cardioprotective effects during IR [67, 68], as well as vasodilator effects on the systemic and pulmonary circulation [69]. Fang et al. reported that pretreatment of human umbilical vein endothelial cells with PGE1 significantly protected those cells from H<sup>2</sup>

**Figure 4.** Representative real-time and simultaneous measurement of nitric oxide and oxygen tension in (A) control and (B) remote ischemic preconditioning (RIPC) groups during myocardial ischemia-reperfusion in Langendorff-perfused

induced cell death [66]. And this effect might depend, at least in part, on the upregulation of NO expression [66]. On the other hand, Huk et al. reported that PGE1 prevents the excessive

injury [70]. Though it is known that PGE1 has cardioprotective effects against IR injury, its mechanism and the correlation between NO and PGE1 are not yet clear. Lee et al. monitored

[67]. They investigated the correlation between endogenous NO and PGE1 in the ischemic episode, as well as oxygen recovery in the post-ischemic myocardium [67]. For statistical and

by PGE1 pretreatment, utilizing sol-gel-modified amperometric NO and O<sup>2</sup>

quantitative analysis, they utilized analytical parameters such as %NO and %*p*O2

defined as the percentage of normalized NO (Eq. (1)) and *p*O2

which trigger a cascade of events leading to IR

(Eq. (2)), respectively:

levels in the myocardium during myocardial IR that were induced

ation in post-ischemic myocardia after reperfusion [65].

82 Nitric Oxide Synthase - Simple Enzyme-Complex Roles

**5.4. Effect of prostaglandin E1**

the changes in NO and O2

generation of NO, superoxide, and ONOO<sup>−</sup>

rat hearts. Reproduced from Kang et al. [65]. © 2013 Elsevier B.V.

in

O2 -

microsensors

, which are

**Figure 5.** The correlation between ischemia-evoked nitric oxide concentration and reoxygenation parameters of the postischemic myocardium in two groups. Error bars represent standard deviation of the mean (n = 5 per group). Reproduced from Ref. [65], Kang SW et al., Anal. Chim. Acta 802, 74 (2013). © 2013 Elsevier B.V.

%NO <sup>=</sup> NO level on ischemic or reperfusion period (nM) \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ NO level on pre‐ischemic period (mM) <sup>×</sup> <sup>100</sup> (1)

$$\% \text{NO} = \frac{\text{NO level on isomeric or peripheral period (mM)}}{\text{NO level on pre-ischemic period (mM)}} \times 100\tag{1}$$

$$\% \text{pO}\_2 = \frac{\text{pO}\_2 \text{level on isomeric or peripheral period (mmHg)}}{\text{pO}\_2 \text{ level on pre-ischemic period (mmHg)}} \times 100\tag{2}$$

As a result, there were significant differences in the NO dynamics during ischemia and reperfusion between the control and PGE1-treated rat hearts (**Table 1** and **Figure 6**) as follows [67]: In the control group, the initial decrease in %NO was 56.0 ± 12.9, and the maximum NO level was 86.7 ± 18.5 during the ischemic period. However, in the PGE1 group, %NO rapidly declined to 19.9 ± 5.8% of the pre-ischemic levels, and this was maintained throughout the 30 min ischemic episode. In addition, after the onset of reperfusion, NO level inclined to a maximum of 82.0 ± 6.4 but did not exceed the pre-ischemic basal NO level. In the control group, the maximum %NO response (164.9 ± 41.0) to reperfusion was approximately double than that of the PGE1 group (p < 0.01, n = 5). They suggest that the cardioprotective effect of PGE1 might be attributed to a reduction in excessive NO production during early reperfusion.


**Table 1.** Changes in %NO level during myocardial ischemia-reperfusion of the control and PGE1-treated groups.

**Figure 6.** The correlation between the maximum %NO and restoration %*p*O2 during 60 min of reperfusion in the two groups. Error bars represent the standard deviation from the mean (n = 5 per group). Reproduced from Kang et al. [67]. © 2014 Elsevier B.V.

#### **6. Conclusions**

[67]: In the control group, the initial decrease in %NO was 56.0 ± 12.9, and the maximum NO level was 86.7 ± 18.5 during the ischemic period. However, in the PGE1 group, %NO rapidly declined to 19.9 ± 5.8% of the pre-ischemic levels, and this was maintained throughout the 30 min ischemic episode. In addition, after the onset of reperfusion, NO level inclined to a maximum of 82.0 ± 6.4 but did not exceed the pre-ischemic basal NO level. In the control group, the maximum %NO response (164.9 ± 41.0) to reperfusion was approximately double than that of the PGE1 group (p < 0.01, n = 5). They suggest that the cardioprotective effect of PGE1 might be attributed to a reduction in excessive NO production during early reperfusion.

**Parameters Control group (n = 5) PGE1 group (n = 5)** *p* **value** Level of baseline during pre-ischemic period 101.4 ± 0.9 101.0 ± 2.9 0.767

Maximum level during ischemia 86.7 ± 18.5 26.0 ± 13.1 <0.001 Maximum level during the reperfusion period 164.9 ± 41.0 82.0 ± 6.4 0.01 Restoration level after 60 min of reperfusion 98.7 ± 42.1 45.6 ± 10.1 0.046

**Table 1.** Changes in %NO level during myocardial ischemia-reperfusion of the control and PGE1-treated groups.

Reproduced from Ref. [67], Kang et al., Sensor. Actuat. B – Chem. 203, 245 (2014). © 2014 Elsevier B.V.

56.0 ± 12.9 19.9 ± 5.8 <0.001

**Figure 6.** The correlation between the maximum %NO and restoration %*p*O2

© 2014 Elsevier B.V.

Level of initial decrement after the onset of

84 Nitric Oxide Synthase - Simple Enzyme-Complex Roles

ischemia

groups. Error bars represent the standard deviation from the mean (n = 5 per group). Reproduced from Kang et al. [67].

during 60 min of reperfusion in the two

NO plays important roles in the cardiovascular system by mediating various physiological and pathophysiological processes. From the real-time measurement of endogenous NO dynamics in the myocardium, we summarize as follows: (1) NO concentration was definitely decreased after myocardial ischemia; (2) there was endogenous NO formation as a protective response against ischemia during the ischemic episode, but it was not enough to restore pre-ischemic NO level; (3) the promotion of endogenous formation and inhibition of the timecourse alteration of NO during an ischemic episode might be helpful as a therapeutic strategy for protecting the myocardium from ischemic injury; and (4) the reduction of excessive NO production in early reperfusion period might also be helpful as a therapeutic strategy to protect the myocardium from IR injury. And NO permselective microsensors have good sensitivity and specificity for detecting biologically released NO dynamics in vivo and can be applied in real-time monitoring of NO dynamics in various organs.

### **Acknowledgements**

This study was supported by the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2013R1A1A2065149 and 2016R1D1A1A09919833) and the Ministry of Science, ICT, and Future Planning (NRF-2015M3A9E2029188).

#### **Author details**

Gi-Ja Lee1, 2, Young Ju Lee<sup>1</sup> and Hun-Kuk Park1, 2\*

\*Address all correspondence to: sigmoidus@khu.ac.kr

1 Department of Biomedical Engineering & Healthcare Industry Research Institute, College of Medicine, Kyung Hee University, Seoul, South Korea

2 Department of Medical Engineering, Graduate School, Kyung Hee University, Seoul, South Korea

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## **Role of Endothelial Nitric Oxide Synthase in Glucocorticoid-Induced Hypertension: An Overview of Experimental Data**

Kinga G. Blecharz-Lang and Malgorzata Burek

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/67025

#### **Abstract**

Imbalances in the synthesis or in the bioavailability of nitric oxide (NO), the freely dif‐ fusible vasodilator, in myocardial endothelial cells were demonstrated to be crucial in the development of hypertension. Glucocorticoids (GCs) are widely used as immuno‐ modulators. One of the numerous side effects of GC therapy is hypertension arising from reduced release of the endothelium‐derived NO. GCs can modulate NO synthesis by targeting the genes involved in it, like nitric oxide synthase (NOS) and guanosine tri‐ phosphate (GTP) cyclohydrolase‐1 (GTPCH‐1). This chapter will give an overview on the impact of GCs on NO synthesis and signalling in animal models as well as in in vitro cell culture models. Moreover, strategies for preventing or neutralizing side effects of long‐term GC therapy will be discussed.

**Keywords:** myocardial endothelial cells, nitric oxide, nitric oxide synthase, glucocorticoids, glucocorticoid receptor

#### **1. Introduction**

Hypertension mediated by glucocorticoids (GCs) is a frequent clinical problem. Among oth‐ ers it results from the fact that GCs remain as one of the most commonly prescribed drugs for many conditions, such as inflammatory diseases, asthma, multiple sclerosis, chronic obstruc‐ tive pulmonary disease (COPD) and many more. Either the pharmacological administration of GCs or intrinsic GC excess in humans (caused that is by Cushing syndrome) may result in hypertension. The problem was originally discussed to originate from sodium excess or from

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

an impairment of water reabsorption by the renal mineralocorticoid receptor [1, 2]. However, extensive experimental and clinical data begun to unravel the complex molecular mecha‐ nisms inducing the onset and leading to a persistence of pathologically high blood pressure induced by GCs. In addition, recent research pointed to the role of extra‐renal tissues, such as the vascular endothelium, that have not been taken into account so far in the regulation of blood pressure. The importance of glucocorticoid receptor (GR) signalling became more and more obvious in this process [3, 4].

Effects of GCs on vessel vasodilatation and nitric oxide (NO) synthesis as well as imbal‐ ances between NO and superoxide were examined extensively in numerous in vitro and in vivo studies. Imbalances in the bioavailability of endothelium‐derived NO play a cru‐ cial role in diverse cardiovascular diseases including hypertension, arteriosclerosis and hypercholesterolemia [5–11]. NO is also known to influence circulating platelets and white blood cells and modulates various cellular events, such as platelet activation, mitochon‐ drial function, ion transport, inflammation, angiogenesis and cell proliferation, all pro‐ cesses being essential in cardiovascular homeostasis. It also contributes to the control of the heart rate and its contractility. Although its role in these processes is not fully known, the amount of bioactive NO has been suggested to be involved in the control of cardiac contractility. While lower NO levels seem to promote contractility, higher levels have the opposite effects [12]. It limits cardiac remodelling after ischemic injury. NO is known to be constitutively generated in cardiomyocytes by two different NO synthase (NOS) isoforms, the endothelial and neuronal one indicating different roles of these isoforms in cardiac function [13]. In this chapter, we summarized the state of knowledge from available experi‐ mental data considering the influence of externally administered GCs on the synthesis of the vasodilatator NO in hypertension. Hypertension arising from intrinsic GC excess will not be discussed here.

#### **2. Glucocorticoids and the glucocorticoid receptor**

GCs are steroid hormones synthesized in the adrenal glands. They are essential for life and involved in various cellular processes, such as in the regulation of metabolic and immunologi‐ cal pathways, and the control of cellular homeostasis. Cortisol is the main form of GCs pro‐ duced in the human body. Multiple naturally occurring as well as synthetic compounds, like dexamethasone, are being used in the treatment of diverse inflammatory and immunological diseases. The cellular effects mediated by GCs, including genomic and non‐genomic ones, are exerted by binding to their receptor, the GR. In its inactive state, the GR is kept in the cytosol fraction by chaperon proteins, such as the heat shock protein 90 (HSP90). The lipophilic GCs diffuse through the cell membrane into the cytosol; they bind to the GR which in that moment is released by the HSPs and is then activated (**Figure 1**). After dimerization, the GR‐GC com‐ plex enters the nucleus where it binds to so‐called glucocorticoid response elements (GREs) located in the promoter regions of target genes. By this binding, the GR activates or represses the expression of its specific targets [14]. Moreover, by binding to other transcription factors, the GR can indirectly influence the expression of certain other genes [14].

Role of Endothelial Nitric Oxide Synthase in Glucocorticoid-Induced Hypertension: An Overview of Experimental Data http://dx.doi.org/10.5772/67025 95

an impairment of water reabsorption by the renal mineralocorticoid receptor [1, 2]. However, extensive experimental and clinical data begun to unravel the complex molecular mecha‐ nisms inducing the onset and leading to a persistence of pathologically high blood pressure induced by GCs. In addition, recent research pointed to the role of extra‐renal tissues, such as the vascular endothelium, that have not been taken into account so far in the regulation of blood pressure. The importance of glucocorticoid receptor (GR) signalling became more and

Effects of GCs on vessel vasodilatation and nitric oxide (NO) synthesis as well as imbal‐ ances between NO and superoxide were examined extensively in numerous in vitro and in vivo studies. Imbalances in the bioavailability of endothelium‐derived NO play a cru‐ cial role in diverse cardiovascular diseases including hypertension, arteriosclerosis and hypercholesterolemia [5–11]. NO is also known to influence circulating platelets and white blood cells and modulates various cellular events, such as platelet activation, mitochon‐ drial function, ion transport, inflammation, angiogenesis and cell proliferation, all pro‐ cesses being essential in cardiovascular homeostasis. It also contributes to the control of the heart rate and its contractility. Although its role in these processes is not fully known, the amount of bioactive NO has been suggested to be involved in the control of cardiac contractility. While lower NO levels seem to promote contractility, higher levels have the opposite effects [12]. It limits cardiac remodelling after ischemic injury. NO is known to be constitutively generated in cardiomyocytes by two different NO synthase (NOS) isoforms, the endothelial and neuronal one indicating different roles of these isoforms in cardiac function [13]. In this chapter, we summarized the state of knowledge from available experi‐ mental data considering the influence of externally administered GCs on the synthesis of the vasodilatator NO in hypertension. Hypertension arising from intrinsic GC excess will

GCs are steroid hormones synthesized in the adrenal glands. They are essential for life and involved in various cellular processes, such as in the regulation of metabolic and immunologi‐ cal pathways, and the control of cellular homeostasis. Cortisol is the main form of GCs pro‐ duced in the human body. Multiple naturally occurring as well as synthetic compounds, like dexamethasone, are being used in the treatment of diverse inflammatory and immunological diseases. The cellular effects mediated by GCs, including genomic and non‐genomic ones, are exerted by binding to their receptor, the GR. In its inactive state, the GR is kept in the cytosol fraction by chaperon proteins, such as the heat shock protein 90 (HSP90). The lipophilic GCs diffuse through the cell membrane into the cytosol; they bind to the GR which in that moment is released by the HSPs and is then activated (**Figure 1**). After dimerization, the GR‐GC com‐ plex enters the nucleus where it binds to so‐called glucocorticoid response elements (GREs) located in the promoter regions of target genes. By this binding, the GR activates or represses the expression of its specific targets [14]. Moreover, by binding to other transcription factors,

more obvious in this process [3, 4].

94 Nitric Oxide Synthase - Simple Enzyme-Complex Roles

not be discussed here.

**2. Glucocorticoids and the glucocorticoid receptor**

the GR can indirectly influence the expression of certain other genes [14].

**Figure 1.** Glucocorticoid receptor signalling. In its inactive state, the glucocorticoid receptor (GR) is kept in the cytosol by chaperon proteins, such as the heat shock protein 90 (HSP90). The lipophilic GCs diffuse through the cell membrane into the cytosol, bind to the GR resulting in release of GR from the HSP complex and its activation. After dimerization, the GR‐GC complex enters the nucleus where it binds to so‐called glucocorticoid response elements (GREs) located in the promoter regions of target genes. The protein amount of the GR correlates with its transcriptional activity. Under physiological conditions and excess GC treatment, the recycling process of receptor/transcriptional DNA complexes is regulated by the 26S proteasome, GR ubiquitination and degradation.

The GR is conserved among all species and its expression was confirmed in a wide range of tissues including the vascular endothelium. It was also shown to be expressed in microvas‐ cular endothelial cells of the myocardium and of the blood‐brain barrier [15–19]. The protein amount of the GR correlates with its transcriptional activity. Under physiological conditions, the GC turnover and the recycling process of receptor/transcriptional DNA complexes is regulated by the 26S proteasome as well as by the degradation of the GR (**Figure 1**) [20, 21].

Although GCs are the most widely prescribed medicine, systemic GC administration, especially for a long period, is associated with numerous detrimental side effects [22, 23]. GC‐induced hypertension is one of the common undesirable effects. Tissue‐specific GR knockout mice shed light on the molecular mechanisms behind GC‐induced hypertension. Against expectations, a GR deletion in the distal nephron did not protect mice against hypertension caused by GC administration [24]. Also, the lack of GR in vascular smooth muscle cells has only delayed the onset of hypertension in comparison to wild‐type mice [25]. Finally, mice with a tissue‐spe‐ cific GR knockout in the vascular endothelium were resistant to GC‐induced hypertension [26]. These facts underlined the pivotal role of the GR expressed in vascular endothelial cells in generation and maintenance of GC‐mediated hypertension demonstrating that cell‐specific actions of the GR are responsible for the phenotype of a whole organism.

#### **3. Glucocorticoid effects on the NO synthesis and signalling pathway**

#### **3.1. Influence on eNOS**

At cellular level, GCs negatively influence the synthesis of NO causing endothelial dys‐ function. NOS is expressed in three isoforms: neuronal NOS (nNOS, NOS1), inducible NOS (iNOS, NOS2) and endothelial NOS (eNOS, NOS3) [27, 28]. Under physiological cir‐ cumstances, all NOS isoforms can catalyse the conversion of l‐arginine to l‐citrulline and NO. Endothelial NOS constitutively produces NO in vascular endothelial cells. Cofactors involved in this process, such as 5,6,7,8,‐tetrahydrobiopterin (BH<sup>4</sup> ), are limiting elements in the synthesis of NO. It has been observed that the absence of BH<sup>4</sup> or l‐arginin leads to eNOS uncoupling and to the production of reactive oxygen species (ROS) instead of NO. In consequence, this results in endothelial dysfunction followed by severe vascular disor‐ ders, that is, of cardiovascular nature [29]. The pivotal role of eNOS and its product NO in regulating the blood pressure and blood flow was demonstrated in eNOS knockout and eNOS over‐expressing mice. While mice lacking eNOS were hypertensive, animals over‐ expressing eNOS in the vascular tissue became hypotensive and suffered from decreased vasoreactivity [30–32]. These mice models confirmed that blood pressure is regulated by eNOS‐derived NO. Therefore, first efforts to explain the molecular mechanism of GC‐ mediated hypertension focussed on the regulation of eNOS.

GCs lead to a down‐regulation of eNOS mRNA and protein in cultured endothelial cells, in the aorta and in organs such as kidney and liver of GC‐treated rats [33]. In vivo, disrup‐ tion of the eNOS gene preserved mice from hypertension [34]. In vitro studies with human umbilical vein endothelial cells(HUVECs) have demonstrated that endogenous GR acts as a negative regulator of both eNOS and iNOS. It was shown that a GR knockdown in HUVECs increased eNOS mRNA and protein levels [35]. An endothelial deletion of the GR in mice resulted in an accelerated expression of eNOS in septic mice [35]. Moreover, other animal studies demonstrated that following GC treatment, the expression of eNOS was significantly down‐regulated [33, 36]. The observed eNOS reduction could be further confirmed at pro‐ moter level after identification of a potential GRE in the promoter region of this gene [37]. This study which has been performed in HUVECs demonstrated the suppression of the eNOS promoter activity in response to cortisol application. In line with these results, the binding of the GR to the suppressive GRE at −111 to −105 bp located on the NOS promoter was tested by chromatin immunoprecipitation [37]. Recently, a dexamethasone‐mediated regulation of the eNOS promoter in microvascular myocardial endothelial cells derived from mice has been shown. Concordantly with previous results, a GC‐mediated suppression of the eNOS promoter could be measured [19]. The GR can inhibit eNOS and other forms of NOS by trans‐repressive interactions with other transcription factors, such as NFκB or AP‐1 [38, 39]. Moreover, recent reports revealed the meaning of the molar ratio between BH<sup>4</sup> and eNOS rather than the absolute amounts of the cofactor and its enzyme. As we know by now, GCs cause a reduction of both BH<sup>4</sup> and eNOS. They also inhibit NOS phosphorylation without an evident uncoupling of eNOS after GCs treatment [40].

### **3.2. Effects on l‐arginine and BH4**

hypertension is one of the common undesirable effects. Tissue‐specific GR knockout mice shed light on the molecular mechanisms behind GC‐induced hypertension. Against expectations, a GR deletion in the distal nephron did not protect mice against hypertension caused by GC administration [24]. Also, the lack of GR in vascular smooth muscle cells has only delayed the onset of hypertension in comparison to wild‐type mice [25]. Finally, mice with a tissue‐spe‐ cific GR knockout in the vascular endothelium were resistant to GC‐induced hypertension [26]. These facts underlined the pivotal role of the GR expressed in vascular endothelial cells in generation and maintenance of GC‐mediated hypertension demonstrating that cell‐specific

actions of the GR are responsible for the phenotype of a whole organism.

involved in this process, such as 5,6,7,8,‐tetrahydrobiopterin (BH<sup>4</sup>

mediated hypertension focussed on the regulation of eNOS.

in the synthesis of NO. It has been observed that the absence of BH<sup>4</sup>

**3.1. Influence on eNOS**

96 Nitric Oxide Synthase - Simple Enzyme-Complex Roles

**3. Glucocorticoid effects on the NO synthesis and signalling pathway**

At cellular level, GCs negatively influence the synthesis of NO causing endothelial dys‐ function. NOS is expressed in three isoforms: neuronal NOS (nNOS, NOS1), inducible NOS (iNOS, NOS2) and endothelial NOS (eNOS, NOS3) [27, 28]. Under physiological cir‐ cumstances, all NOS isoforms can catalyse the conversion of l‐arginine to l‐citrulline and NO. Endothelial NOS constitutively produces NO in vascular endothelial cells. Cofactors

eNOS uncoupling and to the production of reactive oxygen species (ROS) instead of NO. In consequence, this results in endothelial dysfunction followed by severe vascular disor‐ ders, that is, of cardiovascular nature [29]. The pivotal role of eNOS and its product NO in regulating the blood pressure and blood flow was demonstrated in eNOS knockout and eNOS over‐expressing mice. While mice lacking eNOS were hypertensive, animals over‐ expressing eNOS in the vascular tissue became hypotensive and suffered from decreased vasoreactivity [30–32]. These mice models confirmed that blood pressure is regulated by eNOS‐derived NO. Therefore, first efforts to explain the molecular mechanism of GC‐

GCs lead to a down‐regulation of eNOS mRNA and protein in cultured endothelial cells, in the aorta and in organs such as kidney and liver of GC‐treated rats [33]. In vivo, disrup‐ tion of the eNOS gene preserved mice from hypertension [34]. In vitro studies with human umbilical vein endothelial cells(HUVECs) have demonstrated that endogenous GR acts as a negative regulator of both eNOS and iNOS. It was shown that a GR knockdown in HUVECs increased eNOS mRNA and protein levels [35]. An endothelial deletion of the GR in mice resulted in an accelerated expression of eNOS in septic mice [35]. Moreover, other animal studies demonstrated that following GC treatment, the expression of eNOS was significantly down‐regulated [33, 36]. The observed eNOS reduction could be further confirmed at pro‐ moter level after identification of a potential GRE in the promoter region of this gene [37]. This study which has been performed in HUVECs demonstrated the suppression of the eNOS promoter activity in response to cortisol application. In line with these results, the binding of the GR to the suppressive GRE at −111 to −105 bp located on the NOS promoter was tested

), are limiting elements

or l‐arginin leads to

GCs are known to alter various molecules in the NOS‐mediated biosynthesis of NO. l‐arginine is an essential substrate for all NOS isoforms. In GC‐induced hypertension, systemic levels of l‐arginine and l‐citrulline have been observed to be diminished reflecting the importance of upstream NOS regulators in this clinical condition [41]. l‐arginine supplementation could partially reverse hypertension in GC‐treated rats [41, 42]. BH<sup>4</sup> is an essential cofactor of eNOS in the biosynthesis of NO [43]. It can be produced by two different strategies. The first is via the conversion from GTP catalysed by the GTP cyclohydrolase 1 (GTPCH‐1), which is the rate‐limiting enzyme involved in its synthesis. The second way is the salvage production path‐ way, where the precursor protein sepiapterin is converted into the intermediate molecule BH<sup>2</sup> and then into BH<sup>4</sup> [44]. The importance of the bioavailability of BH<sup>4</sup> was validated in ex vivo studied vessel pieces. Here, a 30‐fold increase of NOS activity in the presence of BH<sup>4</sup> compared with BH<sup>4</sup> ‐free preparations could be determined [45, 46]. Limited amounts of BH<sup>4</sup> can lead to an uncoupling of eNOS and further to endothelial dysfunction [47, 48]. Uncoupled eNOS overproduces ROS which in turn enhance the oxidation of BH<sup>4</sup> to BH<sup>2</sup> resulting in BH<sup>4</sup> defi‐ ciency [49–52]. BH<sup>2</sup> can be reconverted to BH<sup>4</sup> by dihydrofolate reductase (DHFR) [53]. A GC supplementation of cultured human endothelial cells was shown to reduce mRNA and protein levels of DHFR [40]. Furthermore, administration of external BH<sup>4</sup> led to an increase of NO levels following GC‐treatment, as it was demonstrated in animal studies as well as in coronal endothelial cells [51, 54]. However, BH<sup>4</sup> application or treatment with sepiapterin, a precursor of BH<sup>4</sup> , did not alter systolic blood pressure although total concentrations of plasma BH<sup>4</sup> were elevated in GC‐induced hypertensive rats [55, 56]. GC effects on BH<sup>4</sup> and NO down‐regulation have been proven to be genomic by the usage of specific GR antagonists. One of such antago‐ nistic compounds is RU486, the application of which could reverse the decreasing effects on NO synthesis caused by GC treatment [34, 57].

#### **3.3. Control of GTP cyclohydrolase 1**

As the rate‐limiting enzyme involved in the de novo biosynthesis of BH<sup>4</sup> , GTPCH‐1 shows a decreased expression in response to GC treatment. As a result of its decrease, a signifi‐ cantly lowered BH<sup>4</sup> and NO production contributed to impaired endothelium‐dependent vaso‐relaxation [55, 58, 59]. In vitro studies with human and mouse endothelial cells demon‐ strated genomic effects of GCs on GTPCH‐1 by GC‐mediated down‐regulation of GTPCH‐1 mRNA and promoter activity [19, 40]. It was also shown that dexamethasone mediated a dense down‐regulation of both GTPCH‐1 and eNOS after a long‐term treatment period [19]. Elsewhere it was shown that a gene transfer of human GTPCH‐1 restored vascular BH<sup>4</sup> con‐ centrations and positively influenced endothelial function in hypertonic rats [60]. The sum‐ mary of this data clearly displays that GCs influence the whole multistep cascade of NO production mediated by eNOS.

### **4. Strategies to reduce glucocorticoid‐induced hypertension**

Since mechanisms behind GC‐induced side reactions, such as hypertension, are complex and in part controversial, a basic knowledge of the pharmacology, clinical guidelines and the adverse effects of these substances is imperative. Once increased blood pressure as an unwanted effect of GC treatment has been detected, the use of appropriate anti‐hypertensive compounds, such as NO‐generating compounds and direct vasodilators and hybrid drugs possessing a NO‐releasing moiety including diuretics, calcium channel blockers, angiotensin‐ converting enzyme (ACE) inhibitors, angiotensin receptor blockers, β‐blockers, α‐adrenergic receptor antagonists, centrally acting α‐2 receptor agonists, should be considered. There are different strategies and natural as well as synthetic compounds that have been discussed to reduce or prevent GC‐induced imbalances occurring in the production of NO. A brief over‐ view on the most important ones that directly influence NO availability or signalling will be given below.

#### **4.1. Reducing glucocorticoid toxicity**

First of all, the side effects of a drug administration should be considered in terms of the drug duration/dosage, which in turn should take into account the risk/benefit ratio. GC‐induced side effects are usually more severe following systemic than topical application. As an impor‐ tant parameter, a definite indication to start GC treatment should be considered. Moreover, short‐ and intermediate‐acting ones should be preferred over longer‐acting GCs and a mini‐ mum necessary dose and duration of treatment should be chosen. Furthermore, a once‐a‐day morning administration should be preferred over a divided dose therapy. Necessarily, the body weight, blood and ocular pressure, the development of cataracts, serum lipids, blood and urine glucose concentration should be monitored during the complete GC therapy.

#### **4.2. NO‐generating agents**

Several drugs act as NO‐releasing agents. Well known are organic nitrates and nitrites, includ‐ ing nitroglycerin acting as anti‐anginal drug. However, the use of these agents has been asso‐ ciated with some limitations and some of the compounds are still tested in clinical trials.

Pulmonary rather than cardiac hypertension can be reduced by a direct delivery of NO into the lung though inhalation. Reaching well‐ventilated areas of the lungs, inhaled NO increases blood oxygenation and improves ventilation‐perfusion. However, its effect on the systemic arterial tone is minor since NO is rapidly scavenged and acutely inactivated after reacting with circulating haemoglobin [61]. Therefore, effects of distal NO rather occur due to the formation of other bioactive nitrogen oxides, including inorganic nitrate and *S*‐nitrosothiols acting in an endocrine manner [62].

Nitrite therapy also contributes to the regulation of blood flow and blood pressure through its reduction to NO catalysed by metalloprotein oxidoreductases. The bioconversion of nitrite into NO in the vascular system is regulated by a reductive reaction with deoxyhaemoglobin and couples hypoxic sensing with NO production and therefore augmented under physi‐ ological or pathological hypoxia [63–65]. Inhaled nitrite causes pulmonary vasodilatation and induces protective remodelling in animal studies [66]. Moreover, nitrite reduces blood pres‐ sure and improves cell and organ viability after ischemia reoxygenation as it was shown in the heart, liver and lungs [67, 68]. Either oral or inhaled delivery of nitrite has been shown to exhibit a rapid and efficient systemic absorption and is still tested for further effects in various clinical studies.

Nitrate can be considered as a pro‐drug that is further metabolized to nitrite and other nitro‐ gen oxides after application. Due to its long half‐life, the enterosalivary circulation and ref‐ ormation, nitrite ensures a low‐grade NO‐like bioactivity over a longer period of time [69]. Nitrate has been shown to possess a robust NO‐like activity in the cardiovascular system and can be assimilated from nitrate‐rich diet, especially in green leafy vegetables, or as a salt [70]. Animal models revealed compensating effects of dietary nitrite and nitrate on the lack of eNOS expression [71]. Similar to nitrite, nitrate has also cardio‐ and vascular‐ and renoprotec‐ tive properties and both molecules act as a blood pressure‐lowering and anti‐inflammatory agent in different animal models of cardiovascular diseases [72–74].

Regulatory functions for the cardiovascular system have also been demonstrated for nitrated fatty acids [75]. These electrophilic compounds can be generated in the reaction of unsatu‐ rated fatty acids with NO‐derived reactive species and act via NO‐dependent and NO‐inde‐ pendent pathways [76]. Besides positive effects on vascular relaxation, nitrated fatty acids have shown anti‐oxidative and anti‐inflammatory effects in blood vessels [77, 78]. Despite the cardio‐protective action of nitrated fatty acids proven in animal models, the first human clini‐ cal trial is still ongoing and safety is being tested.

#### **4.3. l‐arginine and l‐citrulline administration**

dense down‐regulation of both GTPCH‐1 and eNOS after a long‐term treatment period [19]. Elsewhere it was shown that a gene transfer of human GTPCH‐1 restored vascular BH<sup>4</sup>

centrations and positively influenced endothelial function in hypertonic rats [60]. The sum‐ mary of this data clearly displays that GCs influence the whole multistep cascade of NO

Since mechanisms behind GC‐induced side reactions, such as hypertension, are complex and in part controversial, a basic knowledge of the pharmacology, clinical guidelines and the adverse effects of these substances is imperative. Once increased blood pressure as an unwanted effect of GC treatment has been detected, the use of appropriate anti‐hypertensive compounds, such as NO‐generating compounds and direct vasodilators and hybrid drugs possessing a NO‐releasing moiety including diuretics, calcium channel blockers, angiotensin‐ converting enzyme (ACE) inhibitors, angiotensin receptor blockers, β‐blockers, α‐adrenergic receptor antagonists, centrally acting α‐2 receptor agonists, should be considered. There are different strategies and natural as well as synthetic compounds that have been discussed to reduce or prevent GC‐induced imbalances occurring in the production of NO. A brief over‐ view on the most important ones that directly influence NO availability or signalling will be

First of all, the side effects of a drug administration should be considered in terms of the drug duration/dosage, which in turn should take into account the risk/benefit ratio. GC‐induced side effects are usually more severe following systemic than topical application. As an impor‐ tant parameter, a definite indication to start GC treatment should be considered. Moreover, short‐ and intermediate‐acting ones should be preferred over longer‐acting GCs and a mini‐ mum necessary dose and duration of treatment should be chosen. Furthermore, a once‐a‐day morning administration should be preferred over a divided dose therapy. Necessarily, the body weight, blood and ocular pressure, the development of cataracts, serum lipids, blood and urine glucose concentration should be monitored during the complete GC therapy.

Several drugs act as NO‐releasing agents. Well known are organic nitrates and nitrites, includ‐ ing nitroglycerin acting as anti‐anginal drug. However, the use of these agents has been asso‐ ciated with some limitations and some of the compounds are still tested in clinical trials.

Pulmonary rather than cardiac hypertension can be reduced by a direct delivery of NO into the lung though inhalation. Reaching well‐ventilated areas of the lungs, inhaled NO increases blood oxygenation and improves ventilation‐perfusion. However, its effect on the systemic arterial tone is minor since NO is rapidly scavenged and acutely inactivated after reacting with circulating haemoglobin [61]. Therefore, effects of distal NO rather occur due to the

**4. Strategies to reduce glucocorticoid‐induced hypertension**

production mediated by eNOS.

98 Nitric Oxide Synthase - Simple Enzyme-Complex Roles

given below.

**4.1. Reducing glucocorticoid toxicity**

**4.2. NO‐generating agents**

con‐

A direct and very simple way to enhance NO availability is l‐arginine delivery. As a sub‐ strate for all NOSs, this amino acid is present in sufficiently high concentrations in most cells. However, l‐arginine administration was observed to even increase NO generation [79]. Dietary l‐arginine possesses protective effects on the cardiac vascularity, as it was shown in a number of studies and is therefore frequently supplemented in food. However, mixed results of l‐arginine supplementation have been shown in multiple clinical trials, that is, no significant effects of l‐arginine on NO availability and on various cardiovascular parameters followed by even detrimental effects, such as a higher incidence of myocardial infarction after l‐arginine administration, were shown in clinical trials [80]. However, studies focusing on effects on blood pressure have shown blood pressure‐lowering effects [81]. The partially con‐ verse effects may partly result from the unspecific binding of this amino acid to all three NOS isoforms. Moreover, the molecule mediates many other effects than those associated with NO metabolism, that is, it is a precursor of other amino acids or is a substrate for arginase, a key enzyme in the urea cycle [82]. In this context, it was identified that l‐arginine also interacts with the asymmetric dimethylarginine (ADMA) that acts as an endogenous NOS inhibitor. Since high ADMA levels are generated in cardiovascular diseases, l‐arginine sup‐ plementation seems to normalize but not improve NO levels and endothelial function in such individuals [83].

A problem arising from l‐arginine administration might also be that this amino acid is exten‐ sively metabolized by intestinal bacteria and arginase in the liver before reaching the circula‐ tion [84]. Compared with l‐arginine, l‐citrulline has been shown to be effectively transported into endothelial cells where it is enzymatically converted into l‐arginine [84]. However, despite potential advantages shown for this molecule, no clinical trial has been initiated to study the effects of l‐citrulline on cardiovascular function.

#### **4.4. BH4 supplementation**

BH<sup>4</sup> is an important factor controlling the activity of all NOS isoforms. Concerning the sal‐ vage pathway by which this factor can be produced endogenously, the external supplementa‐ tion of BH<sup>4</sup> has been considered as a strategy improving NO synthesis and reducing eNOS uncoupling. Intracellular levels of BH<sup>4</sup> can be restored by exposing cells to sepiapterin, being converted to BH<sup>4</sup> through sepiapterin reductase. Satisfying results on superoxide formation and vascular function have been achieved by the administration of sepiapterin in various models of hypertension, that is, in spontaneously hypertensive or nephrectomised animals [85, 86]. Endothelial dysfunction has also been restored by sepiapterin in dexamethasone‐ incubated aorta rings ex vivo [55]. There are also in vitro studies applying aortic endothelial cells showing an improvement of NO release and decrease of ROS production [58]. There is one main difference between a direct BH<sup>4</sup> and a sepiapterin administration. While sepiapterin is absorbed by cells and converted to intracellular BH<sup>4</sup> , a direct BH<sup>4</sup> administration acts extra‐ cellularly [43]. Despite this seemingly important difference, there is only one report describ‐ ing sepiapterin administration, whereas a direct BH<sup>4</sup> administration has not been described in the context of GC‐induced hypertension [56]. The low number of experimental data might be due to the fact that sepiapterin did not affect dexamethasone‐mediated hypertension in rats although plasma levels of BH<sup>4</sup> were significantly increased. As the authors explained, the uncoupling of eNOS seems not to play a major role in the pathogenesis of GC‐induced hypertension [56].

#### **4.5. Response to antioxidants and anti‐inflammatory agents**

Antioxidants and anti‐inflammatory agents such as tempol, apocynin, N‐acetylcysteine, folic acid and atorvastatin can prevent and partially reverse GCs‐induced hypertension in vivo [87– 92]. Therefore, these substances represent promising candidates for prevention and treatment of increased systolic blood pressure induced by long‐term and high‐dosage GC treatment.

Tempol, 4‐hydroxy‐2,2,6,6‐tetramethyl piperidinoxyl, is a superoxide scavenger with either water‐soluble or membrane‐permeable characteristics. It is known to normalize blood pressure, as it was shown in numerous animal models of differently induced hypertension [90, 93–95]. Interesting findings have been reported by Zhang et al. They showed that tempol did not have an influence on oxidative stress measured in rat plasma. However, they reported tempol to have prevented and partially reversed dexamethasone‐induced hypertension independently of systemic ROS.

The hydrophilic antioxidant N‐acetylcysteine is already widely used in the clinic, that is, as a mucolytic agent or in ischemia‐reperfusion injury [96, 97]. In dexamethasone‐induced hypertension among rats, this agent was shown to lower systolic blood pressure as well as restore plasma NO levels, without any effect on oxidative markers in the plasma. Similar experimental settings revealed N‐acetylcysteine treatment having partially preventing but not reversing properties on GC‐induced hypertension in comparison to what has been shown for tempol [91].

The same laboratory tested folic acid in hypertension induced by synthetic GCs [87]. Folic acid is a B group vitamin that is known from its ROS decreasing properties [98]. It also lowers the concentration of homocysteine that reduces intracellular BH<sup>4</sup> levels, thereby increasing BH<sup>4</sup> bioavailability [99]. Patients suffering from Cushing syndrome tend to have higher systemic homocysteine and reduced folate levels than healthy controls [100]. It is therefore not surprising that the supplementation of folic acid might prevent and partially revers GC‐induced hyper‐ tension by increasing BH<sup>4</sup> amounts, although the detailed mechanism remains elusive [87].

The same group has demonstrated that antioxidant apocynin, which is a specific NAD(P)H oxidase inhibitor, is able either to reverse or to prevent dexamethasone‐caused hypertension [101]. Interestingly, the same study has shown that the NO precursor l‐arginine did not have the same effects as those being observed for apocynin in dexamethasone‐induced hyperten‐ sive rats.

Among its pleiotropic functions, atorvastatin has been reported to improve endothelial func‐ tion through increased bioavailability of NO and ameliorated ROS and pro‐inflammatory cytokine production in different models of hypertension [92, 102, 103]. Atorvastatin could lower the blood pressure of dexamethasone‐treated hypertensive rats due to its positive influ‐ ence on the expression of eNOS measured in aortic samples and due to the lowered levels of superoxide in plasma. However, there was no significant effect of this compound on vaso‐ relaxation although the endothelial function was markedly improved [92].

#### **4.6. Influencing the GC signalling pathway**

NO metabolism, that is, it is a precursor of other amino acids or is a substrate for arginase, a key enzyme in the urea cycle [82]. In this context, it was identified that l‐arginine also interacts with the asymmetric dimethylarginine (ADMA) that acts as an endogenous NOS inhibitor. Since high ADMA levels are generated in cardiovascular diseases, l‐arginine sup‐ plementation seems to normalize but not improve NO levels and endothelial function in such

A problem arising from l‐arginine administration might also be that this amino acid is exten‐ sively metabolized by intestinal bacteria and arginase in the liver before reaching the circula‐ tion [84]. Compared with l‐arginine, l‐citrulline has been shown to be effectively transported into endothelial cells where it is enzymatically converted into l‐arginine [84]. However, despite potential advantages shown for this molecule, no clinical trial has been initiated to

 is an important factor controlling the activity of all NOS isoforms. Concerning the sal‐ vage pathway by which this factor can be produced endogenously, the external supplementa‐

and vascular function have been achieved by the administration of sepiapterin in various models of hypertension, that is, in spontaneously hypertensive or nephrectomised animals [85, 86]. Endothelial dysfunction has also been restored by sepiapterin in dexamethasone‐ incubated aorta rings ex vivo [55]. There are also in vitro studies applying aortic endothelial cells showing an improvement of NO release and decrease of ROS production [58]. There is

cellularly [43]. Despite this seemingly important difference, there is only one report describ‐

in the context of GC‐induced hypertension [56]. The low number of experimental data might be due to the fact that sepiapterin did not affect dexamethasone‐mediated hypertension in

the uncoupling of eNOS seems not to play a major role in the pathogenesis of GC‐induced

Antioxidants and anti‐inflammatory agents such as tempol, apocynin, N‐acetylcysteine, folic acid and atorvastatin can prevent and partially reverse GCs‐induced hypertension in vivo [87– 92]. Therefore, these substances represent promising candidates for prevention and treatment of increased systolic blood pressure induced by long‐term and high‐dosage GC treatment.

Tempol, 4‐hydroxy‐2,2,6,6‐tetramethyl piperidinoxyl, is a superoxide scavenger with either water‐soluble or membrane‐permeable characteristics. It is known to normalize blood pressure, as it was shown in numerous animal models of differently induced hypertension [90, 93–95].

has been considered as a strategy improving NO synthesis and reducing eNOS

through sepiapterin reductase. Satisfying results on superoxide formation

can be restored by exposing cells to sepiapterin, being

and a sepiapterin administration. While sepiapterin

were significantly increased. As the authors explained,

administration acts extra‐

administration has not been described

, a direct BH<sup>4</sup>

study the effects of l‐citrulline on cardiovascular function.

individuals [83].

**4.4. BH4**

tion of BH<sup>4</sup>

converted to BH<sup>4</sup>

hypertension [56].

BH<sup>4</sup>

 **supplementation**

100 Nitric Oxide Synthase - Simple Enzyme-Complex Roles

uncoupling. Intracellular levels of BH<sup>4</sup>

one main difference between a direct BH<sup>4</sup>

rats although plasma levels of BH<sup>4</sup>

is absorbed by cells and converted to intracellular BH<sup>4</sup>

ing sepiapterin administration, whereas a direct BH<sup>4</sup>

**4.5. Response to antioxidants and anti‐inflammatory agents**

As it was shown in a transgenic animal model, an endothelial GR knockout was protected against GC‐induced hypertension [26]. Targeting GR in endothelial cells could therefore con‐ stitute a preventive treatment strategy directed against GC‐induced hypertension. A recent study by Blecharz et al. provided new insights into the GC‐mediated disturbances of NO production in the mouse microvascular endothelial cell line MyEND [19]. They observed negative effects on NO‐synthesis either after a short treatment period of 24 and 48 h or after a long‐term GC administration persisting 30 days. These effects were accompanied by lowered NO concentrations and eNOS activity in response to dexamethasone application. However, instead of reduced eNOS protein expression, a decrease of GTPCH‐1 protein they documented a decrease of GTPCH‐1 protein. Interestingly, the observed changes in enzymes and cofactors involved in NO synthesis were accompanied by a significantly lowered immune reactivity of the GR in MyEND cells. Ligand‐dependent down‐regulation of the GR, associated with the loss of functional GC responsiveness, was referred in numerous publications [20, 21, 104, 105]. It was also shown that the expression of the GR is lower in endothelial cells derived from the mouse myocardium than in those isolated from the blood‐brain barrier [106]. Although the degradation of the receptor by the proteasome can be observed in capillaries from both organs, brain endothelial cells remain responsive to GCs due to a sufficient expression of the GR [17]. The complete loss of GC responsiveness verified in MyEND cells in contrast to endothelium of the central nervous system hinders the translocation of the GC‐GR complex into the nucleus, thereby hindering the transactivation of the GC target gene GTPCH‐1 (as depicted in **Figure 1**) [19]. As it was shown, blocking the proteasomal GR degradation by calpain inhibitor I or by over‐expressing a nondegradable GR isoform rescued either NO or BH<sup>4</sup> levels, as well as eNOS activity in MyEND cells after GC treatment. Hence, these data provide the biochemical evidence that GC‐induced disturbed vasodilatation may result from the involvement of the proteasome in the failure of GC responsiveness rather than from GC‐ mediated trans‐repression of NO‐synthesizing molecules, as it was thought before [19, 57]. On the other hand, together with data gained in brain endothelial cells, the results support the endocrine heterogeneity of different vascular beds and a blockage of the proteasomal sig‐ nalling pathway may be a potential future option for neutralizing or reducing unfavourable effects of long‐term GC therapy [17, 107].

#### **5. Conclusions**

GC‐induced hypertension remains a severe clinical problem, since GCs are the widest pre‐ scribed drugs by clinicians. Despite numerous basic and clinical studies, the complexity of the induction and persistence of hypertension remains not entirely understood. A robust explana‐ tion for GC hypertension is that GCs reduce the activity and expression of eNOS. Moreover, enzymes and cofactors upstream of eNOS are negatively influenced. The effects mediated by GCs on these molecules can be genomic or non‐genomic. Therefore, further examinations are necessary to learn about this very frequent and serious clinical issue. On one hand, a detailed comprehension of the multistep GC/GR signalling cascade in the vasculature as well as in other non‐renal tissues is required. Moreover, the regulation of molecules upstream and downstream of eNOS by GCs in a genomic and non‐genomic manner should be clarified, given the fact that these effects might differ depending on the tissue. This might be explained by the evidence that the expression of the GR differs in a tissue‐specific manner. In addition, it will be necessary to develop new in vivo models mimicking this complex disease and helping to understand the interplay between organs such as the kidney, the liver, the cardiovascu‐ lar and the central nervous system. These animal models should be supported by single‐ or multi‐tissue culture models in vitro. They would accelerate and promote the development of new compounds, such as synthetic and natural antioxidants lowering GC‐induced pathologi‐ cally increased blood pressure, and enable the identification of potential therapeutic targets.

#### **Author details**

a decrease of GTPCH‐1 protein. Interestingly, the observed changes in enzymes and cofactors involved in NO synthesis were accompanied by a significantly lowered immune reactivity of the GR in MyEND cells. Ligand‐dependent down‐regulation of the GR, associated with the loss of functional GC responsiveness, was referred in numerous publications [20, 21, 104, 105]. It was also shown that the expression of the GR is lower in endothelial cells derived from the mouse myocardium than in those isolated from the blood‐brain barrier [106]. Although the degradation of the receptor by the proteasome can be observed in capillaries from both organs, brain endothelial cells remain responsive to GCs due to a sufficient expression of the GR [17]. The complete loss of GC responsiveness verified in MyEND cells in contrast to endothelium of the central nervous system hinders the translocation of the GC‐GR complex into the nucleus, thereby hindering the transactivation of the GC target gene GTPCH‐1 (as depicted in **Figure 1**) [19]. As it was shown, blocking the proteasomal GR degradation by calpain inhibitor I or by over‐expressing a nondegradable GR isoform rescued either NO or

 levels, as well as eNOS activity in MyEND cells after GC treatment. Hence, these data provide the biochemical evidence that GC‐induced disturbed vasodilatation may result from the involvement of the proteasome in the failure of GC responsiveness rather than from GC‐ mediated trans‐repression of NO‐synthesizing molecules, as it was thought before [19, 57]. On the other hand, together with data gained in brain endothelial cells, the results support the endocrine heterogeneity of different vascular beds and a blockage of the proteasomal sig‐ nalling pathway may be a potential future option for neutralizing or reducing unfavourable

GC‐induced hypertension remains a severe clinical problem, since GCs are the widest pre‐ scribed drugs by clinicians. Despite numerous basic and clinical studies, the complexity of the induction and persistence of hypertension remains not entirely understood. A robust explana‐ tion for GC hypertension is that GCs reduce the activity and expression of eNOS. Moreover, enzymes and cofactors upstream of eNOS are negatively influenced. The effects mediated by GCs on these molecules can be genomic or non‐genomic. Therefore, further examinations are necessary to learn about this very frequent and serious clinical issue. On one hand, a detailed comprehension of the multistep GC/GR signalling cascade in the vasculature as well as in other non‐renal tissues is required. Moreover, the regulation of molecules upstream and downstream of eNOS by GCs in a genomic and non‐genomic manner should be clarified, given the fact that these effects might differ depending on the tissue. This might be explained by the evidence that the expression of the GR differs in a tissue‐specific manner. In addition, it will be necessary to develop new in vivo models mimicking this complex disease and helping to understand the interplay between organs such as the kidney, the liver, the cardiovascu‐ lar and the central nervous system. These animal models should be supported by single‐ or multi‐tissue culture models in vitro. They would accelerate and promote the development of new compounds, such as synthetic and natural antioxidants lowering GC‐induced pathologi‐ cally increased blood pressure, and enable the identification of potential therapeutic targets.

BH<sup>4</sup>

effects of long‐term GC therapy [17, 107].

102 Nitric Oxide Synthase - Simple Enzyme-Complex Roles

**5. Conclusions**

Kinga G. Blecharz‐Lang<sup>1</sup> and Malgorzata Burek<sup>2</sup> \*

\*Address all correspondence to: Burek\_M@ukw.de


#### **References**


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108 Nitric Oxide Synthase - Simple Enzyme-Complex Roles

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1650S–1655S.


**Nitric Oxide Synthase in Reproductive System**

## **Nitric Oxide Synthase in Male Urological and Andrologic Functions**

Qingfeng Yu, Tieqiu Li, Jingping Li, Liren Zhong and

Xiangming Mao

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/67562

#### **Abstract**

Nitric oxide (NO), a crucial signaling molecule, is synthesized by the nitric oxide synthase (NOS) enzyme. The significant effects of NOS are under exploration, and the roles of potential therapy targets for diseases of NOS are widely accepted. In this chapter, we summarized the important roles of NOS mainly on pathogenesis of prostate diseases, male infertility, erectile dysfunction and, addition, the potential therapeutic efficacies of NOS for those diseases.

**Keywords:** nitric oxide synthase, nitric oxide, prostate cancer, male infertility, erectile dysfunction, male reproduction

#### **1. Introduction**

Urology and andrology are the branches of medicine that focus on urinary tract system and male reproductive organs. In recent years, incidences of diseases in urology and andrology system such as prostate cancer and male infertility are increasing and causing heavy burden to our society. Growing studies have been demonstrating that nitric oxide synthase (NOS), which synthesized nitric oxide (NO) by converting L-arginine to L-citrulline, locates in tissues of urinary and male reproductive system and acts as key regulators for sexual function, male reproduction, cancer progression and so on [1–3]. The aims of this chapter are to present the roles of NOS and the recent advances of regulation and therapy function with regard to sexual function, male infertility, prostate carcinoma, Peyronie disease, priapism and cryptorchidism.

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

### **2. NOS and male sexual function**

#### **2.1. NOS and erectile dysfunction**

Erectile dysfunction (ED) refers to the symptom that the penis cannot reach and (or) maintain the adequate erection to complete the satisfaction of sexual intercourse, and the course of disease will last at least 6 months or more. Penile erection is an integrated process of artery blood supply and cavernous blood storage launched by nerve, and during this process, neurotransmitter plays an important role [4]. NO is a main messenger, which involves in the induction and maintenance of erection through hemangiectasis and corpus cavernosum relaxation [5]. It has been clear that NO penetrates the smooth muscle cell membrane and catalyzes the formation of cGMP after combining with the ornithine enzyme on the iron ring and then changing the intracellular calcium concentration of smooth muscle to cause relaxation.

#### *2.1.1. NOS in penile tissue*

The nNOS and iNOS were found in the central nervous system, especially the hypothalamic area, such as paraventricular nucleus and the medial optic zone, that control the erectile and sexual behavior and also regulate penile erection through spinal nerve centers [6, 7] (**Figure 1**). Specially, nNOS mainly distributes in the penile and pelvic nerve plexus in adult rats, whereas eNOS is in the penis and pelvic area of the urethra but less in the body part of the penis [8, 9].

**Figure 1.** Role of NOS and NO in penile erection. The nNOS and iNOS regulate penile erection through NO/cGMP/ PKG pathway. Abbreviations: eNOS, endothelial nitric oxide synthase; nNOS, neuronal nitric oxide synthase; NO, nitric oxide; GTP, guanosine triphosphate; cGMP, cyclic guanosine monophosphate; PKG, protein kinase G.

#### *2.1.2. Current reviews on the effects of NOS on disease-related ED*

#### *2.1.2.1. Diabetes-related ED*

**2. NOS and male sexual function**

114 Nitric Oxide Synthase - Simple Enzyme-Complex Roles

Erectile dysfunction (ED) refers to the symptom that the penis cannot reach and (or) maintain the adequate erection to complete the satisfaction of sexual intercourse, and the course of disease will last at least 6 months or more. Penile erection is an integrated process of artery blood supply and cavernous blood storage launched by nerve, and during this process, neurotransmitter plays an important role [4]. NO is a main messenger, which involves in the induction and maintenance of erection through hemangiectasis and corpus cavernosum relaxation [5]. It has been clear that NO penetrates the smooth muscle cell membrane and catalyzes the formation of cGMP after combining with the ornithine enzyme on the iron ring and then changing

The nNOS and iNOS were found in the central nervous system, especially the hypothalamic area, such as paraventricular nucleus and the medial optic zone, that control the erectile and sexual behavior and also regulate penile erection through spinal nerve centers [6, 7] (**Figure 1**). Specially, nNOS mainly distributes in the penile and pelvic nerve plexus in adult rats, whereas eNOS is in the penis and pelvic area of the urethra but less in the body part

**Figure 1.** Role of NOS and NO in penile erection. The nNOS and iNOS regulate penile erection through NO/cGMP/ PKG pathway. Abbreviations: eNOS, endothelial nitric oxide synthase; nNOS, neuronal nitric oxide synthase; NO, nitric

oxide; GTP, guanosine triphosphate; cGMP, cyclic guanosine monophosphate; PKG, protein kinase G.

the intracellular calcium concentration of smooth muscle to cause relaxation.

**2.1. NOS and erectile dysfunction**

*2.1.1. NOS in penile tissue*

of the penis [8, 9].

A possible percentage ranges from 50% to 90% of diabetes patients suffer from ED [10], which counts around three times more than that in healthy cohort. Also, previous report showed that diabetes patients who firstly suffered from ED had a younger age in average compared with people without diabetes and more severe appeared of their symptoms [11]. It has now reached a consensus on the relationship between diabetes and ED, the damage of endothelial cell, ultrastructure changes in cavernous smooth muscle and matrix fibrosis are the common factors affecting the cavernous diastolic function and resulting in ED [12], and damage of eNOS-NO-cGMP pathway was considered to be the main molecular mechanism [13]. The expression of Recombinant Nitric Oxide Synthase Trafficker (NOSTRIN) in Dulbecco's modified eagle medium (DMED) corpus cavernosum increased while the expression of eNOS decreased. It is theorized that increased NOSTRIN may be an important mechanism for the reduction of eNOS expression, while further study is still needed [14].

#### *2.1.2.2. Benign prostatic hyperplasia-related ED*

Approximately 49% of BPH patients suffer from ED. A significant correlation was reported between BPH/lower urinary tract obstruction (LUTS) and ED after excluding the effects of age and other etiologies on ED [15, 16]. BPH/LUTS seems to be one of the most harmful factors contribute to ED compared with diabetes, hypertension and (or) heart disease [17]. In fact, both parasympathetic innervation of prostate and cavernous nerve of penis are from the pelvic plexus [18]. Pathophysiology studies also showed that the mechanisms of BPH are similar to those of sexual dysfunction which include decrease of the ratio of endothelial NOS/NO, enhancement of endothelial presenilin-1 contraction effect, overreaction of autonomic nervous of bladder, prostate and penis, enhancement of signaling pathway Rho kinase expression/activity and (or) pelvic vascular sclerosis [19]. Since NOS has been found to play significant effects on BPH patients with ED, new treatment by exogenous NO donor and NOS activating enzyme would be promising [20]. However, only few animal experiments have been under exploration up to date, and the mechanisms for the occurrence of ED in BPH patients are still needed to be identified.

#### *2.1.2.3. Hypertension-related ED*

Hypertension is another important risk factor for ED. Jensen et al. reported that nearly 27% of hypertensive patients suffered from ED [21]. The increase of plasma asymmetrical dimethylarginine (ADMA) concentration caused reduction of the NO expression in penile tissue by inhibiting the NOS activity, which might be a possible mechanism for hypertension-related ED [22].

#### *2.1.3. Possible therapy strategies of NOS on ED*

Increasing evidence has been indicating that L-Arg-NO-cGMP pathway might be a crucial mechanism of penile erection [23]. As a key enzyme in the synthesis of NO, NOS has always been one of the research focuses. Specially, nNOS acts as a key role in erection launch, whereas eNOS enables cavernous body dilate and maintains the status of erection [24]. Although the effect of iNOS was absent in the direct regulation of penile erection, a special "double effect" in the elderly and the pathological state was reported [25]. Since the reduction of NOSs or the decrease of its activity might contribute to ED, the treatment on L-Arg-NO-cGMP for ED might be revolutionary breakthrough, as phosphodiesterase type 5 inhibitors (PDE5Is) was found to improve the erectile function by increasing the NO concentration but reducing the eGMP degradation [26]. However, nearly 20% of patients with ED still showed little benefit after receiving PDE5Iss, especially in patients with diabetes or prostate cancer (Prostate carcinoma) after radical mastectomy [27]. Future NOSs gene transfer therapy from the molecular level would be another choice [28], which might have long-term curative effect, little side effect to the body and, even, completely cure ED. Therapies including increasing expression of NOSs (nNOS, iNOS, eNOS) or inhibiting the expression of protein inhibitor of NOS (PIN) might be promising and worthwhile exploration [29]. However, shortcomings such as short effect duration, possibility of inducing abnormal erection and other potential unknown side effects from the long-term excessive expression of NO are addressed.

#### **2.2. NOS and libido**

ED may cause low sexual desire or loss of libido in men [30]. Previous study reported that treatment for ED could somehow retrieve sexual desire [31]. It is believed that NO and NOS are beneficial for penile erection, and consequently, NO and NOS may enhance sexual motivation in indirect ways. NO could also affect libido in the direct ways.

Areas for male sexual behavior in brain distribute NO responsive guanylyl cyclase, which involves cellular events of NO [32], and previous studies showed that the NOS inhibitor NG-nitro-L-arginine methyl ester (NAME) administered to medial preoptic area by reverse dialysis caused reduced mounting of male rat [32, 33]. Chu et al. further reported that Impaza, a stimulator of eNOS, could raise the sexual incentive motivation rates of male rats through the NO-guanylyl cyclase pathway [34], and nNOS was also considered to affect the male sexual behavior by activating the cyclic guanosine monophosphate (cGMP) [35]. However, adverse result was reported by other researchers, and the conclusion was still controversial [36].

#### **3. NOS and male infertility**

Approximately 15% of couples suffer from infertility while male cause contributed to nearly 50% in these infertile couples [37]. Male reproduction is known to involve complicated aspects such as spermatogenesis, sperm dynamics, sperm morphology and acrosome reaction. Increasing evidences have been indicating that NOS and NO are associated with male infertility [38].

#### **3.1. NOS and male reproductive system**

The hypothalamic-pituitary axis plays core roles in reproduction and steroid hormone production in man. Gonadotropin-releasing hormone (GnRH), also known as luteinizing hormone-releasing hormone (LHRH), which is produced and secreted by the arcuate nucleus of the hypothalamus, could stimulate the anterior pituitary to episodically release folliclestimulating hormone (FSH) and luteinizing hormone (LH). LH stimulates the Leydig cells to produce testosterone, and FSH exerts its effect directly on the Sertoli cells to promote spermatogenesis (**Figure 2**).

In vitro studies have shown that NO stimulates LHRH secretion from the hypothalamus and modulates LH release from the pituitary [39, 40]. Ceccatelli [41] reported that sodium nitroprusside, a NO donor, suppressed GnRH-stimulated LH release from pituitaries in male rats. Chatterjee et al. [42] showed that NOS inhibitor p-nitro-L-arginine methyl ester (L-NAME) enhanced GnRH-induced LH release from pituitaries in rats. Decreased level of GnRH and gonadotropin in chronic NO deficiency rats were also observed [43].

#### *3.1.1. Testis*

eNOS enables cavernous body dilate and maintains the status of erection [24]. Although the effect of iNOS was absent in the direct regulation of penile erection, a special "double effect" in the elderly and the pathological state was reported [25]. Since the reduction of NOSs or the decrease of its activity might contribute to ED, the treatment on L-Arg-NO-cGMP for ED might be revolutionary breakthrough, as phosphodiesterase type 5 inhibitors (PDE5Is) was found to improve the erectile function by increasing the NO concentration but reducing the eGMP degradation [26]. However, nearly 20% of patients with ED still showed little benefit after receiving PDE5Iss, especially in patients with diabetes or prostate cancer (Prostate carcinoma) after radical mastectomy [27]. Future NOSs gene transfer therapy from the molecular level would be another choice [28], which might have long-term curative effect, little side effect to the body and, even, completely cure ED. Therapies including increasing expression of NOSs (nNOS, iNOS, eNOS) or inhibiting the expression of protein inhibitor of NOS (PIN) might be promising and worthwhile exploration [29]. However, shortcomings such as short effect duration, possibility of inducing abnormal erection and other potential unknown side

ED may cause low sexual desire or loss of libido in men [30]. Previous study reported that treatment for ED could somehow retrieve sexual desire [31]. It is believed that NO and NOS are beneficial for penile erection, and consequently, NO and NOS may enhance sexual moti-

Areas for male sexual behavior in brain distribute NO responsive guanylyl cyclase, which involves cellular events of NO [32], and previous studies showed that the NOS inhibitor NG-nitro-L-arginine methyl ester (NAME) administered to medial preoptic area by reverse dialysis caused reduced mounting of male rat [32, 33]. Chu et al. further reported that Impaza, a stimulator of eNOS, could raise the sexual incentive motivation rates of male rats through the NO-guanylyl cyclase pathway [34], and nNOS was also considered to affect the male sexual behavior by activating the cyclic guanosine monophosphate (cGMP) [35]. However, adverse result was reported by other researchers, and the conclusion was still controversial [36].

Approximately 15% of couples suffer from infertility while male cause contributed to nearly 50% in these infertile couples [37]. Male reproduction is known to involve complicated aspects such as spermatogenesis, sperm dynamics, sperm morphology and acrosome reaction. Increasing evidences have been indicating that NOS and NO are associated with male infertility [38].

The hypothalamic-pituitary axis plays core roles in reproduction and steroid hormone production in man. Gonadotropin-releasing hormone (GnRH), also known as luteinizing hormone-releasing hormone (LHRH), which is produced and secreted by the arcuate nucleus

effects from the long-term excessive expression of NO are addressed.

vation in indirect ways. NO could also affect libido in the direct ways.

**2.2. NOS and libido**

116 Nitric Oxide Synthase - Simple Enzyme-Complex Roles

**3. NOS and male infertility**

**3.1. NOS and male reproductive system**

#### *3.1.1.1. Testicular microcirculation*

The testis has a rich vascular system that plays a very important role in maintaining the normal functions and stable inner environment of the testis [44]. The regulation of testicular blood microcirculation is very complex, including self-regulation, neural regulation and humoral regulation [45]. NO is the major physiological regulator of basal blood vessel tone and is continually released from endothelium of testicular arteries [46]. A study showed that the regulation effect of NO on testicular blood flow was limited under basal conditions, but this limitation could be significant reversed after HCG treatment; in this case, NO showed

**Figure 2.** Regulation of hypothalamic-pituitary axis. Abbreviations: LHRH, luteinizing hormone-releasing hormone; GnRH, gonadotropin-releasing hormone; LH, luteinizing hormone; FSH, follicle-stimulating hormone.

the effects of increasing blood flow and inhibiting leukocyte accumulation on rat testicular arteries [46]. NO is also an important factor in regulating testicular vessel tension at different temperatures, at 34–37°C, disturbance of testicular arteries reaction appeared after L-NAME treatment [47]. Interestingly, NO content and NOS activity could be significantly increased at abnormal high temperatures caused by varicose spermatic veins in varicocele patients [48].

#### *3.1.1.2. Leydig cells*

Leydig cells, also known as interstitial cells, are adjacent to the seminiferous tubules in the testicle. Leydig cells produce and release testosterone under the control of LH and act as autocrine and/or paracrine hormones in gonad under the modulation of NO [49]. An immunohistochemistry study demonstrated that eNOS, nNOS and iNOS all expressed in cytoplasm of Leydig cells in rat testis [50]. Interestingly, a testis-specific subclass of nNOS, known as the truncated form of nNOS (TnNOS), has been recently identified as a major contributor to the formation of NO [51]. TnNOS has been found to be localized solely in the Leydig cells of the testes but neither in the Sertoli nor germ cells [51], which enable us to predict that NO may associate with functions of Leydig cells. Kozieł et al. found that NOS was able to act directly within the male gonad by means of regulating androgen secretion though Leydig cells [52]. Another study showed that stress-induced stimulation of the testicular NO signaling pathway leaded to the inhibition of steroidogenic enzymes [53]. But NOS seemingly exerted a biphasic effect on testosterone secretion [54]. At low concentrations, NO exerted a transient stimulatory effect on testosterone secretions mediated by cyclic GMP, whereas at high concentrations, it inhibited steroidogenesis by Leydig cells.

#### *3.1.1.3. Sertoli cells*

Spermatogenesis is a complex process in which Sertoli cells closely involve, and NOS also plays a crucial role in this process through Sertoli cells. Zini et al. [55] showed that eNOS protein located in Sertoli cells and some parts of germ cells in seminiferous tubules, especially in degenerating germ cells and spermatids in histologically normal testes. The iNOS was also found in Sertoli cells, as well as a small subset of pachytene spermatocytes and elongated spermatids in the normal testis [56]. However, iNOS expression could be very intense in Sertoli cells in pathological conditions, for example, the absence of seminiferous tubules [57]. iNOS involves in germ cell death in testicular ischemia-reperfusion injury model, and inhibition of iNOS could improve impaired spermatogenesis [58]. In cryptorchidism model, the transgene expression of eNOS increased testicular germ cell apoptosis. In iNOS⁄mice, the numbers of spermatocytes, spermatids and Sertoli cells per tubule were significantly more than those with wild-type testes [59]. A possible conclusion could be drawn that NO plays an important role in both numerical and functional regulation of key somatic cells in the testis, which in turn impacts on germ cells and their survivals during the process of daily sperm production.

#### *3.1.1.4. Epididymis*

The main functions of the epididymis are promoting spermatozoa mature and storing spermatozoa [60]. An immunostaining study in human epididymis showed that NOS almost exclusively located in the epithelium [55], and the greatest concentration was in the adluminal region [55]. It suggests that NOS may involve secretion and/or absorption of epididymal fluids, or in another way diffuse into the tubule lumen to affect nearby spermatozoa. Another study showed a similar distribution of NOS protein in rat epididymis, speculating that epididymal NOS protein might contribute to spermatozoa maturation [61].

#### **3.2. NOS and sperm function**

the effects of increasing blood flow and inhibiting leukocyte accumulation on rat testicular arteries [46]. NO is also an important factor in regulating testicular vessel tension at different temperatures, at 34–37°C, disturbance of testicular arteries reaction appeared after L-NAME treatment [47]. Interestingly, NO content and NOS activity could be significantly increased at abnormal high temperatures caused by varicose spermatic veins in varicocele patients [48].

Leydig cells, also known as interstitial cells, are adjacent to the seminiferous tubules in the testicle. Leydig cells produce and release testosterone under the control of LH and act as autocrine and/or paracrine hormones in gonad under the modulation of NO [49]. An immunohistochemistry study demonstrated that eNOS, nNOS and iNOS all expressed in cytoplasm of Leydig cells in rat testis [50]. Interestingly, a testis-specific subclass of nNOS, known as the truncated form of nNOS (TnNOS), has been recently identified as a major contributor to the formation of NO [51]. TnNOS has been found to be localized solely in the Leydig cells of the testes but neither in the Sertoli nor germ cells [51], which enable us to predict that NO may associate with functions of Leydig cells. Kozieł et al. found that NOS was able to act directly within the male gonad by means of regulating androgen secretion though Leydig cells [52]. Another study showed that stress-induced stimulation of the testicular NO signaling pathway leaded to the inhibition of steroidogenic enzymes [53]. But NOS seemingly exerted a biphasic effect on testosterone secretion [54]. At low concentrations, NO exerted a transient stimulatory effect on testosterone secretions mediated by cyclic GMP, whereas at high con-

Spermatogenesis is a complex process in which Sertoli cells closely involve, and NOS also plays a crucial role in this process through Sertoli cells. Zini et al. [55] showed that eNOS protein located in Sertoli cells and some parts of germ cells in seminiferous tubules, especially in degenerating germ cells and spermatids in histologically normal testes. The iNOS was also found in Sertoli cells, as well as a small subset of pachytene spermatocytes and elongated spermatids in the normal testis [56]. However, iNOS expression could be very intense in Sertoli cells in pathological conditions, for example, the absence of seminiferous tubules [57]. iNOS involves in germ cell death in testicular ischemia-reperfusion injury model, and inhibition of iNOS could improve impaired spermatogenesis [58]. In cryptorchidism model, the transgene expression of eNOS increased testicular germ cell apoptosis. In iNOS⁄mice, the numbers of spermatocytes, spermatids and Sertoli cells per tubule were significantly more than those with wild-type testes [59]. A possible conclusion could be drawn that NO plays an important role in both numerical and functional regulation of key somatic cells in the testis, which in turn impacts on germ cells and their survivals during the process of daily sperm production.

The main functions of the epididymis are promoting spermatozoa mature and storing spermatozoa [60]. An immunostaining study in human epididymis showed that NOS almost exclusively located in the epithelium [55], and the greatest concentration was in the adluminal

centrations, it inhibited steroidogenesis by Leydig cells.

*3.1.1.2. Leydig cells*

118 Nitric Oxide Synthase - Simple Enzyme-Complex Roles

*3.1.1.3. Sertoli cells*

*3.1.1.4. Epididymis*

Approximately 15% of couples suffer from infertility while male cause contributed to nearly 50% in these infertile couples [37]. Male reproduction is known to involve complicated aspects, such as spermatogenesis, sperm dynamics, sperm morphology and acrosome reaction. Increasing evidences have been indicating that NOS and NO are associated with male infertility [38].

#### *3.2.1. Sperm motility, morphology and viability*

Sperm motility is an essential factor for male fertility. Low sperm motility, also referred as asthenozoospermia, is one of the major causes to male infertility [62]. Previous study indicated that nearly 80% of semen samples from infertile males were defective in sperm motility [63]. Hellstrom et al. reported for the first time that sodium nitroprusside, a NO releaser, was beneficial for maintenance of thaw-sperm motility by reducing lipid peroxidative damage to sperm membranes. Significantly improved motion parameters of sperm were observed in semen samples treated with sodium nitroprusside in concentrations of 50 and 100 nM compared to control samples, and this beneficial effect maintained for 5–6 hours after thaw [64]. However, NO concentration in normozoospermic fertile men was observed to be significantly lower than those of asthenospermia infertile men [65]. In fact, the effect of NO seems to be double-sided, low concentration of NO improves sperm motility, while high concentration contributes to adverse effect [66]. Herrero et al. reported that a significant decrease on sperm motility was observed in semen samples treated with sodium nitroprusside in a higher concentration of 300 mM, and this effect could be blocked by hemoglobin, a scavenger of NO, as sperm motility in samples furtherly treated with hemoglobin was significantly higher than those without. While when the incubating concentration of sodium nitroprusside reduced to 150 mM, no modifications of sperm motility were found [67]. Besides, the other NO releaser, S-nitroso-N-acetylpenicillamine (0.012–0.6 mM), along with sodium nitroprusside (0.25–2.5 mM), was found to decrease percentage of forward progressive sperm motility and straight line velocity in a concentration-dependent manner [68].

As to sperm morphology and viability, the effects of NO reveal controversial contributions. a positive correlation between NO with defects in sperm morphology has been found in male with normal sperm rate ≥14% but a negative correlation with defects in sperm morphology in male with normal sperm rate <14% [69]. However, later study failed to find any significant association between NO production and sperm morphology [70]. Researchers reported that semen treated with 0.25–2.5 mM sodium nitroprusside revealed significantly less sperm bound to the zona pellucida compared with the control group which treated without NO [71], whereas some other researchers reported no any significant effect of NO on sperm viability [66, 69, 72]. And meanwhile, low concentration of NO also plays a role in the maintenance of sperm viability after cryopreservation and post-thaw sperm [65, 66, 68].

#### *3.2.2. Capacitation, hyperactivation and acrosome reaction*

Capacitation is a process in which spermatozoa acquire the ability to bind to the egg's zone pellucida and fertilize an oocyte during their transit in the female genital tract [73, 74]. Capacitation involves in some molecular events, and it was clear that low level of NO from NO-releasing agents induces human sperm capacitation [75]. Indeed, it has been reported that NO-releasing compounds significantly benefit the capacitation, whereas NO inhibitors decrease this process [76]. In fact, NO produced by spermatozoa involves in a cascade of molecular events of capacitation, which is needed over the course of this process [77–79].

Hyperactivation can be treated as a subcategory of capacitation. Hyperactivation of spermatozoa exhibits high amplitude and asymmetric flagellar movement, non-linear motility and penetrate the oocyte with strong propulsive force [70, 80]. The effect of NO on hyperactivation was found to be similar to which on sperm motility, little concentrations of NO increased spermatozoa hyperactivation, whereas excessive concentrations decreased the hyperactivated spermatozoa motility [81, 82].

Acrosome reaction denotes the process that capacitated and hyperactivated motility sperm binds to the zona pellucida and continues to pass through the exocytotic release of proteolytic enzymes from the acrosome so that to bind to the mature ovum. The amount of NO influenced the acrosome reaction, and increased amount of sperm was observed to undergo the acrosome reaction with the presence of NO donor compound [83]. Meanwhile, significantly increased amount of sperm was found to bind to membrane of the ovum with their plasma membrane [84].

#### *3.2.3. Sperm mitochondria*

Mitochondria in sperm activate as a generator which supply sperm with energy for the process of motility, acrosome reaction, oocyte fusion, fertilization and so on [85]. NO has been reported to involve in functions of mitochondrial that include biogenesis, remodeling and mitochondrial respiration [86–88]. Specially, different levels of NO could cause different sperm mitochondrial functions, low concentrations of NO enhanced the sperm motility, while NO with higher levels cause mitochondrial hyperpolarization and sperm apoptosis [64, 89]. This might explain the adverse effects of various concentrations NO on sperm motility.

#### **3.3. Single-nucleotide polymorphisms of NOS and male infertility**

Genetic variations are crucial etiological factors contribute to male infertility. Up to date, some single-nucleotide polymorphisms (SNPs) have been identified to involve in sperm defects and male infertility in ethnic populations. Polymorphisms T786C and G894T of eNOS were reported to decrease sperm motility and quantity by increasing the seminal oxidative stress in Egyptian infertile male population [90]. Similar results of G894T were reported in Italian and Iranian infertile male populations [91, 92], and this SNP also found to be associated with higher level of sperm DNA fragmentation in Chinese infertile males [93]. The polymorphism 4a4b, which refers to a sequence variant with variable sequence of tandem 4a4b repeats in intron 4, was found to be associated with poor sperm morphology and male infertility in a Korean and Chinese population [94, 95]. Associations between SNPs of NOS and male infertility are under exploration, which would be promising tools for diagnosis or further curing male infertility.

#### **3.4. Possible therapy strategies of NOS on male infertility**

Increasing evidences has been showing that inappropriate concentration level of NO may contribute to male infertility in some extend by means of decreasing sperm motility and normal sperm morphology, reducing efficiency of capacitation and acrosome action. It is reasonable to consider possible therapy strategies to the utilization of NOS donors or inhibitors so that to adjust the concentration of NO to the "right" level. In fact, significantly higher fertile rate was observed in animal experiment in vitro, and further researches would be needed to warrant the potential benefits for human beings.

#### **4. NOS and prostate carcinoma**

*3.2.2. Capacitation, hyperactivation and acrosome reaction*

120 Nitric Oxide Synthase - Simple Enzyme-Complex Roles

vated spermatozoa motility [81, 82].

*3.2.3. Sperm mitochondria*

Capacitation is a process in which spermatozoa acquire the ability to bind to the egg's zone pellucida and fertilize an oocyte during their transit in the female genital tract [73, 74]. Capacitation involves in some molecular events, and it was clear that low level of NO from NO-releasing agents induces human sperm capacitation [75]. Indeed, it has been reported that NO-releasing compounds significantly benefit the capacitation, whereas NO inhibitors decrease this process [76]. In fact, NO produced by spermatozoa involves in a cascade of molecular events of capacitation, which is needed over the course of this process [77–79]. Hyperactivation can be treated as a subcategory of capacitation. Hyperactivation of spermatozoa exhibits high amplitude and asymmetric flagellar movement, non-linear motility and penetrate the oocyte with strong propulsive force [70, 80]. The effect of NO on hyperactivation was found to be similar to which on sperm motility, little concentrations of NO increased spermatozoa hyperactivation, whereas excessive concentrations decreased the hyperacti-

Acrosome reaction denotes the process that capacitated and hyperactivated motility sperm binds to the zona pellucida and continues to pass through the exocytotic release of proteolytic enzymes from the acrosome so that to bind to the mature ovum. The amount of NO influenced the acrosome reaction, and increased amount of sperm was observed to undergo the acrosome reaction with the presence of NO donor compound [83]. Meanwhile, significantly increased amount of

Mitochondria in sperm activate as a generator which supply sperm with energy for the process of motility, acrosome reaction, oocyte fusion, fertilization and so on [85]. NO has been reported to involve in functions of mitochondrial that include biogenesis, remodeling and mitochondrial respiration [86–88]. Specially, different levels of NO could cause different sperm mitochondrial functions, low concentrations of NO enhanced the sperm motility, while NO with higher levels cause mitochondrial hyperpolarization and sperm apoptosis [64, 89]. This might explain the adverse effects of various concentrations NO on sperm motility.

Genetic variations are crucial etiological factors contribute to male infertility. Up to date, some single-nucleotide polymorphisms (SNPs) have been identified to involve in sperm defects and male infertility in ethnic populations. Polymorphisms T786C and G894T of eNOS were reported to decrease sperm motility and quantity by increasing the seminal oxidative stress in Egyptian infertile male population [90]. Similar results of G894T were reported in Italian and Iranian infertile male populations [91, 92], and this SNP also found to be associated with higher level of sperm DNA fragmentation in Chinese infertile males [93]. The polymorphism 4a4b, which refers to a sequence variant with variable sequence of tandem 4a4b repeats in intron 4, was found to be associated with poor sperm morphology and male infertility in a

sperm was found to bind to membrane of the ovum with their plasma membrane [84].

**3.3. Single-nucleotide polymorphisms of NOS and male infertility**

Prostate carcinoma is one of the most common cancers among men and second in cancerrelated deaths in the United States. An estimated study predicted that there will be 180, 890 new prostate carcinoma cases and 26, 120 deaths due to the disease in the country in 2016 [96]. Etiological studies implicated that multiple reasons involved in prostate carcinoma susceptibility, such as dietary, environment, hormone status and genetic factors [97]. Growing studies indicated that NOS and NO system play crucial roles in progression of human prostate carcinoma [98–100].

The physiological functions of NO are dependent primarily on concentrations. Low concentration of NO acted as a signal transducer and affects many physiological processes including blood flow regulation, platelet activity, iron homeostasis, cell proliferation and neurotransmission, whereas, in high concentrations, it exerted a cytotoxic protective effect, for example, to against pathogens and perhaps tumors [101, 102].

#### **4.1. Role of nitric oxide synthase in cancer biology**

The roles of NOS and NO on DNA damage, apoptosis, cell cycle, enhancement of cell proliferation, angiogenesis and metastasis are currently viewed, and NO was found to be associated with tumor environment, for example, the vasculature cells and other stromal cells [103–105]. Research also indicated that NOS2 expression was correlated with tumor vascularization, accumulations of p53 mutations and activation of epidermal growth factor receptor, even could be treated as an independent predictor of poor survival in women with estrogen receptor (ER)-negative breast tumors [106]. Low concentrations of NO acted as a promotional role in angiogenesis which stimulates tumor progression by providing blood flow access to the tumor and subsequently resulting in cell proliferation. On the contrary, high levels of NO tend to be cytotoxic to cancer cells [107]. While in animal models, iNOS overexpression produced either pro-tumor or anti-tumor effect on tumor growth, these alterable effects seem to be dependent on the tumor microenvironment and the tumor type itself [104, 108]. The effects of NO possibly differ in expression level of iNOS, duration and timing of NO delivery, the microenvironment, the genetic background and the cell type (**Figure 3**) [109].

#### **4.2. NOS and proliferation of prostate carcinoma**

NO generated by eNOS or iNOS might be involved in prostate proliferation. At low concentrations, NO acted as a signaling molecule regulating smooth muscle relaxation and blood flow, neurotransmission, platelet activity, iron homeostasis, cell survival and proliferation, while at high concentrations acted as modulating immune-mediated anti-tumor activities [110, 111]. Concentration of NO less than 100nM had an effect of preventing certain cell types from apoptosis and thereby favors tumorigenesis and progression [112]. Higher expression of iNOS was detected in cancer specimens than that in normal tissues of prostate carcinoma patient. Aaltoma et al. also demonstrated a positive association between expression level of iNOS and rapid cancer cell proliferation rate, dedifferentiation and advanced stage cancer [113]. A recent study has shown that NO also regulated cell proliferation in a pathway of CPD-Arg-NO [114].

#### **4.3. Nitric oxide synthase and angiogenesis of prostate carcinoma**

Angiogenesis is a critical molecular event in tumor progression [115, 116]. Epidermal growth factor receptor (EGFR) signaling pathway, tumor suppressor p53 and VEGF, which are collective mediators that exacerbate angiogenesis can be stimulated by NO [115, 117]. The involvement of eNOS in the NO-induced human endothelial and prostate carcinoma cell migration was further warranted [116]. Recent research also reported that NO played vital roles in maintaining blood supply for prostate carcinoma, and an anti-tumor vascular activity effect revealed with presence in inhibition of NOS [115].

#### **4.4. Single-nucleotide polymorphisms of NOS and susceptibility of prostate carcinoma**

Several studies suggested that polymorphisms of some NOS genes were genetic susceptibility factors for prostate carcinoma, especially for aggressive diseases [118, 119]. A plethora of metaanalyses has identified eNOS gene polymorphisms as strong susceptibility factors for the progression toward prostate carcinoma [120]. Another study also reported that NOS3 gene

**Figure 3.** Roles of NO in prostate cancer. Abbreviation: NO, nitric oxide.

polymorphisms were genetic susceptibility factors for the progression of prostate carcinoma and poor patient outcomes [121]. A meta-analysis conducted by Zhao et al. suggested that eNOS gene 894G > T polymorphism contributed to aggravate the onset of prostate carcinoma in males [122]. Nikolic et al [123] also corroborated the involvement of eNOS or NOS3 gene in the pathogenesis of prostate carcinoma. NOS3 rs1799983 polymorphism augmented the risk of prostate carcinoma in various populations. As one of the possible mechanisms, the involvement of NO receptor component, sGC-1, in mediating the proliferation of prostate carcinogenesis, has been surmised [124].

#### **4.5. Possible therapy strategies of NOS on prostate carcinoma**

Anti-cancer agents such as gold lotion have successfully demonstrated their anti-carcinogenic potential through the regulation of both iNOS and eNOS [125–127]. Yu et al. [128] also elucidated the significance of eNOS as a seemingly promising strategy for targeting anti-androgen resistant prostate carcinoma. Arginine-releasing compounds such as carboxypeptidase-D increased NO production, which slackened progression of prostate carcinoma so that prolonged survival time [129]. NO-donor drugs also have been under increasing explorations. A few NO-donor drugs have been confirmed to have favorable anticancer activity and could be potential anticancer therapies [3, 130]. GIT-27NO, a novel NO donor, inhibited the growth of PC3 and LnCap prostate carcinoma cells xenografted into nude mice in a concentration-dependent manner [131]. And DETA-NONOate was revealed to inhibit epithelial-mesenchymal transition (EMT) and invasion of human prostate metastatic cells by producing large amount of NO [132]. It is sensible that novel NOS-based therapeutics may prove valuable in the future treatment of prostate carcinoma.

#### **5. NOS and other urinary and male reproductive diseases**

#### **5.1. Peyronie disease**

of NO possibly differ in expression level of iNOS, duration and timing of NO delivery, the

NO generated by eNOS or iNOS might be involved in prostate proliferation. At low concentrations, NO acted as a signaling molecule regulating smooth muscle relaxation and blood flow, neurotransmission, platelet activity, iron homeostasis, cell survival and proliferation, while at high concentrations acted as modulating immune-mediated anti-tumor activities [110, 111]. Concentration of NO less than 100nM had an effect of preventing certain cell types from apoptosis and thereby favors tumorigenesis and progression [112]. Higher expression of iNOS was detected in cancer specimens than that in normal tissues of prostate carcinoma patient. Aaltoma et al. also demonstrated a positive association between expression level of iNOS and rapid cancer cell proliferation rate, dedifferentiation and advanced stage cancer [113]. A recent study has shown that NO also regulated cell proliferation in a pathway of CPD-Arg-NO [114].

Angiogenesis is a critical molecular event in tumor progression [115, 116]. Epidermal growth factor receptor (EGFR) signaling pathway, tumor suppressor p53 and VEGF, which are collective mediators that exacerbate angiogenesis can be stimulated by NO [115, 117]. The involvement of eNOS in the NO-induced human endothelial and prostate carcinoma cell migration was further warranted [116]. Recent research also reported that NO played vital roles in maintaining blood supply for prostate carcinoma, and an anti-tumor vascular activity effect

**4.4. Single-nucleotide polymorphisms of NOS and susceptibility of prostate carcinoma**

Several studies suggested that polymorphisms of some NOS genes were genetic susceptibility factors for prostate carcinoma, especially for aggressive diseases [118, 119]. A plethora of metaanalyses has identified eNOS gene polymorphisms as strong susceptibility factors for the progression toward prostate carcinoma [120]. Another study also reported that NOS3 gene

microenvironment, the genetic background and the cell type (**Figure 3**) [109].

**4.3. Nitric oxide synthase and angiogenesis of prostate carcinoma**

**4.2. NOS and proliferation of prostate carcinoma**

122 Nitric Oxide Synthase - Simple Enzyme-Complex Roles

revealed with presence in inhibition of NOS [115].

**Figure 3.** Roles of NO in prostate cancer. Abbreviation: NO, nitric oxide.

Peyronie disease (PD) is an intractable, sexually dysfunctional disease resulting in penile curvature, penile pain, penile deformity, difficulty with coitus, shortening, hinging, narrowing and ED. Mechanisms of PD have not been fully elucidated. A recent hypothesis was that the recurrent microtrauma of the tunica albuginea caused small damages that activated processes of wound healing and fibrotic plaque development during sexual intercourse [133]. Inflammatory cells and iNOS accumulated in the process of wound healing, the increased NO then led to the myofibroblasts and proliferation of fibroblasts and redundant collagen between the layers of the tunica albuginea (penile plaque) [134]. Although surgical therapy is now the first-option for PD patients, researchers are focusing on the nonsurgical treatments of PD, and NOs inhibitors might be a promising choice [135].

#### **5.2. Priapism**

Priapism is defined as a persistent and painful erection that lasts longer than 4 hours without sexual stimulation and can lead to ED [136]. The relation between penile erection and production of NOS has been well investigated: nNOS and eNOS were the causes of both the initiation and maintenance phases of penile erection [137]. However, decreased function in NO generated by decreased activation of eNOS resulted in PDE5 downregulation that was thought to be a derivate of NO and, therefore, reduced basal levels of PDE5 and caused priapism [138].

#### **5.3. Cryptorchidism**

Cryptorchidism denotes failure of the movement of the testis to the scrotum, and in most cases, it raises risk of testicular germ cell cancer and subfertility later in patients' life course. Testicular germ cell apoptosis which causes by exposure of testicular in elevated temperature and oxidative stress is the primary etiology of infertility. Animal models with cryptorchidism induced by surgery revealed that eNOS played a significant role in mouse spermatogenesis in cryptorchidisminduced apoptosis [139]. Contemporaneously, reduced rate of testicular atrophy was observed in heterozygous Hoxa 11 knockout mice which had congenital bilateral cryptorchidism when early treated with Nomega-nitro-L-arginine methyl ester (L-NAME), a NOS inhibitor [140].

#### **6. Conclusion**

It has been becoming evident that redox regulation driven by NOS and NO represents a promising tool for exploring fundamental diseases process and new development of strategies to treat urinary, male reproductive and sexual diseases.

#### **Acknowledgements**

This work was supported by Foundation of Science and Technology Projects of Shenzhen (Grant Number: JCYJ20140415162542992).

#### **Author details**

Qingfeng Yu1,2, Tieqiu Li<sup>3</sup> , Jingping Li<sup>4</sup> , Liren Zhong<sup>5</sup> and Xiangming Mao<sup>1</sup> \*

\*Address all correspondence to: dr.xiangmingmao@gmail.com

1 Department of Urology, Zhujiang Hospital, Southern Medical University, Guangzhou, PR China

2 Department of Urology, Ludwig-Maximilians University, Munich, Germany

3 Department of Urology, Hunan Provincial People's Hospital, The First Affiliated Hospital of Hunan Normal University, Changsha, PR China

4 Department of Reproductive Endocrinology, Women's Hospital, School of Medicine, Reproductive Medicine Center, Zhejiang University, Hangzhou, Zhejiang, PR China

5 Department of Urology, Southern Medical University affiliated Nanfang Hospital, Guangzhou, PR China

#### **References**

and maintenance phases of penile erection [137]. However, decreased function in NO generated by decreased activation of eNOS resulted in PDE5 downregulation that was thought to be a derivate of NO and, therefore, reduced basal levels of PDE5 and caused priapism [138].

Cryptorchidism denotes failure of the movement of the testis to the scrotum, and in most cases, it raises risk of testicular germ cell cancer and subfertility later in patients' life course. Testicular germ cell apoptosis which causes by exposure of testicular in elevated temperature and oxidative stress is the primary etiology of infertility. Animal models with cryptorchidism induced by surgery revealed that eNOS played a significant role in mouse spermatogenesis in cryptorchidisminduced apoptosis [139]. Contemporaneously, reduced rate of testicular atrophy was observed in heterozygous Hoxa 11 knockout mice which had congenital bilateral cryptorchidism when early treated with Nomega-nitro-L-arginine methyl ester (L-NAME), a NOS inhibitor [140].

It has been becoming evident that redox regulation driven by NOS and NO represents a promising tool for exploring fundamental diseases process and new development of strate-

This work was supported by Foundation of Science and Technology Projects of Shenzhen

, Liren Zhong<sup>5</sup>

1 Department of Urology, Zhujiang Hospital, Southern Medical University, Guangzhou, PR

3 Department of Urology, Hunan Provincial People's Hospital, The First Affiliated Hospital of

4 Department of Reproductive Endocrinology, Women's Hospital, School of Medicine, Repro-

5 Department of Urology, Southern Medical University affiliated Nanfang Hospital, Guangzhou,

2 Department of Urology, Ludwig-Maximilians University, Munich, Germany

ductive Medicine Center, Zhejiang University, Hangzhou, Zhejiang, PR China

and Xiangming Mao<sup>1</sup>

\*

gies to treat urinary, male reproductive and sexual diseases.

, Jingping Li<sup>4</sup>

\*Address all correspondence to: dr.xiangmingmao@gmail.com

Hunan Normal University, Changsha, PR China

**5.3. Cryptorchidism**

124 Nitric Oxide Synthase - Simple Enzyme-Complex Roles

**6. Conclusion**

**Acknowledgements**

**Author details**

China

PR China

Qingfeng Yu1,2, Tieqiu Li<sup>3</sup>

(Grant Number: JCYJ20140415162542992).


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#### **Chapter 8**

### **Nitric Oxide: Key Features in Spermatozoa**

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Florentin-Daniel Staicu and Carmen Matas Parra

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/67551

#### **Abstract**

Several *in vitro* studies have pointed to the importance of nitric oxide (NO) in the female and male reproductive system in mammals. Its functions vary from preventing oocyte aging, improving the integrity of the microtubular spindle apparatus in aged oocytes, modulating the contraction of the oviduct, to regulating sperm physiology by affecting the motility, inducing chemotaxis in spermatozoa, regulating tyrosine phosphorylation, enhancing the sperm-zona pellucida binding ability, and modulating the acrosomal reaction. In spermatozoa, NO exerts its functions in different ways, which involve key elements such as the soluble isoform of guanylate cyclase, cyclic guanosine monophosphate (cGMP), cyclic adenosine monophosphate (cAMP), protein kinase A (PKA), adenylate cyclase, and the extracellular signal-regulated kinase (ERK) pathway. Furthermore, NO leads to the S-nitrosylation of several sperm proteins, among them a substantial group associated with energy generation and cell movement, but also with signal transduction, suggesting a role for S-nitrosylation in sperm motility and in modulating the sperm function, respectively. In this chapter, an overview of how NO modulates the sperm physiology is presented, based on the knowledge acquired to this day.

**Keywords:** nitric oxide, nitric oxide synthase, S-nitrosylation, spermatozoa, fertilization

#### **1. Introduction**

NO is a small hydrophobic molecule which can easily diffuse through biological membranes [1]. *In vivo*, it is synthesized during the conversion of L-arginine to L-citrulline by nitric oxide synthase, with the help of co-factors such as the reduced form of nicotinamide adenine dinucleotide phosphate, flavin mononucleotide, flavin adenine dinucleotide, and tetrahydrobiopterin [2].

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The nitric oxide synthase (NOS) may be found in three different isoforms. Two of them, the endothelial and the neuronal NOS (eNOS, nNOS), require calcium/calmodulin to be activated and are responsible for the continuous basal release of NO. The third isoform, known as inducible NOS (iNOS), is calcium independent [3, 4]. Since NOS activity depends on the availability of its substrate and its co-factors, all these elements jointly determine the cellular rates of NO synthesis [5].

Substantial evidence indicates that NO is a crucial biological messenger involved in a wide variety of physiological and pathological processes in different systems in mammals, including the vascular, nervous, and reproductive system [1, 6].

#### **2. NOS/NO duo in the reproductive system**

The NOS/NO duo regulates key functions in both the female and male reproductive systems [6].

All three isoforms of NOS have been identified in the oviduct [7, 8], oocytes, and cumulus and corona cells [9, 10] of several species [7, 11, 12]. The expression of NOS isoforms differs during the estrous cycle in the follicles as well as in the oviduct [13]. Tao *et al*. [10] showed that the immunoreactivity of eNOS in early antral follicles was restricted to the oocyte and increased from small and medium to large follicle-enclosed oocytes. In contrast, no immunoreactivity for iNOS was found in primordial, early antral follicle, or the cumulus-oocyte complexes aspirated from small and medium follicles.

In the oviduct, the endogenous basal release of NO regulates its contraction and the ciliary beating of the ciliated epithelial cells and induces chemotaxis in human spermatozoa via activation of the nitric oxide/soluble isoform of guanylate cyclase/cyclic guanosine monophosphate pathway (NO/sGC/cGMP) [14–16]. NO forms a vital component of the oocyte microenvironment and has been positively implicated in meiotic resumption [17], in preventing oocyte aging and improving the integrity of the microtubular spindle apparatus in aged oocytes [18]. It may also contribute as an anti-platelet agent during implantation [19].

As far as the male gamete is concerned, research was first concentrated on determining the effects of NO-releasing compounds on sperm motility and viability. Low concentrations of sodium nitroprusside (SNP), an NO-releasing compound, stimulated sperm hyperactivation in mouse, fish, and hamster [20–22] and were beneficial to the maintenance of post-thaw human sperm motility [23]. On the other hand, high concentrations of NO-releasing compounds decreased sperm motility [20, 24–26].

Numerous studies have also been conducted to determine the presence and localization of NOS in sperm from several species (**Table 1**). For example, Herrero *et al*. [27] located nNOS in the head of freshly ejaculated human spermatozoa, with a more concentrated fluorescent staining toward the equatorial region. O'Bryan *et al*. [28] described the pattern of eNOS expression in human spermatozoa, finding that morphologically normal spermatozoa exhibited post-acrosomal and equatorial eNOS immunostaining. Interestingly, though, abnormally shaped sperm cells exhibited aberrant staining, especially in the midpiece and/or head region, which correlated negatively with the percentage of motile sperm.


The nitric oxide synthase (NOS) may be found in three different isoforms. Two of them, the endothelial and the neuronal NOS (eNOS, nNOS), require calcium/calmodulin to be activated and are responsible for the continuous basal release of NO. The third isoform, known as inducible NOS (iNOS), is calcium independent [3, 4]. Since NOS activity depends on the availability of its substrate and its co-factors, all these elements jointly determine the cellular rates of NO synthesis [5]. Substantial evidence indicates that NO is a crucial biological messenger involved in a wide variety of physiological and pathological processes in different systems in mammals, includ-

The NOS/NO duo regulates key functions in both the female and male reproductive systems [6]. All three isoforms of NOS have been identified in the oviduct [7, 8], oocytes, and cumulus and corona cells [9, 10] of several species [7, 11, 12]. The expression of NOS isoforms differs during the estrous cycle in the follicles as well as in the oviduct [13]. Tao *et al*. [10] showed that the immunoreactivity of eNOS in early antral follicles was restricted to the oocyte and increased from small and medium to large follicle-enclosed oocytes. In contrast, no immunoreactivity for iNOS was found in primordial, early antral follicle, or the cumulus-oocyte complexes aspi-

In the oviduct, the endogenous basal release of NO regulates its contraction and the ciliary beating of the ciliated epithelial cells and induces chemotaxis in human spermatozoa via activation of the nitric oxide/soluble isoform of guanylate cyclase/cyclic guanosine monophosphate pathway (NO/sGC/cGMP) [14–16]. NO forms a vital component of the oocyte microenvironment and has been positively implicated in meiotic resumption [17], in preventing oocyte aging and improving the integrity of the microtubular spindle apparatus in aged

As far as the male gamete is concerned, research was first concentrated on determining the effects of NO-releasing compounds on sperm motility and viability. Low concentrations of sodium nitroprusside (SNP), an NO-releasing compound, stimulated sperm hyperactivation in mouse, fish, and hamster [20–22] and were beneficial to the maintenance of post-thaw human sperm motility [23]. On the other hand, high concentrations of NO-releasing com-

Numerous studies have also been conducted to determine the presence and localization of NOS in sperm from several species (**Table 1**). For example, Herrero *et al*. [27] located nNOS in the head of freshly ejaculated human spermatozoa, with a more concentrated fluorescent staining toward the equatorial region. O'Bryan *et al*. [28] described the pattern of eNOS expression in human spermatozoa, finding that morphologically normal spermatozoa exhibited post-acrosomal and equatorial eNOS immunostaining. Interestingly, though, abnormally shaped sperm cells exhibited aberrant staining, especially in the midpiece and/or head region,

oocytes [18]. It may also contribute as an anti-platelet agent during implantation [19].

ing the vascular, nervous, and reproductive system [1, 6].

**2. NOS/NO duo in the reproductive system**

rated from small and medium follicles.

138 Nitric Oxide Synthase - Simple Enzyme-Complex Roles

pounds decreased sperm motility [20, 24–26].

which correlated negatively with the percentage of motile sperm.

**Table 1.** Summary of *in vitro* studies and the techniques used to identify NOS isoforms in different species.

NOSs were revealed in mature mouse spermatozoa by means of biochemical techniques and Western blot. Herrero *et al*. [29] showed that mouse spermatozoa can synthesize L-citrulline, depending on the concentration of L-arginine present in the incubation medium while different concentrations of N(G)-nitro-L-arginine methyl ester (L-NAME) inhibit the formation of the amino acid. Furthermore, when sperm protein extracts were incubated under denaturing and nonreducing conditions and then subjected to immunoblotting assay, a protein fraction of 140 kDa was recognized by the three anti-NOS antibodies.

Bull spermatozoa were examined for the presence of constitutive NOS [30]. NO generation seemed to be enhanced by L-arginine and abolished by the NOS-inhibitor, L-NAME. In addition, Meiser and Schulz [30] verified the presence of NOS in bull sperm cells by immunohistochemistry, which was confirmed by Western blot. Confocal laser microscopy localized nNOS-related immunofluorescence at the acrosome cap and the main part of the flagellum. The same technique also identified eNOS staining spread over the spermatozoan head. Moreover, when these findings were confirmed by Western blot, immunoreactive bands at 161 kDa (nNOS) and 133 kDa (eNOS) were identified.

Hou *et al*. [31] investigated whether boar sperm can generate NO, finding that porcine spermatozoa synthesized low levels of NO under noncapacitating conditions, but that the NO concentration almost doubled when sperms were capacitated. Furthermore, NO production was significantly inhibited when capacitated sperms were treated with L-NAME. In another study [32], Western blot analysis was performed to identify NOS enzymes in boar sperm samples. The immunoblots showed three distinct bands: ~160, ~130, and ~135 kDa, corresponding to nNOS, iNOS, and eNOS, respectively.

NO production was evaluated in stallion spermatozoa before and after freezing/thawing [33] by means of flow cytometry, after loading the sperm suspension with an NO detection probe. NO synthesis was positively correlated with sperm motility after thawing and, interestingly, the presence of egg yolk in the semen extender radically reduced the amount of NO produced. The authors further investigated in fresh and frozen/thawed stallion sperm the presence of NOS enzymes by Western blot, using anti-nNOS, anti-eNOS, and anti-universal NOS antibodies. Two bands of approximately 83 kDa and 96 kDa were labeled by the antibodies anti-nNOS and anti-eNOS, respectively. Moreover, the other antibody, which recognized an epitope present in all the NOS isoforms described so far, showed two similar bands of 84 and 92 kDa.

Recently, Liman and Alan [34] investigated the localization of NOS isoforms in spermatozoa within the intratesticular and excurrent duct systems of adult domestic cats. Overall, the spermatozoa head did not exhibit immunoreactivity. On the other hand, immunoreactivity for all three isoforms was observed in the flagellum, in the proximal cytoplasmic droplets of spermatozoa (located in the neck region) within the lumen of the intratesticular and efferent ducts, in the epididymal duct of the caput epididymis, and in the distal cytoplasmic droplets of spermatozoa (located at the mid-principal piece junction of the tail) within the lumen of corpus and cauda epididymis and the vas deferens.

#### **3. Role of NO on sperm functionality**

Several *in vitro* studies were conducted in order to determine the effects that NO has on sperm physiology (**Figure 1**). It has been shown that NO affects sperm motility [28, 35, 36], acts as chemoattractant [16, 37], regulates the tyrosine phosphorylation of different sperm proteins [38, 39], enhances the sperm-zona pellucida binding ability [40], and modulates the acrosomal reaction [41, 42].

In detail, NO seems to play an important role in the maintenance of sperm motility at physiological levels. A study [36] showed that the basal release of NO by spermatozoa from normozoospermic samples tended to be greater than that from asthenozoospermic samples, suggesting a physiological and beneficial role for endogenous NO in the preservation of sperm motility. These observations agree with a previous report that normozoospermic spermatozoa express more NOS and generate more nitrite than asthenozoospermic spermatozoa [35]. On the other hand, as previously mentioned, it has been shown that spermatozoa with an abnormal morphology show aberrant staining for eNOS, which was negatively correlated with the motility [28]. A detrimental effect on motility has also been reported by Rosselli *et al*. [24] and Weinberg *et al*. [25] when millimolar concentrations of exogenous NO donors were added to sperm samples.

was significantly inhibited when capacitated sperms were treated with L-NAME. In another study [32], Western blot analysis was performed to identify NOS enzymes in boar sperm samples. The immunoblots showed three distinct bands: ~160, ~130, and ~135 kDa, corresponding

NO production was evaluated in stallion spermatozoa before and after freezing/thawing [33] by means of flow cytometry, after loading the sperm suspension with an NO detection probe. NO synthesis was positively correlated with sperm motility after thawing and, interestingly, the presence of egg yolk in the semen extender radically reduced the amount of NO produced. The authors further investigated in fresh and frozen/thawed stallion sperm the presence of NOS enzymes by Western blot, using anti-nNOS, anti-eNOS, and anti-universal NOS antibodies. Two bands of approximately 83 kDa and 96 kDa were labeled by the antibodies anti-nNOS and anti-eNOS, respectively. Moreover, the other antibody, which recognized an epitope present in all the NOS isoforms described so far, showed two similar bands

Recently, Liman and Alan [34] investigated the localization of NOS isoforms in spermatozoa within the intratesticular and excurrent duct systems of adult domestic cats. Overall, the spermatozoa head did not exhibit immunoreactivity. On the other hand, immunoreactivity for all three isoforms was observed in the flagellum, in the proximal cytoplasmic droplets of spermatozoa (located in the neck region) within the lumen of the intratesticular and efferent ducts, in the epididymal duct of the caput epididymis, and in the distal cytoplasmic droplets of spermatozoa (located at the mid-principal piece junction of the tail) within the lumen of

Several *in vitro* studies were conducted in order to determine the effects that NO has on sperm physiology (**Figure 1**). It has been shown that NO affects sperm motility [28, 35, 36], acts as chemoattractant [16, 37], regulates the tyrosine phosphorylation of different sperm proteins [38, 39], enhances the sperm-zona pellucida binding ability [40], and modulates the acrosomal

In detail, NO seems to play an important role in the maintenance of sperm motility at physiological levels. A study [36] showed that the basal release of NO by spermatozoa from normozoospermic samples tended to be greater than that from asthenozoospermic samples, suggesting a physiological and beneficial role for endogenous NO in the preservation of sperm motility. These observations agree with a previous report that normozoospermic spermatozoa express more NOS and generate more nitrite than asthenozoospermic spermatozoa [35]. On the other hand, as previously mentioned, it has been shown that spermatozoa with an abnormal morphology show aberrant staining for eNOS, which was negatively correlated with the motility [28]. A detrimental effect on motility has also been reported by Rosselli *et al*. [24] and Weinberg *et al*. [25] when millimolar concentrations of exogenous NO donors were

to nNOS, iNOS, and eNOS, respectively.

140 Nitric Oxide Synthase - Simple Enzyme-Complex Roles

corpus and cauda epididymis and the vas deferens.

**3. Role of NO on sperm functionality**

of 84 and 92 kDa.

reaction [41, 42].

added to sperm samples.

**Figure 1.** Some aspects of the sperm physiology modulated by the NOS/NO system. At physiological levels, endogenous NO has a beneficial role in maintaining sperm motility, enhances tyrosine phosphorylation, which, in turn, promotes the capacitation process. NO also increases the sperm-zona pellucida binding ability and leads to a rise in the percentage of reacted spermatozoa, especially in the presence of follicular fluid or protein-enriched extracts of follicular fluid.

It has been suggested that, upon approaching and entering the cumulus oophorus, both NO and progesterone, which are synthesized by the cumulus cells [9, 10, 43–45], provide a synergistic stimulus to mobilize stored calcium in the sperm neck/midpiece [46]. As a consequence, they can modulate flagellar activity and contribute to the hyperactivation that is vital for penetration of the oocyte vestments [47].

Interestingly, it has also been suggested that NO may exert a chemoattractant effect on spermatozoa. In fact, the percentage of mouse sperm migrating toward the medium containing an NO donor increased significantly [37]. Similar results were obtained when human spermatozoa were exposed to an NO donor [16]. In the latter case, the signal transduction pathway was also studied. It was proposed that NO exerts its chemoattractant effect through the activation of the NO/sGC/cGMP pathway, since the use of an NO scavenger and/or an sGC and cGMPdependent protein kinase inhibitor reverted the NO donor-induced migration of sperm.

Since tyrosine phosphorylation in different sperm proteins is associated with the capacitation process [48], this aspect was investigated in order to further define the involvement of NO in capacitation. Herrero *et al*. [38] observed an increase in tyrosine phosphorylation when human sperm capacitation was accelerated by an NO-releasing compound. On the other hand, when sperm capacitation was inhibited by L-NAME, there was an attenuation in the tyrosine phosphorylation of sperm proteins. In addition, Thundathil *et al*. [39] reported that L-NAME prevented, and a NO donor promoted, the increase in threonine, glutamine, and tyrosine phosphorylation in human spermatozoa. Furthermore, the addition of L-arginine reversed the inhibitory effect of L-NAME on the capacitation and the associated increase in phosphorylation.

The correlation between NO and sperm-zona pellucida binding ability was investigated by Sengoku *et al*. [40], who reported that when treated with low concentrations of a NO donor, the number of spermatozoa which binds to the hemizona is higher than in sperm treated with a higher concentration. Additionally, a NO quencher lowered the enhancement of sperm binding by the NO donor.

NO also seems to modulate the acrosome reaction. The percentage of acrosome loss induced by human follicular fluid or by calcium ionophore was studied when human spermatozoa were capacitated in the presence/absence of NO-releasing compounds or NOS inhibitors [38]. NO donors induced sperm cells to respond faster to human follicular fluid, whereas NOS inhibitors decreased the percentage of acrosome reaction. Similar results were obtained by Revelli *et al*. [41], who showed that different NO-releasing compounds were able to increase the percentage of reacted spermatozoa in the presence of protein-enriched extracts of human follicular fluid. Also, hemoglobin, a NO scavenger, inhibited the follicular fluid–induced acrosomal reaction. In an in-depth analysis of the signaling pathway of the nitric oxide–induced acrosome reaction in human spermatozoa [42], the authors suggested that the acrosome reactioninducing effect of exogenous NO on capacitated human spermatozoa is accomplished via the NO/sGC/cGMP pathway, which leads to the activation of cGMP-dependent protein kinase (PKG). In fact, both the intracellular cGMP levels and the percentage of reacted spermatozoa were significantly increased after incubation with SNP. Furthermore, the SNP-induced acrosome reaction was significantly reduced in the presence of sGC inhibitors, a reduction that was reversed by the addition of a cell-permeating cGMP analogue to the incubation medium. Finally, PKG inhibition reduced the SNP-induced acrosome reaction.

#### **4. NOS-activating molecules**

As previously stated, NOS activity depends on the availability of its substrate and co-factors [5]. However, the scientific literature does not include many studies on the molecules present in the female reproductive tract which may activate, in one way or another, NOS enzymes in spermatozoa.

Starting from follicular fluid samples, Revelli *et al*. [41] obtained a protein-enriched follicular fluid solution (PFF), which was then used to study its effects on NOS activity, citrulline synthesis, and acrosome reaction in human sperm. Interestingly, this study showed for the first time that the endogenous NOS activity of human sperm may be increased by PFF. Moreover, the authors demonstrated that PFF-mediated induction of sperm NOS activity leads to acrosome reaction in the same cells, thereby, establishing a link between follicle-derived substances, the activation of NO synthesis in sperm and biological responses.

Furthermore, the increase in NO synthesis mediated by PFF was not associated with a rise in the expression of NOS catalytic units, which is not surprising since specialized cells possess very poor, if any, transcriptional activity [41]. The authors hypothesized that PFF first determines the transient enzyme activation of sperm NOS, which is subsequently strengthened by a more stable modification of the enzyme.

However, more studies should be performed in order to identify the NOS-activating molecule(s) in the follicular fluid.

#### **5. NO pathway in spermatozoa**

NO in capacitation. Herrero *et al*. [38] observed an increase in tyrosine phosphorylation when human sperm capacitation was accelerated by an NO-releasing compound. On the other hand, when sperm capacitation was inhibited by L-NAME, there was an attenuation in the tyrosine phosphorylation of sperm proteins. In addition, Thundathil *et al*. [39] reported that L-NAME prevented, and a NO donor promoted, the increase in threonine, glutamine, and tyrosine phosphorylation in human spermatozoa. Furthermore, the addition of L-arginine reversed the inhibitory effect of L-NAME on the capacitation and the associated increase in phosphorylation. The correlation between NO and sperm-zona pellucida binding ability was investigated by Sengoku *et al*. [40], who reported that when treated with low concentrations of a NO donor, the number of spermatozoa which binds to the hemizona is higher than in sperm treated with a higher concentration. Additionally, a NO quencher lowered the enhancement of sperm

NO also seems to modulate the acrosome reaction. The percentage of acrosome loss induced by human follicular fluid or by calcium ionophore was studied when human spermatozoa were capacitated in the presence/absence of NO-releasing compounds or NOS inhibitors [38]. NO donors induced sperm cells to respond faster to human follicular fluid, whereas NOS inhibitors decreased the percentage of acrosome reaction. Similar results were obtained by Revelli *et al*. [41], who showed that different NO-releasing compounds were able to increase the percentage of reacted spermatozoa in the presence of protein-enriched extracts of human follicular fluid. Also, hemoglobin, a NO scavenger, inhibited the follicular fluid–induced acrosomal reaction. In an in-depth analysis of the signaling pathway of the nitric oxide–induced acrosome reaction in human spermatozoa [42], the authors suggested that the acrosome reactioninducing effect of exogenous NO on capacitated human spermatozoa is accomplished via the NO/sGC/cGMP pathway, which leads to the activation of cGMP-dependent protein kinase (PKG). In fact, both the intracellular cGMP levels and the percentage of reacted spermatozoa were significantly increased after incubation with SNP. Furthermore, the SNP-induced acrosome reaction was significantly reduced in the presence of sGC inhibitors, a reduction that was reversed by the addition of a cell-permeating cGMP analogue to the incubation medium.

As previously stated, NOS activity depends on the availability of its substrate and co-factors [5]. However, the scientific literature does not include many studies on the molecules present in the female reproductive tract which may activate, in one way or another, NOS enzymes in

Starting from follicular fluid samples, Revelli *et al*. [41] obtained a protein-enriched follicular fluid solution (PFF), which was then used to study its effects on NOS activity, citrulline synthesis, and acrosome reaction in human sperm. Interestingly, this study showed for the first time that the endogenous NOS activity of human sperm may be increased by PFF. Moreover,

Finally, PKG inhibition reduced the SNP-induced acrosome reaction.

binding by the NO donor.

142 Nitric Oxide Synthase - Simple Enzyme-Complex Roles

**4. NOS-activating molecules**

spermatozoa.

In spermatozoa, NO acts via three main pathways (**Figure 2**) [13]. First, NO is able to activate sGC, leading to a rise in the intracellular levels of cGMP [49]. The latter activates the cyclic nucleotide-gated channels (CNG) localized in the flagellum of mammalian spermatozoa [50, 51]. These channels seem to play an important role in the sperm motility control, by allowing the entry of Ca2+ ions to the cytoplasm during the capacitation process of mammal sperm [50]. Their activation is one of the first events that occur during capacitation in the mouse spermatozoa [52]. cGMP also activates PKG [53, 54], which is involved in the serine/threonine phosphorylation of proteins that promote sperm capacitation and the acrosome reaction [55, 56]. Furthermore, since cGMP and cAMP compete for the catalytic sites of phosphodiesterases [57, 58], an increase in the intracytoplasmic cGMP concentration may inhibit cAMP degradation via cyclic nucleotide phosphodiesterase type 3 [59], thus increasing cAMP intracellular levels and activating protein kinase A (PKA). The latter leads to an increase in protein tyrosine phosphorylation [60].

Second, NO is directly involved in tyrosine phosphorylation by modulating the cAMP/PKA and the extracellular signal-regulated kinase (ERK) pathways. The cAMP/PKA pathway can be influenced by NO via activation of sGC (as described above), but it can also be regulated directly. In fact, S-nitrosylation of adenylate cyclase (AC) has been suggested as a possible mechanism of action of NO [61]. Low levels of NO may activate AC, consequently increasing the cAMP concentration and activating PKA [62]. However, high levels of NO can inhibit AC [61]. As far as the ERK pathway is concerned, NO reacts with the cysteine residues of the RAS protein, inducing its activation [35]. In turn, RAS triggers the RAF, MEK, and ERK1/2 complex, necessary for tyrosine phosphorylation [63].

Third, NO regulates the post-translational protein modification in spermatozoa via S-nitrosylation [64], a process similar to phosphorylation and acetylation [65, 66]. S-nitrosylation consists of the covalent incorporation of NO into thiol groups (-SH) to form S-nitrosothiols (S-NO), a modification that is selective and reversible [13].

**Figure 2.** Representation of the main pathways through which NO acts in spermatozoa. NO leads to an increase in the intracellular levels of cyclic guanosine monophosphate (cGMP) by activating the soluble isoform of guanylate cyclase (sGC). The cGMP can activate the cyclic nucleotide-gated channels (CNG) localized in the flagellum of mammalian spermatozoa, which regulate the influx of Ca2+ ions to the cytoplasm during the capacitation process and also activates the cGMP-dependent protein kinase (PKG), leading to the serine/threonine phosphorylation of different proteins. It can also inhibit cyclic adenosine monophosphate (cAMP) degradation via cyclic nucleotide phosphodiesterase (PDE), which leads to the activation of cAMP-dependent protein kinase A (PKA) and tyrosine phosphorylation. Furthermore, NO is involved in the tyrosine phosphorylation process in a direct manner, by activating adenylate cyclase (AC) and the extracellular signal-regulated kinase (ERK) pathway. Finally, NO determines post-translational protein modification in spermatozoa via S-nitrosylation.

#### **6. Function of S-nitrosoproteins in spermatozoa**

An extensive study by Lefièvre *et al*. [64] described a large number of proteins present in the sperm of normozoospermic men, which can be subjected to S-nitrosylation in the presence of NO donors. Although the function of some nitrosylated proteins remains to be discovered, a considerable group of them are known to be metabolic proteins and proteins associated with energy generation and cell movement, suggesting a role for S-nitrosylation in sperm motility. This agrees with a previous proteomic analysis [67], in which the most abundant group was also involved in energy production.

Other considerable groups of proteins were those involved in signal transduction, which agrees with a role for S-nitrosylation in modulating the sperm function [64]. Interestingly, since sperms are generally assumed to be transcriptionally inactive, a small percentage of the S-nitrosylated proteins identified by Lefièvre *et al*. [64] was related to transcription. Previous proteomic studies in sperm also observed the presence of proteins involved in transcription [67, 68]. However, when comparing the human sperm S-nitrosoproteome with proteins identified during a proteomic study of sperm-oocyte interaction, only three proteins were found in common, suggesting that S-nitrosylation is not a regulatory mechanism employed during fertilization [64, 69].

It is known that the mobilization of Ca2+ stored in the sperm neck/midpiece is necessary for the hyperactivation process [46]. The Ca2+ store in the neck of the sperm coincides with the region occupied by the redundant nuclear envelope (RNE) [70] and in order to mobilize Ca2+ from this site, ryanodine receptors (RyRs), which are intracellular Ca2+-release channels involved in regulation of cytosolic calcium levels [71], need to be activated. These proteins contain a large number of thiol groups and are thus prone to S-nitrosylation by NO [64, 72, 73]. S-nitrosylation can potentiate the opening of RyRs [74–79], probably through the generation of the membrane permanent product S-nitrosocysteine [80]. It has been shown that an increase in Ca2+ induced by NO is accompanied by an increase in S-nitrosylation levels of endogenous RyRs [81, 82] while these Ca2+ channels may be inhibited under strongly nitrosylating conditions or at high doses of NO (**Figure 3**) [76, 79, 82]. Furthermore, progesterone acts synergistically with NO to mobilize Ca2+ in the sperm neck/midpiece by activation of RyRs [47], contributing to the hyperactivation process.

**6. Function of S-nitrosoproteins in spermatozoa**

also involved in energy production.

spermatozoa via S-nitrosylation.

144 Nitric Oxide Synthase - Simple Enzyme-Complex Roles

An extensive study by Lefièvre *et al*. [64] described a large number of proteins present in the sperm of normozoospermic men, which can be subjected to S-nitrosylation in the presence of NO donors. Although the function of some nitrosylated proteins remains to be discovered, a considerable group of them are known to be metabolic proteins and proteins associated with energy generation and cell movement, suggesting a role for S-nitrosylation in sperm motility. This agrees with a previous proteomic analysis [67], in which the most abundant group was

**Figure 2.** Representation of the main pathways through which NO acts in spermatozoa. NO leads to an increase in the intracellular levels of cyclic guanosine monophosphate (cGMP) by activating the soluble isoform of guanylate cyclase (sGC). The cGMP can activate the cyclic nucleotide-gated channels (CNG) localized in the flagellum of mammalian spermatozoa, which regulate the influx of Ca2+ ions to the cytoplasm during the capacitation process and also activates the cGMP-dependent protein kinase (PKG), leading to the serine/threonine phosphorylation of different proteins. It can also inhibit cyclic adenosine monophosphate (cAMP) degradation via cyclic nucleotide phosphodiesterase (PDE), which leads to the activation of cAMP-dependent protein kinase A (PKA) and tyrosine phosphorylation. Furthermore, NO is involved in the tyrosine phosphorylation process in a direct manner, by activating adenylate cyclase (AC) and the extracellular signal-regulated kinase (ERK) pathway. Finally, NO determines post-translational protein modification in

Other considerable groups of proteins were those involved in signal transduction, which agrees with a role for S-nitrosylation in modulating the sperm function [64]. Interestingly, since sperms are generally assumed to be transcriptionally inactive, a small percentage of the

**Figure 3.** S-nitrosylation process. NO acts on the thiol groups (-SH) of the cysteines in proteins to form S-nitrosothiols (S-NO). At the sperm neck/midpiece, the S-nitrosylation occurs in ryanodine receptors (RyRs) allowing the release of calcium from the redundant nuclear envelope (RNE), which is required for sperm hyperactivation. Adapted and modified from López-Úbeda and Matás [13].

Other examples of proteins which can undergo S-nitrosylation in sperm and have a known biological significance are the A-kinase anchoring proteins (AKAPs) [64]. Both AKAP3 and AKAP4 are present in the fibrous sheath of the sperm flagellum, control PKA activity and undergo phosphorylation during the capacitation process [83–85]. AKAP complexes also modulate the motility of sperm. In fact, phosphodiesterase inhibitors were seen to significantly increase sperm motility [86], whereas PKA-anchoring inhibitor peptides arrested sperm motility [87]. Since the effects of NO on sperm motility are well established, the S-nitrosylation of AKAPs would be an interesting subject for additional studies.

A number of heat shock proteins (HSPs) may also be targets of S-nitrosylation in sperm [64], and some of them have been reported to act as important modulators of sperm capacitation. For instance, Asquith *et al*. [88] reported that heat shock protein 1 and endoplasmin undergo tyrosine phosphorylation during mouse sperm capacitation, whereas Nixon *et al*. [89] suggested that they form part of a zona pellucida complex, allowing successful spermegg interaction in the same species. Heat shock 70 kDa protein 8 and heat shock protein 90α also undergo tyrosine phosphorylation during human sperm capacitation [83], but whether they function in a zona receptor complex is still unknown [64]. Furthermore, HspA2 has been shown to be a marker of sperm maturity [90], its expression in infertile men with idiopathic oligoteratozoospermia being lower than in normozoospermic men [91].

#### **7. Concluding remarks**

In recent years, our knowledge of the involvement of the NOS/NO system in mammalian fertilization has grown, and there is clear evidence that NO acts as a significant modulator of the male and female gamete. However, many aspects regarding the NOS/NO duo, such as the presence of NOS-activating molecule(s) in the fertilization site or how the biological function of the S-nitrosylated proteins changes, remain to be discovered. Shedding light on these mechanisms will increase our understanding of the etiopathology of subfertility/infertility problems and how such problems can be overcome.

#### **Acknowledgements**

This work was supported by H2020 MSC-ITN-EJD 675526 REP-BIOTECH, the Spanish Ministry of Economy and Competitiveness (MINECO) and the European Regional Development Fund (FEDER), Grants AGL2015–66341-R.

#### **Author details**

Florentin-Daniel Staicu and Carmen Matas Parra\*

\*Address all correspondence to: cmatas@um.es

Department of Physiology, Faculty of Veterinary Science, University of Murcia, Spain

#### **References**

Other examples of proteins which can undergo S-nitrosylation in sperm and have a known biological significance are the A-kinase anchoring proteins (AKAPs) [64]. Both AKAP3 and AKAP4 are present in the fibrous sheath of the sperm flagellum, control PKA activity and undergo phosphorylation during the capacitation process [83–85]. AKAP complexes also modulate the motility of sperm. In fact, phosphodiesterase inhibitors were seen to significantly increase sperm motility [86], whereas PKA-anchoring inhibitor peptides arrested sperm motility [87]. Since the effects of NO on sperm motility are well established, the S-nitrosylation

A number of heat shock proteins (HSPs) may also be targets of S-nitrosylation in sperm [64], and some of them have been reported to act as important modulators of sperm capacitation. For instance, Asquith *et al*. [88] reported that heat shock protein 1 and endoplasmin undergo tyrosine phosphorylation during mouse sperm capacitation, whereas Nixon *et al*. [89] suggested that they form part of a zona pellucida complex, allowing successful spermegg interaction in the same species. Heat shock 70 kDa protein 8 and heat shock protein 90α also undergo tyrosine phosphorylation during human sperm capacitation [83], but whether they function in a zona receptor complex is still unknown [64]. Furthermore, HspA2 has been shown to be a marker of sperm maturity [90], its expression in infertile men with idiopathic

In recent years, our knowledge of the involvement of the NOS/NO system in mammalian fertilization has grown, and there is clear evidence that NO acts as a significant modulator of the male and female gamete. However, many aspects regarding the NOS/NO duo, such as the presence of NOS-activating molecule(s) in the fertilization site or how the biological function of the S-nitrosylated proteins changes, remain to be discovered. Shedding light on these mechanisms will increase our understanding of the etiopathology of subfertility/infertility

This work was supported by H2020 MSC-ITN-EJD 675526 REP-BIOTECH, the Spanish Ministry of Economy and Competitiveness (MINECO) and the European Regional Development Fund

Department of Physiology, Faculty of Veterinary Science, University of Murcia, Spain

of AKAPs would be an interesting subject for additional studies.

oligoteratozoospermia being lower than in normozoospermic men [91].

problems and how such problems can be overcome.

Florentin-Daniel Staicu and Carmen Matas Parra\*

\*Address all correspondence to: cmatas@um.es

**7. Concluding remarks**

146 Nitric Oxide Synthase - Simple Enzyme-Complex Roles

**Acknowledgements**

**Author details**

(FEDER), Grants AGL2015–66341-R.


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## **From Nitric Oxide Toward S-Nitrosylation: Expanding Roles in Gametes and Embryos**

Ješeta Michal, Marketa Sedmikova and Jean-François Bodart

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/67270

#### **Abstract**

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1791–1794. DOI: http://dx.doi.org/10.1093/humrep/del055.

DOI: http://dx.doi.org/10.1016/S0009–8981(98)00060–6.

Nitric oxide (NO) is a gasotransmitter involved in various aspects of reproduction. The observational data from different species, such as sea urchin, ascidians, amphibians, rodents, porcine, bovine, and human, suggest that NO might have a significant role in reproduction through several mechanisms. This proposed role might appear preserved through evolution; however, the effects of NO also depend on the species or stages considered. There has been debate over the physiological relevance of NO, though the benefits of its use in assisted reproduction are now widely recognized. Over the past years, S-nitrosylation has provided a new angle to decipher the mechanisms through which NO exerts its actions. This chapter summarizes, in a nonexhaustive manner, research that explores the role of NO in gametes and embryos.

**Keywords:** nitric oxide, gamete, meiosis, oocyte, spermatozoa, nitrosylation, cell cycle, embryo, reproduction

#### **1. Introduction**

Nitric oxide (NO) is a gaseous free radical that plays a key role both in intra- and extracellular signaling pathways in a wide variety of organisms. The role of NO has been emphasized in many physiological processes including reproduction. NO is generated by nitric oxide synthases (NOS), whose isoforms have been detected in a variety of mammalian reproductive tissues such as ovary, uterus, testis, or epididymis. Nitric oxide has been involved in the regulation of follicle growth and ovulation in mice, spermatogenesis in humans, embryo implantation in rats, and meiosis in pigs and in mice. Data collected from the various abovementioned

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

species suggest that NO might have a significant role in reproduction through mechanisms preserved through evolution; however, one cannot discard that the effects of NO may also be dependent on the species or stages considered. Therefore, nitric oxide could be considered as a gasotransmitter ruling out several aspects of reproduction, from gametes to early embryogenesis, though there have been a debate over the physiological relevance of NO. This chapter summarizes, in a nonexhaustive manner, the research that explores the role of NO in gametes and embryos.

#### **2. NO in sea urchin**

Nitric oxide was first reported to trigger parthenogenetic activation in sea urchin oocytes and was suggested as a potential physiological regulator for egg activation. Twenty years ago, an increase in NO levels at fertilization was reported [1, 2], and NO was hypothesized as the primary activator for sea urchin egg activation [3]. Enthusiasm has been lately shaded by a report of NO increases occurring lately during fertilization in comparison to the rise of intracellular calcium level [4, 5]. From this observation, it has been suggested that the role of NO could be limited to sustaining the duration of the calcium transient [4]. NO increases are likely correlated to the calcium increases but not by being its primary activator [4, 5]. Nevertheless, NO increases may play a role in the hardening of the fertilization envelope surrounding the fertilized egg, thereby protecting the embryo from severe environmental conditions during its early development [5].

#### **3. NO in ascidians oocytes, eggs, and embryos**

In contrast to the observations performed in sea urchins, Hyslop et al. [6] reported that NO was not likely to be involved in the physiological process of fertilization of *Ascidiella aspersa* eggs. This report was in contrast to a previous study examining the inward current induced by NO, sharing similarities with the ascidians sperm current, which suggested that fertilization in ascidians could use NO as a second messenger [7]. Though nitric oxide donors induce increases in free calcium in ascidians and sea urchin eggs at the intracellular level, NOS inhibitor l-NAME (N(G)-nitro-l-arginine methyl ester) did not prevent sperm-induced fertilization, and not so much as a discrete increase in NO preceded the calcium wave [6].

However, NO pathways and production were related to metamorphosis, notochord, and tail regression in *Ciona intestinalis*, which are correlated to caspase-dependent apoptosis [8]. The nitric oxide synthase spatial pattern expression was highly dynamic during this larval development [8]. Experimental increases and decreases in NO levels can drive delays or accelerations in tail regression, respectively, with NO changes being related to the modulation of Caspase 3-like activity [8]. In most of the considered marine larvae of ascidians, NO acted as a repressor of the initiation of metamorphosis [9, 10]. Further works had been undertaken in *C. intestinalis* to unravel the role of NO during larval development. Mitogen-activated protein kinase (MAPK) and extracellular-regulated kinase (ERK) phosphorylations levels appeared closely related to NO levels [11].

In conclusion, the fine tuning of NO pathways and levels are physiologically involved in ascidian larvae development and metamorphosis. However, NO did not seem to be required at very early steps of development.

#### **4. NO in amphibian oocytes, eggs, and embryos**

species suggest that NO might have a significant role in reproduction through mechanisms preserved through evolution; however, one cannot discard that the effects of NO may also be dependent on the species or stages considered. Therefore, nitric oxide could be considered as a gasotransmitter ruling out several aspects of reproduction, from gametes to early embryogenesis, though there have been a debate over the physiological relevance of NO. This chapter summarizes, in a nonexhaustive manner, the research that explores the role of NO in gametes

Nitric oxide was first reported to trigger parthenogenetic activation in sea urchin oocytes and was suggested as a potential physiological regulator for egg activation. Twenty years ago, an increase in NO levels at fertilization was reported [1, 2], and NO was hypothesized as the primary activator for sea urchin egg activation [3]. Enthusiasm has been lately shaded by a report of NO increases occurring lately during fertilization in comparison to the rise of intracellular calcium level [4, 5]. From this observation, it has been suggested that the role of NO could be limited to sustaining the duration of the calcium transient [4]. NO increases are likely correlated to the calcium increases but not by being its primary activator [4, 5]. Nevertheless, NO increases may play a role in the hardening of the fertilization envelope surrounding the fertilized egg, thereby protecting the embryo from severe environmental

In contrast to the observations performed in sea urchins, Hyslop et al. [6] reported that NO was not likely to be involved in the physiological process of fertilization of *Ascidiella aspersa* eggs. This report was in contrast to a previous study examining the inward current induced by NO, sharing similarities with the ascidians sperm current, which suggested that fertilization in ascidians could use NO as a second messenger [7]. Though nitric oxide donors induce increases in free calcium in ascidians and sea urchin eggs at the intracellular level, NOS inhibitor l-NAME (N(G)-nitro-l-arginine methyl ester) did not prevent sperm-induced fertilization, and not so much as a discrete increase in NO preceded the calcium wave [6].

However, NO pathways and production were related to metamorphosis, notochord, and tail regression in *Ciona intestinalis*, which are correlated to caspase-dependent apoptosis [8]. The nitric oxide synthase spatial pattern expression was highly dynamic during this larval development [8]. Experimental increases and decreases in NO levels can drive delays or accelerations in tail regression, respectively, with NO changes being related to the modulation of Caspase 3-like activity [8]. In most of the considered marine larvae of ascidians, NO acted as a repressor of the initiation of metamorphosis [9, 10]. Further works had been undertaken in *C. intestinalis* to unravel the role of NO during larval development. Mitogen-activated

and embryos.

**2. NO in sea urchin**

156 Nitric Oxide Synthase - Simple Enzyme-Complex Roles

conditions during its early development [5].

**3. NO in ascidians oocytes, eggs, and embryos**

Amphibian oocytes offer a typical playground for deciphering the biochemical mechanisms underlying cell cycle transition. Meiosis or the M-phase–promoting factor (MPF) was discovered in amphibian oocytes [12] and characterized in this model to be made up of at least two subunits: Cdk1 (catalytic) and Cyclin B (regulatory) [13]. In *Xenopus* oocytes, NO-scavenging did not appear to impair the progression of M-phase entry and meiotic maturation because CPTIO (nitric oxide scavenger)-treated oocytes resumed and completed meiosis after hormonal stimulation by progesterone [14]. If NO is not required for meiotic progression, an excess in NO provided by a donor, such as S-nitroso-N-acetyl penicillamine (SNAP), leads to meiotic maturation inhibition. Under these conditions, SNAP lead heterogeneous response at the biochemical level with respect to the two pathways involved in meiotic resumption (MPF and MAPK). SNAP hindered the all-or-none response of MPF and MAPK pathways, especially in *Xenopus* oocytes; most oocytes exhibited partially active MPF and MAPK in the absence of any external signs of meiotic resumption [15]. Noticeably, SNAP altered meiotic spindle formation, therefore impairing proper genomic transmission [15]. This observation corroborates reports of mitosis inhibition through Tyrosine residue nitrosylation in plant models, and Alteration of cross walls orientation, presumably by impairing microtubules organization [16, 17]. Nevertheless, the mechanisms hindered by NO in vertebrate oocyte spindle formations remain undetermined.

Studies carried out in amphibians reported parthenogenetic activation of *Xenopus* eggs with nitric oxide donor SNAP. This parthenogenetic activation induced M-phase exit through an atypical mechanism involving calcium-dependent pathway and MAPK inactivation, while MPF activity was maintained [14] (**Figure 1A**).

During early development, the *Xenopus* nitric oxide synthase 1 (XNOS1) accounts for most of the NOS detected, being expressed in oocytes and eggs during segmentation [18]. At later stages, XNOS1 expression is restricted to the notochord, eyes, and developing neural system [18]. Exploration of NO function in *Xenopus* oocytes indicates that NO increased cell proliferation but impaired cell movements at gastrulation. Inhibition of NOS affected cell movements both in neural and mesodermal extension, through cGMPSindependent pathways involving dishevelled (Dsh) and the central components of the planary cell polarity (PCP) pathway [18]. Cell division during early development has been proposed according to this model to be impacted by NO through the ROCK-cGMP pathway (**Figure 1B**).

**Figure 1.** (A) Proposed mechanisms for NO action for parthenogenetic activation in *Xenopus* eggs. (B) Schematic representation of the pathways mediating the action of nitric oxide during early development in *Xenopus* embryos. Adapted from reference [18]. NO, nitric oxide; XNOS, *Xenopus* nitric oxide synthase; sGC, soluble guanylyl cyclase; cGMP, cyclic guanosine monophosphate; CaMK3, calmodulin kinase 3; Dsh, disheveled; PKG, protein kinase G.

#### **5. NO in rodent oocytes, eggs, and embryos**

Observations gathered in *Xenopus* oocytes may be to put in perspective with earlier reports endothelial NOS (eNOs) knock-out mice, in which meiotic abnormalities suggested that eNOS-derived NO is a modulator of oocyte meiotic maturation [19]. Indeed, the ovulation rates decreased in eNOS nullizygous mice, and oocytes often exhibited blocks during metaphase I or indicated various meiotic abnormalities with degenerative/atypical morphology of meiotic stages [19]. One should also note that in such conditions, oocytes from eNos nullizygous mice exhibit a higher rate of cell death than those in control ones. The importance of NOS for rodent oocyte meiotic maturation was confirmed by the expression of NOS isoforms in mouse or rat oocytes [20–22]. If NO was acknowledged to be important for meiotic maturation, high concentrations of NO can damage mouse oocyte and impair their further development [23]. Positive effects of low doses of NO donor SNAP during *in vitro* maturation was observed in mouse [24] and rat [25] oocytes. NO has been reported to play a dual role in oocyte meiotic maturation in mice, depending on its concentrations; however, the mechanism by which it influences oocyte maturation has not been fully clarified. In addition to these results, reports have shown that NO was most likely not essential for mouse oocyte fertilization [6].

Though NO is not a primary stimulus for oocyte activation through calcium mobilization, as observed in sea urchin and ascidians, it has been reported to play several potential roles during embryogenesis. The NO donor SNP brought about the arrest of embryonic development at early stages in mice; only half of the treated embryos reached blastocysts stages, with concentrations ranging from 10 nM to 1 mM [26]. Inhibition of NO production also suggested that NO plays a role in preimplantation embryos, which can be achieved through oxygen consumption limitation and mitochondrial activity or apoptosis modulation [27, 28]. Recently, it has been suggested that NO, through the use of the NO donor SNP and NOS inhibitor l-NAME, may regulate blastocyst hatching, which is a crucial step for embryo survival and implantation [29].

#### **6. NO in porcine oocytes, eggs, and embryos**

**5. NO in rodent oocytes, eggs, and embryos**

158 Nitric Oxide Synthase - Simple Enzyme-Complex Roles

Observations gathered in *Xenopus* oocytes may be to put in perspective with earlier reports endothelial NOS (eNOs) knock-out mice, in which meiotic abnormalities suggested that eNOS-derived NO is a modulator of oocyte meiotic maturation [19]. Indeed, the ovulation rates decreased in eNOS nullizygous mice, and oocytes often exhibited blocks during metaphase I or indicated various meiotic abnormalities with degenerative/atypical morphology of meiotic stages [19]. One should also note that in such conditions, oocytes from eNos nullizygous mice exhibit a higher rate of cell death than those in control ones. The importance of NOS for rodent oocyte meiotic maturation was confirmed by the expression of NOS isoforms in mouse or rat oocytes [20–22]. If NO was acknowledged to be important for meiotic maturation, high concentrations of NO can damage mouse oocyte and impair their further development [23]. Positive effects of low doses of NO donor SNAP during *in vitro* maturation was observed in mouse [24] and rat [25] oocytes. NO has been reported to play a dual role in oocyte meiotic maturation in mice, depending on its concentrations; however, the mechanism by which it influences oocyte maturation has not been fully clarified. In addition to these results, reports have shown that NO was most likely not essential for mouse oocyte fertilization [6].

**Figure 1.** (A) Proposed mechanisms for NO action for parthenogenetic activation in *Xenopus* eggs. (B) Schematic representation of the pathways mediating the action of nitric oxide during early development in *Xenopus* embryos. Adapted from reference [18]. NO, nitric oxide; XNOS, *Xenopus* nitric oxide synthase; sGC, soluble guanylyl cyclase; cGMP, cyclic guanosine monophosphate; CaMK3, calmodulin kinase 3; Dsh, disheveled; PKG, protein kinase G.

Though NO is not a primary stimulus for oocyte activation through calcium mobilization, as observed in sea urchin and ascidians, it has been reported to play several potential roles during embryogenesis. The NO donor SNP brought about the arrest of embryonic development at early stages in mice; only half of the treated embryos reached blastocysts stages, with concentrations ranging from 10 nM to 1 mM [26]. Inhibition of NO production also suggested that NO plays a role in preimplantation embryos, which can be achieved through oxygen consumption The production of low concentrations of NO concentrations has been reported to be necessary for meiotic maturation in porcine oocytes, whereas high concentrations of NO damage oocytes integrity [30]. Inhibition of NO synthase suppressed *in vitro* maturation of porcine oocytes, as attested by the absence of GVBD or meiosis I to meiosis II transitions [31]. NO synthases were detected in porcine oocytes [32, 33], but the involvement of NO/NOS in oocyte development has not been fully elucidated. Previously, it was assumed that NO acted *via* the cGMP cascade, similar to mechanisms observed for muscle contractions [19], or *via* the cascade-activating kinases, which are necessary for the resumption of meiotic maturation [34] (**Figure 2**). Nitric oxide had been reported to act on NO-sensitive guanyl cyclases, but new articles suggest that NO could also exert its functions by non-cGMP-mediated pathways by protein S-nitrosylation. It was reported that NOS inhibition decreases the amount of S-nitrosylated proteins in porcine oocytes [35].

**Figure 2.** Scheme of NO/GMPc/PKG pathway in porcine oocytes and likely impact on basic regulation factor of meiosis, MPF. Nitric oxide synthase (NOS) increase enhances the concentration of nitric oxide (NO) in oocyte. NO stimulates the activity of soluble guanylyl cyclase (sGC), which catalyzes the production of cyclic guanosine monophosphate (cGMP). cGMP is necessary for protein kinase G (PKG) activation, which in turn suppresses MPF activation, and therefore, impairs meiosis resumption and maturation.

In porcine oocytes, NO donors are potent to induce parthenogenetic activation, resulting in early embryonic development [36]. NO production is also important for embryonic development since the NOS inhibitor l-NAME strongly decreases the proportion of porcine embryo blastocysts after 6 days of *in vitro* cultivation [37].

#### **7. NO in bovine oocytes, eggs, and embryos**

As well as in porcine oocytes, nitric oxide synthases (NOS) were detected in bovine oocytes [38]. Oocyte maturation was noticeably inhibited by the use of NOS inhibitors [39], which strengthens the hypothesis that NO plays a role during oocyte meiosis in this model. Regarding the molecular mechanisms involved, Bilodeau-Goeseels was first to suggest that the inhibitory effect of NO on bovine oocyte meiotic resumption did not appear to be mediated by the cGMP/ PKG pathway; this is in contrast to previous observations gathered in mice [40].

Inhibition of NOS during maturation of bovine oocytes affected the quality of resulting bovine embryos by increasing the number of apoptotic blastomeres [41]. The importance of NO for correct embryonic development was observed, mainly for transition from morulae to the blastocyst stage. Treatment of embryos with the NOS inhibitor l-NAME reduced blastocysts numbers and hatching rates [42]. Contrarily, exposing bovine mature oocytes to a nitric oxide donor short term did not induce stress tolerance and had no positive effect on the *in vitro* embryo production of bovine embryos, as was expected [43].

#### **8. A role for NO in human follicles?**

Only a limited number of studies have addressed NO in human reproduction. Anteby et al. [44] observed an increase in NO concentrations in follicular fluid from bigger follicles, which positively correlated follicular volume and oestradiol concentrations. NO most likely acts as an important endocrinological regulator of ovulation. NO may be involved in an autocrine/paracrine regulation of the developing follicle and have a direct effect on granulosa cells, theca cells, and the developing oocyte. In a recent study, a possible association between idiopathic recurrent spontaneous abortion and variations in the gene encoding endothelial nitric oxide synthase was proposed [45].

#### **9. NO roles in spermatozoa**

In spermatozoa, NO was described to be involved in the regulation of viability, motility, capacitation, hyperactivation, acrosome reaction, and fusion with oocytes. Thus nitric oxide appears to be crucial for the processes driving successful fertilization (reviewed in [46, 47]). NOS isoforms were found in sperm of different mammal species such as mice [48], bulls [49], boars [50], and humans [51]. The abovementioned and well-known duality of NO's effects, depending upon concentrations, was also described in spermatozoa. Low levels of NO stimulate hyperactivation and increase motility of cryopreserved sperm after thawing [52]; conversely, high concentrations of NO decrease sperm motility [46, 53, 54] and inhibit sperm-oocyte fusion [55].

NO was reported to have a positive impact on sperm motility [55], whereas NO donor increased sperm motility [56, 57], inhibition of nitric oxide synthases by l-NAME negatively affected this motility [58]. Miraglia and colleagues [53] observed the existence of NO signaling pathways in human spermatozoa. NO stimulates sperm motility *via* the activation of soluble guanylate cyclase (sGC), the subsequent synthesis of cGMP, and the activation of cGMPdependent protein kinase. The level of cGMP is modulated by cGMP-dependent phosphodiesterase (PDE). These observations concur with a former report that PDE inhibitor sidenafil citrate increased sperm motility [59]. NO is, on the other hand, considered a major free radical involved in sperm damage at sperm motility level. Nitrosative stress produced by high levels of reactive nitrogen species decreases progressive and total motility, as well as several sperm kinetic parameters, meanwhile, sperm viability is not affected [60, 61].

Inhibition of NOS during maturation of bovine oocytes affected the quality of resulting bovine embryos by increasing the number of apoptotic blastomeres [41]. The importance of NO for correct embryonic development was observed, mainly for transition from morulae to the blastocyst stage. Treatment of embryos with the NOS inhibitor l-NAME reduced blastocysts numbers and hatching rates [42]. Contrarily, exposing bovine mature oocytes to a nitric oxide donor short term did not induce stress tolerance and had no positive effect on the

Only a limited number of studies have addressed NO in human reproduction. Anteby et al. [44] observed an increase in NO concentrations in follicular fluid from bigger follicles, which positively correlated follicular volume and oestradiol concentrations. NO most likely acts as an important endocrinological regulator of ovulation. NO may be involved in an autocrine/paracrine regulation of the developing follicle and have a direct effect on granulosa cells, theca cells, and the developing oocyte. In a recent study, a possible association between idiopathic recurrent spontaneous abortion and variations in the gene encoding endothelial

In spermatozoa, NO was described to be involved in the regulation of viability, motility, capacitation, hyperactivation, acrosome reaction, and fusion with oocytes. Thus nitric oxide appears to be crucial for the processes driving successful fertilization (reviewed in [46, 47]). NOS isoforms were found in sperm of different mammal species such as mice [48], bulls [49], boars [50], and humans [51]. The abovementioned and well-known duality of NO's effects, depending upon concentrations, was also described in spermatozoa. Low levels of NO stimulate hyperactivation and increase motility of cryopreserved sperm after thawing [52]; conversely, high concentrations of NO decrease sperm motility [46, 53, 54] and inhibit

NO was reported to have a positive impact on sperm motility [55], whereas NO donor increased sperm motility [56, 57], inhibition of nitric oxide synthases by l-NAME negatively affected this motility [58]. Miraglia and colleagues [53] observed the existence of NO signaling pathways in human spermatozoa. NO stimulates sperm motility *via* the activation of soluble guanylate cyclase (sGC), the subsequent synthesis of cGMP, and the activation of cGMPdependent protein kinase. The level of cGMP is modulated by cGMP-dependent phosphodiesterase (PDE). These observations concur with a former report that PDE inhibitor sidenafil citrate increased sperm motility [59]. NO is, on the other hand, considered a major free radical involved in sperm damage at sperm motility level. Nitrosative stress produced by high levels

*in vitro* embryo production of bovine embryos, as was expected [43].

**8. A role for NO in human follicles?**

160 Nitric Oxide Synthase - Simple Enzyme-Complex Roles

nitric oxide synthase was proposed [45].

**9. NO roles in spermatozoa**

sperm-oocyte fusion [55].

Sperm capacitation and acrosome reaction of mammal spermatozoa are essential for the fertilization process to occur. Both of them are NO-dependent. The final step in spermatozoa maturation, capacitation, involves a cascade of events such as the removal of cholesterol from plasma membrane, an influx of Ca2+ followed by an increase in intracellular cAMP levels, change of pH, and hyperactivation of sperm [62] (**Figure 3**). Acrosome reaction is a precondition for sperm fusion with the oocyte. It encompasses the release of proteolytic enzymes from the acrosome cap of the spermatozoa and an influx of Ca2+ and phosphorylation of tyrosine residues at the molecular level [63]. It has been reported that NO donors support acrosome reaction and accelerate capacitation and hyperactivation, whereas NOS inhibitors such as l-NAME significantly decrease or block this processes [46, 56, 58]. l-Arginin has similar effects as NO donors. Supplementation by l-arginin increases intracellular NO levels and supports sperm capacitation and acrosomal reaction without decreasing sperm viability [64]. Herrero et al. [65] also reported that capacitation was regulated by NO *via* cAMP levels and protein tyrosine phosphorylation. Moreover, it was proven that exogenous NO induces acrosomal reactions in human spermatozoa, and the process was mediated by the stimulation of a NO-sensitive sGC, cGMP synthesis, and the activation of PKC. However, the presence of extracellular Ca2+ was required for PKC activation in such conditions [66].

**Figure 3.** Scheme of NO/NOS role in sperm capacitation and acrosome reaction; eNOS—endothelial nitric oxide synthase; nNOS—neuronal nitric oxide synthase; iNOS—inducible nitric oxide synthase; sCG—soluble guanylyl cyclase; cGMP—cyclic guanosine monophosphate; PKG—protein kinase G; PDE5—phosphodiesterase 5; CNGC—cyclic nucleotide-gated channels; PMCA4—plasma membrane calcium ATPase 4; ROS—reactive oxygen species.

NO has the ability to improve the quality of freshly ejaculated sperm as well as thawed sperm. The NO donor sodium nitroprusside (SNP) was found to increase motility and viability of sperm after thawing and reduced membrane lipid peroxidation levels [57, 67].

#### **10. NO effects on oocyte aging**

Mammal oocytes are normally fertilized soon after the completion of meiotic maturation during the MII stage. If ovulated *in vitro,* matured oocytes are not fertilized, they undergo a process called aging, which is characterized by numerous changes. Oocyte aging rapidly decreases their quality and capacity to undergo embryonic development after fertilization. Functional and morphological changes associated with oocyte aging include decreased fertilization rates, polyspermy, parthenogenetic activation, apoptosis, chromosomal abnormalities, cortical granules exocytosis, ooplasmic microtubule dynamics, zona pellucida hardening, decreases in MPF and MAPK activities, epigenetic changes, and abnormal or delayed embryo development [24, 68–71]. Pathological conditions of oocyte aging impose limits for assisted reproduction technologies in animals as well as in humans [72].

It has been described that nitric oxide plays a part in oocyte aging, but it appears to do so by mobilizing more than one signaling pathway. NO can act either as a decelerating factor in oocyte aging [24] or, conversely, as an important cause of unwholesome aging-associated changes, which are caused by high ROS production [73].

Although high levels of NO are related mostly to pathological conditions, e.g., poor oocyte quality, increased protein nitration, and resistance to IVM in women with endometriosis [74], supplementing culture medium with low doses of the NO donor S-nitroso acetyl penicillamine (SNAP) delays manifestation of oocyte aging, *i.e*., decrease of spontaneous cortical granule exocytosis, zona pellucida hardening, and the rate of spindle abnormalities [24]. The significance of NO in sustaining oocyte quality was demonstrated by Goud et al. [75]. Exposure of aged oocytes to l-NAME resulted in a significant disruption of fertilization and apoptosis during early embryonic development.

Different NOS isoforms could play different roles in aging. Lower NO levels produced by eNOS and nNOS could participate in delaying oocyte aging through the activation of sGC, which leads to an increased production of cyclic guanosine monophosphate [76]. However, high NO levels generated by iNOS were associated with higher O<sup>2</sup> •− production and promoted oocyte fragmentation and apoptosis [75]. Contrarily, Tripathi and colleagues [73] reported that the generation of NO through iNOS-mediated pathways was associated with the maintenance of meiotic arrest in diplotene-arrested oocytes and the sustained reduction of iNOS expression. Furthermore, they reported that intracellular NO level may induce apoptosis in aged rat oocytes cultured *in vitro*. Similarly, Nevoral et al. [77] described the suppression of apoptosis and lysis after prolonged cultivation of porcine oocytes in media supplemented by the NOS nonspecific inhibitor l-NAME. The decrease of intracellular levels of NO interrupts intracellular signal transduction pathways, especially Ca2+-mediated pathways [75]. Premkumar and Chaube [78] reported that NO increases levels of cytosolic free Ca2+, cGMP, and Wee 1 through an iNOS-mediated pathway. High levels of these signaling molecules trigger parthenogenetic activation of aged oocytes *via* the accumulation of phosphorylated Cdk1 (pThr-14/Tyr-15), a catalytic subunit of MPF. These findings indicate that NO can influence changes associated with oocyte aging in various manners.

#### **11. S-nitrosylation as a posttranslational modification potentially regulating cell cycle**

**10. NO effects on oocyte aging**

162 Nitric Oxide Synthase - Simple Enzyme-Complex Roles

changes, which are caused by high ROS production [73].

apoptosis during early embryonic development.

high NO levels generated by iNOS were associated with higher O<sup>2</sup>

Mammal oocytes are normally fertilized soon after the completion of meiotic maturation during the MII stage. If ovulated *in vitro,* matured oocytes are not fertilized, they undergo a process called aging, which is characterized by numerous changes. Oocyte aging rapidly decreases their quality and capacity to undergo embryonic development after fertilization. Functional and morphological changes associated with oocyte aging include decreased fertilization rates, polyspermy, parthenogenetic activation, apoptosis, chromosomal abnormalities, cortical granules exocytosis, ooplasmic microtubule dynamics, zona pellucida hardening, decreases in MPF and MAPK activities, epigenetic changes, and abnormal or delayed embryo development [24, 68–71]. Pathological conditions of oocyte aging impose limits for assisted reproduction technologies in animals as well as in humans [72].

It has been described that nitric oxide plays a part in oocyte aging, but it appears to do so by mobilizing more than one signaling pathway. NO can act either as a decelerating factor in oocyte aging [24] or, conversely, as an important cause of unwholesome aging-associated

Although high levels of NO are related mostly to pathological conditions, e.g., poor oocyte quality, increased protein nitration, and resistance to IVM in women with endometriosis [74], supplementing culture medium with low doses of the NO donor S-nitroso acetyl penicillamine (SNAP) delays manifestation of oocyte aging, *i.e*., decrease of spontaneous cortical granule exocytosis, zona pellucida hardening, and the rate of spindle abnormalities [24]. The significance of NO in sustaining oocyte quality was demonstrated by Goud et al. [75]. Exposure of aged oocytes to l-NAME resulted in a significant disruption of fertilization and

Different NOS isoforms could play different roles in aging. Lower NO levels produced by eNOS and nNOS could participate in delaying oocyte aging through the activation of sGC, which leads to an increased production of cyclic guanosine monophosphate [76]. However,

moted oocyte fragmentation and apoptosis [75]. Contrarily, Tripathi and colleagues [73] reported that the generation of NO through iNOS-mediated pathways was associated with the maintenance of meiotic arrest in diplotene-arrested oocytes and the sustained reduction of iNOS expression. Furthermore, they reported that intracellular NO level may induce apoptosis in aged rat oocytes cultured *in vitro*. Similarly, Nevoral et al. [77] described the suppression of apoptosis and lysis after prolonged cultivation of porcine oocytes in media supplemented by the NOS nonspecific inhibitor l-NAME. The decrease of intracellular levels of NO interrupts intracellular signal transduction pathways, especially Ca2+-mediated pathways [75]. Premkumar and Chaube [78] reported that NO increases levels of cytosolic free Ca2+, cGMP, and Wee 1 through an iNOS-mediated pathway. High levels of these signaling molecules trigger parthenogenetic activation of aged oocytes *via* the accumulation of phosphorylated Cdk1 (pThr-14/Tyr-15), a catalytic subunit of MPF. These findings indicate

that NO can influence changes associated with oocyte aging in various manners.

•− production and pro-

Though cGMP pathway has been reported to be the main road for NO's involvement, evidences have been raised for c-GMP independent pathway, through protein S-nitrosylation (i.e., in porcine oocytes [35]). S-nitrosylation is an established posttranslation modification, whose potential spectra of involvements in cancer cell lines, oocytes, and embryos ranges from cell cycle regulation to embryo implantation [18, 79–81]. The effects of NO may differ among the cellular models considered. For instance, whereas low concentrations of NO donors DETA-NO promote cell proliferation in promyelotic HL-60 cells [82], nitric oxide synthase inhibition drives cell proliferation at blastula stages in *Xenopus* embryos [18]. Such a duality in the effects certainly rely on the diversity of S-nitrosylated proteins.

S-nitrosylation has been reported to modify several regulators of cell cycle progression (**Figure 4**), such as cyclin-dependent kinases (CDKs). CDKs' S-nitrosylation was observed for CDK2, CDK5, and CDK6. While CDK2-nitrosylation enhances its activity independently of any effects on protein levels expression [82], the effect of S-nitrosylation on CDK5 and CDK6 remains elusive. Though G2/M arrests might be observed associated with NO release [83, 84], no S-nitrosylation has been so far reported for Cdk1, the catalytic subunit of MPF. S-nitrosylation of cyclin B was seek in HL-60 cells, but not observed [82]. As well, no S-nitrosylations have yet been reported for polo-like kinases (PLKs), anaphase promoting factor/cyclosome (APC/C), Wee1, and Myt1, which are MPF regulators. Nevertheless, the dual specificity phosphatase Cdc25, which is the main activator of MPF, is clearly impacted by its S-nitrosylation because it annihilates its phosphatase activity [82, 87]. Recently, an

**Figure 4.** Comprehensive scheme of nitrosylation pivotal role in cell cycle, apoptosis, survival, matrix remodelling, and trophoblasts invasiveness.

alternate mechanism than direct S-nitrosylation of Cdc25C for its inhibition was proposed since NOAD, a nitric oxide-releasing derivative of oleanolic acid, induced activation of Chk2, resulting in an increase of the inhibitory phosphorylation of Cdc25C on its residue Serine 216 [84]. However, genotoxicity of nitric oxide might account for the activation of Chk2, the latter being involved in DNA damage response machinery. In the same study, the arrest in G2/M was associated with the upregulation of Cdk inhibitors p21WAF1/CIP1 and P27KIP1, without providing evidence for direct S-nitrosylation of these proteins. p21WAF1/CIP1 downregulation by NO was also reported in *Xenopus* embryos, but through nitric oxide modulation of the RhoA-ROCK pathway [18] (**Figure 1B**). Thus, though there are converging evidences for role of NO in cell cycle regulation [85], the exact mechanisms remain to be fully deciphered.

### **12. S-nitrosylation plays pivotal role in modulating trophoblast motility and survival**

As mentioned above, S-nitrosylation has been also called to play a role in preimplantation embryos and implantation (**Figure 4**). Microenvironnemental presence of NO was reported to contribute to the pathologic effects of endometriose on the development potential of embryos. In this context, NO effects on embryos survival could either rely upon S-nitrosylation, NO/GC/cGMP, or peroxynitrite formation. Lee and collaborators [28] suggested that the apoptotic effects of NO excess on mice embryos could be related to S-nitrosylation, in exclusion to other any mechanisms. The latter effects were closely associated with lipid-rich organelles (mitochondria and endoplasmic reticulum) [28, 86]. On the other hand, trophoblasts might also be protected from apoptosis *via* S-nitrosylation of caspase 3 [87].

Moreover, NO was shown to influence trophoblasts motility [88, 89]: it was further proposed that NO effects trophoblasts migration and invasion, which are critical processes for the successful embryonic development. In human trophoblasts, NO was required for outgrowth since l-NAME prevented this phenomenon in a dose-dependent manner [90]. Nevertheless, one shall keep in mind that high concentrations of NO in the environment have deleterious effects on trophoblasts outgrowth [90]. Effects of NO on trophoblasts motility has been proposed to be mediated by nitrosylation of the matrix metalloprotease MMP9 [79], based on (1) iNOS and MMP9 colocalization in front migration in trophoblast (leading edge with lamellipodium) and (2) observations of MMP9 being S-nitrosylated [91]. The dynamics of iNOS and S-nitrosylated proteins accumulation at the leading edge in trophoblasts led the authors to conclude that iNOS was not likely to be passively piling up [79] (**Figure 5**). In these conditions, iNOS also accumulates in aggregates in the cytoplasm. Taken together, colocalization of Actin, MMP9, and iNOS at the leading edge suggested indeed an active role for S-nitrosylation in cell migration. Invasiveness of cytotrophoblast-derived cell lines induced by adrenomedullin was also associated with an increase in S-nitrosylated protein rate [92]. S-nitrosylation of urokinase plasminogen activator (uPA), whose involvement in extracellular matrix is acknowledged, was reported to increase in these conditions [92]; the mechanisms through which S-nitrosylation increased this enzyme activity remain to be clarified.

alternate mechanism than direct S-nitrosylation of Cdc25C for its inhibition was proposed since NOAD, a nitric oxide-releasing derivative of oleanolic acid, induced activation of Chk2, resulting in an increase of the inhibitory phosphorylation of Cdc25C on its residue Serine 216 [84]. However, genotoxicity of nitric oxide might account for the activation of Chk2, the latter being involved in DNA damage response machinery. In the same study, the arrest in G2/M was associated with the upregulation of Cdk inhibitors p21WAF1/CIP1 and P27KIP1, without providing evidence for direct S-nitrosylation of these proteins. p21WAF1/CIP1 downregulation by NO was also reported in *Xenopus* embryos, but through nitric oxide modulation of the RhoA-ROCK pathway [18] (**Figure 1B**). Thus, though there are converging evidences for role of NO in cell cycle regulation [85], the exact mechanisms remain to

**12. S-nitrosylation plays pivotal role in modulating trophoblast** 

might also be protected from apoptosis *via* S-nitrosylation of caspase 3 [87].

S-nitrosylation increased this enzyme activity remain to be clarified.

As mentioned above, S-nitrosylation has been also called to play a role in preimplantation embryos and implantation (**Figure 4**). Microenvironnemental presence of NO was reported to contribute to the pathologic effects of endometriose on the development potential of embryos. In this context, NO effects on embryos survival could either rely upon S-nitrosylation, NO/GC/cGMP, or peroxynitrite formation. Lee and collaborators [28] suggested that the apoptotic effects of NO excess on mice embryos could be related to S-nitrosylation, in exclusion to other any mechanisms. The latter effects were closely associated with lipid-rich organelles (mitochondria and endoplasmic reticulum) [28, 86]. On the other hand, trophoblasts

Moreover, NO was shown to influence trophoblasts motility [88, 89]: it was further proposed that NO effects trophoblasts migration and invasion, which are critical processes for the successful embryonic development. In human trophoblasts, NO was required for outgrowth since l-NAME prevented this phenomenon in a dose-dependent manner [90]. Nevertheless, one shall keep in mind that high concentrations of NO in the environment have deleterious effects on trophoblasts outgrowth [90]. Effects of NO on trophoblasts motility has been proposed to be mediated by nitrosylation of the matrix metalloprotease MMP9 [79], based on (1) iNOS and MMP9 colocalization in front migration in trophoblast (leading edge with lamellipodium) and (2) observations of MMP9 being S-nitrosylated [91]. The dynamics of iNOS and S-nitrosylated proteins accumulation at the leading edge in trophoblasts led the authors to conclude that iNOS was not likely to be passively piling up [79] (**Figure 5**). In these conditions, iNOS also accumulates in aggregates in the cytoplasm. Taken together, colocalization of Actin, MMP9, and iNOS at the leading edge suggested indeed an active role for S-nitrosylation in cell migration. Invasiveness of cytotrophoblast-derived cell lines induced by adrenomedullin was also associated with an increase in S-nitrosylated protein rate [92]. S-nitrosylation of urokinase plasminogen activator (uPA), whose involvement in extracellular matrix is acknowledged, was reported to increase in these conditions [92]; the mechanisms through which

be fully deciphered.

**motility and survival**

164 Nitric Oxide Synthase - Simple Enzyme-Complex Roles

**Figure 5.** Schematic view of iNOS and MMP9 localization in trophoblasts. Adapted from reference [79]. LE, leading edge; TE, trailing edge; N, nucleus; C, cytoplasm; Ag, aggresome (iNOS particle). Actin polymerisation sites were also detected in LE. Noteworthy, Harris and colleagues noted a colocalization of eNOS and actin in trophoblasts.

#### **13. A yin-yang relationship for S-nitrosylation and S-sulfhydration?**

Along with NO, hydrogen sulfide (H<sup>2</sup> S) is another gasotransmitter involved in regulating various aspects of cellular life. Many protein sites have been reported to undergo both S-nitrosylation and S-sulfhydration (brought by H<sup>2</sup> S), such as Actin [93, 94], GAPDH [95], Parkin [96], PTEN [97], and the p65 subunit of NF-κB [98]. If S-sulfhydration and nitrosylation may occur on the same residue [99], reactive site cysteine, they generally promote different and opposing effects. Indeed, S-nitrosylation typically reduces cysteine thiols reactivity, while S-sulfhydration increases cysteine thiols reactivity, thereby making them more nucleophilic. If one wants to compare S-sulhydration and nitrosylation, it has mainly to outline that (1) proteins are rather S-sulhydrated than S-nitrosylated, and (2) nitrosylation rather inhibits and impairs protein functions. In this regard, the case of tensin (PTEN) provides an example of yinyang relationship for S-nitrosylation and S-sulfhydration (**Figure 6**). Gene suppressor PTEN acts as an inhibitor of the PI-3 kinase/Akt signaling pathway, attenuating cellular growth and survival. S-nitrosylation enabled Akt hyperactivity, and thereby is associated with observed neuroprotective effects [100]. Oxidation of the active site cysteine is acknowledged as a common mechanism for regulating protein tyrosine phosphatases [101]. In addition, a NO-mediated PTEN degradation mechanism has been suggested to be common in neurodegenerative conditions where NO exerts a critical physiopathological role [102]. Finally, S-sulfhydration was

**Figure 6.** PTEN (left: phosphatase domain; right: C2 domain) is either S-nitrosylated (SNO) or S-sulfhydrated (SSH). When S-sulfhydrated, PTEN exerts its activity of phosphatase on PIP3 (phosphatidylinositol—3,4,5-triphosphate), in order to generate PIP2 (phosphatidylinositol-4,5-bisphosphate). In the absence of hydrogen sulfide, low concentrations of NO drive S-nitrosylation of PTEN, leading to its inactivation. In these conditions, Akt activity is maintained. If low concentration of NO drives SNO-PTEN, higher concentrations lead to SNO-Akt and abrogation of survival signal.

reported to maintain the activity of this lipid tyrosine-phosphatase, thereby preventing its S-nitrosylation [97]. PTEN structure reports disparate sites for S-nitrosylation (Cys 83 [100]), S-sulfhydration, and hydrogen peroxide-induced disulfide bond formation (Cys 71 and Cys 124 [103]). One should keep in mind that in this particular example, S-nitrosylation targets a different site than the Cys 124, mandatory in the phosphatase activity of PTEN.

It is tantalizing to hypothesize that sequence of S-nitrosylation and S-sulfhydration could provide a way for fine tuning of signaling pathways and cellular functions regulation. Because protein S-nitrosylation can foster intramolecular disulfide bond formation, a protein S-nitrosylation event might promote the formation of a more enduring S-sulfhydration reaction.

#### **14. Conclusion**

While NO is not necessary for meiotic resumption *per se* in amphibians, NO clearly influences meiotic processes in rodents, porcine, and bovine oocytes. During fertilization, the role of nitric oxide evolved from the hypothesis of being a primary activator to being solely correlated to fertilization, and its role was limited to a particular function such as hardening of the fertilization envelope in sea urchins. Therefore, the physiological relevance of NO has been debated. Nevertheless, NO, together with its effects on spermatozoa (viability, motility, capacitation acrosome reaction, and fusion with oocytes) appeared as a modulator of oocyte aging. The benefits of nitric oxide use in assisted reproduction are now well-considered.

Though NO was not reported to be involved in early developmental processes in ascidians, NO positively affects cell proliferation in early *Xenopus* embryos and impairs cell movement during gastrulation in this model. The involvement of NO during segmentation is emphasized in mammalian models, where NO seemed to be requested for segmentation and blastocysts survival (rodents, porcine, and bovine) and for implantation through blastocysts hatching (rodents and bovine) and trophoblasts motility (humans).

One of the main difficulties when considering the effects of NO remains in the existence of the multiple pathways that can be activated by this gasotransmitter: cGMP-dependent pathway, calcium-related pathways, and reactive oxygen species production. Over the past few years, S-nitrosylation has offered a new angle to decipher NO's actions since S-nitrosylation modulates the activities of many key regulators such as members of the RhoA-ROCK pathway, Cdk2, Cc25, or PTEN. PTEN regulation by nitrosylation offers a new paradigm since sulfhydration and nitrosylation, both provided by gasotransmitters, appeared to play reciprocally in a yin-yang manner.

#### **Acknowledgements**

JFB is affiliated with the Site de Recherche Intégrée en Cancérologie (SIRIC ONCOLILLE). We would like to thank the personnel of the BICeL-Lille1-HB Facility for access to the microscopy systems and technical advices. We are indebted to the Research Federation FRABio for providing the scientific and technical environment to achieving our work [14, 15]. MJ work in Department of Obstetrics and Gynaecology, Center of Assisted Reproduction, University Hospital Brno, and Masaryk University. MJ's work was supported by grant MH CZ—DRO (FNBr, 65269705) and funds from the Faculty of Medicine, Masaryk University Brno, Czech Republic. We thank Brian Kavalir for helpful discussions and comments.

#### **Author details**

reported to maintain the activity of this lipid tyrosine-phosphatase, thereby preventing its S-nitrosylation [97]. PTEN structure reports disparate sites for S-nitrosylation (Cys 83 [100]), S-sulfhydration, and hydrogen peroxide-induced disulfide bond formation (Cys 71 and Cys 124 [103]). One should keep in mind that in this particular example, S-nitrosylation targets

**Figure 6.** PTEN (left: phosphatase domain; right: C2 domain) is either S-nitrosylated (SNO) or S-sulfhydrated (SSH). When S-sulfhydrated, PTEN exerts its activity of phosphatase on PIP3 (phosphatidylinositol—3,4,5-triphosphate), in order to generate PIP2 (phosphatidylinositol-4,5-bisphosphate). In the absence of hydrogen sulfide, low concentrations of NO drive S-nitrosylation of PTEN, leading to its inactivation. In these conditions, Akt activity is maintained. If low concentration of NO drives SNO-PTEN, higher concentrations lead to SNO-Akt and abrogation of survival signal.

It is tantalizing to hypothesize that sequence of S-nitrosylation and S-sulfhydration could provide a way for fine tuning of signaling pathways and cellular functions regulation. Because protein S-nitrosylation can foster intramolecular disulfide bond formation, a protein S-nitrosylation

While NO is not necessary for meiotic resumption *per se* in amphibians, NO clearly influences meiotic processes in rodents, porcine, and bovine oocytes. During fertilization, the role of nitric oxide evolved from the hypothesis of being a primary activator to being solely correlated to fertilization, and its role was limited to a particular function such as hardening of the fertilization envelope in sea urchins. Therefore, the physiological relevance of NO has

a different site than the Cys 124, mandatory in the phosphatase activity of PTEN.

event might promote the formation of a more enduring S-sulfhydration reaction.

**14. Conclusion**

166 Nitric Oxide Synthase - Simple Enzyme-Complex Roles

Ješeta Michal<sup>1</sup> , Marketa Sedmikova<sup>2</sup> , and Jean-François Bodart<sup>3</sup> \*

\*Address all correspondence to: jean-francois.bodart@univ-lille1.fr

1 Department of Obstetrics and Gynaecology, Center of Assisted Reproduction, University Hospital Brno and Masaryk University, Brno, Czech Republic

2 Department of Veterinary Sciences, Faculty of Agrobiology, Food and Natural Resources, Czech University of Life Sciences in Prague, Prague-Suchdol, Czech Republic

3 University of Lille, CNRS, UMR 8576 - UGSF - Unité de Glycobiologie Structurale et Fonctionnelle, Lille, France

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**Miscellaneous Roles of Nitric Oxide Synthase**

## **Role of Endothelial Nitric Oxide Synthase in Breast Cancer**

Tupurani Mohini Aiyengar, Padala Chiranjeevi and Hanumanth Surekha Rani

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/67493

#### **Abstract**

Breast cancer (BC) is the most common form of carcinoma and a primary cause of morbidity and mortality globally. Oxidative stress represents as an important factor in carcinogenesis and may play a role in initiation and progression of tumors. Oxidative stress–induced NO• damage to DNA includes a multitude of lesions, many of which are mutagenic and have multiple roles in cancer and aging. It is caused by an unfavorable balance between reactive oxygen species/reactive nitrogen species (ROS/RNS) and antioxidant defenses. ROS/RNS are generated during normal cellular metabolism, as a result of the influence of various environmental factors, as well as during pathological processes. Nitric oxide (NO•) is a ubiquitous, short-lived free radical produced from L-arginine by nitric oxide synthases (NOSs), and isoforms of NOS exist, depending on the site of origin: endothelial (eNOS), neuronal (nNOS), mitochondrial (mtNOS), and inducible (iNOS). eNOS is responsible for the endothelial synthesis of NO• and has shown to modulate cancer-related events such as inflammation, angiogenesis, apoptosis, cell cycle, invasion, and metastasis. Genetic studies also showed that eNOS gene polymorphisms are associated with the development of breast cancer. Therefore, selective targeting of eNOS may prove a potential strategy for prevention and treatment of breast cancer.

**Keywords:** breast cancer, oxidative stress, nitric oxide, endothelial nitric oxide synthase, therapeutics

#### **1. Introduction**

Cancer, a multifaceted disorder, represents one of the most important health problems worldwide, with approximately 14 million new cases and 8.2 million cancer-related deaths every

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

year [1]. The transformation from a normal cell into a tumor cell is a multistage process, typically a progression from a precancerous lesion to malignant tumors.

Cancers originate from a single abnormal cell (clonal origin) with an altered DNA sequence (mutation). Successive rounds of mutation and selective expansion of these cells result in the formation of a tumor mass and leads to tumor growth and progression, which eventually breaks through the basal membrane barrier of surrounding tissues and spreads to other parts of the body (metastasis) (**Figure 1**).

Cancers may be classified by their primary site of origin such as brain cancer, oral cancer, lung cancer, prostate cancer, liver cancer, renal cell carcinoma (kidney cancer), breast cancer, etc.

Breast cancer is defined as a malignant growth that begins in the epithelium of the breast. It is estimated as one of the most commonly diagnosed cancers worldwide (11.9%) [2]. In India, breast cancer has overtaken cervical cancer and has become the most prevailing cancer among women [3]. The most frequent type of breast cancer is ductal carcinoma *in situ* (DCIS), which affects the cells of the milk ducts. Cancer that starts in lobes or lobules is called lobular carcinoma *in situ* (LCIS); it is the second most common type of breast cancer. While rare breast cancer types include tubular, medullary, metaplastic, mucinous carcinoma, and Paget's disease.

Breast cancer is a clinically heterogeneous disease. Breast cancer cells generally overexpress estrogen receptor (ER)/progesterone receptor (PR), and/or human epidermal growth factor-2 (HER-2) receptor and lead to the tumor formation and progression. Thus, breast cancer can be classified into two subgroups on the basis of receptor status namely: (i) ER/PR positive (luminal A/B) and (ii) triple negative (ER, PR, and HER-2 negative) (basal-like) to know the prognosis and clinical outcome of breast cancer (**Figure 2**). Early breast cancer usually does not cause symptoms. As the cancer grows, symptoms may include: lump in the armpit, change in the size, shape, or feel of the breast or nipples, nipple discharge, etc. Symptoms of advanced breast cancer may include: breast pain or discomfort, bone pain, skin ulcers, and weight loss.

Breast cancer etiology is complex and multifactorial where there is a strong interplay between genetic and environmental factors. The strongest nonmodifiable determinants of breast cancer risk are female gender and age. Other risk factors associated with breast cancer can be grouped into three broad determinants: family history (hereditary) factors, hormonal and reproductive factors, and environmental (including lifestyle) factors (**Figure 3**).

**Figure 1.** Carcinogenesis, a multistep process.

Role of Endothelial Nitric Oxide Synthase in Breast Cancer http://dx.doi.org/10.5772/67493 181

**Figure 2.** Classification of breast cancer.

year [1]. The transformation from a normal cell into a tumor cell is a multistage process, typi-

Cancers originate from a single abnormal cell (clonal origin) with an altered DNA sequence (mutation). Successive rounds of mutation and selective expansion of these cells result in the formation of a tumor mass and leads to tumor growth and progression, which eventually breaks through the basal membrane barrier of surrounding tissues and spreads to other parts

Cancers may be classified by their primary site of origin such as brain cancer, oral cancer, lung cancer, prostate cancer, liver cancer, renal cell carcinoma (kidney cancer), breast cancer, etc. Breast cancer is defined as a malignant growth that begins in the epithelium of the breast. It is estimated as one of the most commonly diagnosed cancers worldwide (11.9%) [2]. In India, breast cancer has overtaken cervical cancer and has become the most prevailing cancer among women [3]. The most frequent type of breast cancer is ductal carcinoma *in situ* (DCIS), which affects the cells of the milk ducts. Cancer that starts in lobes or lobules is called lobular carcinoma *in situ* (LCIS); it is the second most common type of breast cancer. While rare breast cancer types include tubular, medullary, metaplastic, mucinous carcinoma, and Paget's disease. Breast cancer is a clinically heterogeneous disease. Breast cancer cells generally overexpress estrogen receptor (ER)/progesterone receptor (PR), and/or human epidermal growth factor-2 (HER-2) receptor and lead to the tumor formation and progression. Thus, breast cancer can be classified into two subgroups on the basis of receptor status namely: (i) ER/PR positive (luminal A/B) and (ii) triple negative (ER, PR, and HER-2 negative) (basal-like) to know the prognosis and clinical outcome of breast cancer (**Figure 2**). Early breast cancer usually does not cause symptoms. As the cancer grows, symptoms may include: lump in the armpit, change in the size, shape, or feel of the breast or nipples, nipple discharge, etc. Symptoms of advanced breast cancer may include: breast pain or discomfort, bone pain, skin ulcers, and weight loss. Breast cancer etiology is complex and multifactorial where there is a strong interplay between genetic and environmental factors. The strongest nonmodifiable determinants of breast cancer risk are female gender and age. Other risk factors associated with breast cancer can be grouped into three broad determinants: family history (hereditary) factors, hormonal and reproduc-

cally a progression from a precancerous lesion to malignant tumors.

tive factors, and environmental (including lifestyle) factors (**Figure 3**).

of the body (metastasis) (**Figure 1**).

180 Nitric Oxide Synthase - Simple Enzyme-Complex Roles

**Figure 1.** Carcinogenesis, a multistep process.

**Figure 3.** Breast cancer etiology.

*Family history (hereditary) factors* are associated primarily with early-onset of breast cancer. Previous genetic analyses of breast cancer–prone families have identified the BRCA1 and BRCA2 genes (breast cancer type 1 and 2 susceptibility protein). Women with mutations in either BRCA1 or BRCA2 (over 250 mutations have been identified) are at a significantly elevated lifetime risk (55–85% compared with 12% for the general population) for developing breast or ovarian cancer [4].

*Reproductive and hormonal factors* may increase the time and/level of steroid hormone exposure, consequently stimulating cell growth, and have been associated with breast cancer susceptibility. Major reproductive factors such as early menarche, late menopause, later age at first full-term pregnancy, and nulliparity are known to be associated with a higher risk of breast cancer [5].

*Environmental (including lifestyle) factors* such as the use of exogenous estrogens, radiation exposure, alcohol consumption, and socioeconomic status are also well-known risk factors for the disease [6]. Physical inactivity is a major risk factor for several types of cancer [7– 9]. Exposure to ionizing radiation such as X-rays at a young age, alcohol consumption, and smoking habits have consistently been shown to increase breast cancer risk.

Radiations are classified into two major types namely: ionizing radiation and nonionizing radiation. The ionizing radiation transmits energy via X-rays and alpha particles disrupt chemical bonds, which results in chemically reactive free radical formation, a phenomenon known as ionization. These ionizing radiations may directly pass through the cell and may cause DNA damage. Such damage if unrepaired can result in nonlethal DNA modifications or cell death. The nonlethal DNA modifications thus eventually may cause malignant transformations. Ionizing radiation is, therefore, a known mutagen and an established breast carcinogen. Nonionizing radiation (e.g., microwaves and extremely low-frequency electric and magnetic fields (ELF-EMF)) does not have enough energy to break chemical bonds and produce ionization [10].

Several epidemiological studies have shown that alcohol consumption has been associated with breast cancer susceptibility. The alcohol metabolism occurs via multiple stages which may increase the risk of carcinogenesis. Alcohol metabolized into an acetaldehyde and other products subsequently damages DNA by inducing DNA modifications. Apart from this, acetaldehyde alone may also cause breast tumorigenesis by interfering with DNA repair mechanisms. Free radicals generated in the second stage of alcohol metabolism are thought to cause DNA damage, strand breakage, and base alterations and have been implicated for their role in alcohol-associated carcinogenesis [11].

There are over 60 carcinogens in cigarette smoke, which have been evaluated by the International Agency for Research on Cancer [12]. Cigarette smoke is rich in carcinogens such as nitrosamines and polycyclic aromatic hydrocarbons and induces oxidative damage. The gas phase of freshly generated cigarette smoke has large amounts of nitric oxide and other unstable oxidants. The presence of such free radicals and oxidants can lead to oxidative DNA damage [13]. The environmental risk factors that alter the levels of free radicals (reactive species) generated in the body are known to react with DNA to cause mutations in critical genes and consequently promote carcinogenesis.

#### **2. Free radicals**

*Family history (hereditary) factors* are associated primarily with early-onset of breast cancer. Previous genetic analyses of breast cancer–prone families have identified the BRCA1 and BRCA2 genes (breast cancer type 1 and 2 susceptibility protein). Women with mutations in either BRCA1 or BRCA2 (over 250 mutations have been identified) are at a significantly elevated lifetime risk (55–85% compared with 12% for the general population) for developing

*Reproductive and hormonal factors* may increase the time and/level of steroid hormone exposure, consequently stimulating cell growth, and have been associated with breast cancer susceptibility. Major reproductive factors such as early menarche, late menopause, later age at first full-term pregnancy, and nulliparity are known to be associated with a higher risk of

*Environmental (including lifestyle) factors* such as the use of exogenous estrogens, radiation exposure, alcohol consumption, and socioeconomic status are also well-known risk factors for the disease [6]. Physical inactivity is a major risk factor for several types of cancer [7– 9]. Exposure to ionizing radiation such as X-rays at a young age, alcohol consumption, and

Radiations are classified into two major types namely: ionizing radiation and nonionizing radiation. The ionizing radiation transmits energy via X-rays and alpha particles disrupt chemical bonds, which results in chemically reactive free radical formation, a phenomenon known as ionization. These ionizing radiations may directly pass through the cell and may cause DNA damage. Such damage if unrepaired can result in nonlethal DNA modifications or cell death. The nonlethal DNA modifications thus eventually may cause malignant transformations. Ionizing radiation is, therefore, a known mutagen and an established breast carcinogen. Nonionizing radiation (e.g., microwaves and extremely low-frequency electric and magnetic fields (ELF-EMF)) does not have enough energy to break chemical bonds and produce ionization [10].

Several epidemiological studies have shown that alcohol consumption has been associated with breast cancer susceptibility. The alcohol metabolism occurs via multiple stages which may increase the risk of carcinogenesis. Alcohol metabolized into an acetaldehyde and other products subsequently damages DNA by inducing DNA modifications. Apart from this, acetaldehyde alone may also cause breast tumorigenesis by interfering with DNA repair mechanisms. Free radicals generated in the second stage of alcohol metabolism are thought to cause DNA damage, strand breakage, and base alterations and have been implicated for their role

There are over 60 carcinogens in cigarette smoke, which have been evaluated by the International Agency for Research on Cancer [12]. Cigarette smoke is rich in carcinogens such as nitrosamines and polycyclic aromatic hydrocarbons and induces oxidative damage. The gas phase of freshly generated cigarette smoke has large amounts of nitric oxide and other unstable oxidants. The presence of such free radicals and oxidants can lead to oxidative DNA damage [13]. The environmental risk factors that alter the levels of free radicals (reactive species) generated in the body are known to react with DNA to cause mutations in critical genes

smoking habits have consistently been shown to increase breast cancer risk.

breast or ovarian cancer [4].

182 Nitric Oxide Synthase - Simple Enzyme-Complex Roles

in alcohol-associated carcinogenesis [11].

and consequently promote carcinogenesis.

breast cancer [5].

Free radicals are molecules with high instability and reactivity due to the presence of an odd number of electrons in the outermost orbit of their atoms; their action derives from their attempts to attain "balance" by binding with electrons of neighboring atoms, giving rise to chain reactions (**Figure 4**) [14].

Free radical can be classified as reactive oxygen species (ROS) and reactive nitrogen species (RNS). ROS include the superoxide anion (O2 − ), hydroxyl radical (OH<sup>−</sup> ), hydrogen peroxide (H2 O2 ), while RNS include nitric oxide (NO**•**<sup>−</sup> ) and peroxynitrite (ONOO<sup>−</sup> ) the radical nitrogen dioxide (NO2 − ), and nitrite (NO2 − ) [15].

Free radicals are key players in the initiation and progression of tumor cells and enhance their metastatic potential. They are now considered a hallmark of cancer.

**Figure 4.** Formation and elimination of ROS.

#### **3. Oxidative stress**

The cell generates free radicals and also degrades, which is strictly necessary to avoid the damage derived from free radicals. However, various intrinsic and extrinsic circumstances and the biochemical activity of the cell can make it lose control over the formation and management of free radicals and results in "oxidative stress." It results from a disturbance in balance between the formation of ROS/RNS and the defense provided by cell antioxidants. This imbalance may cause damage related to various human diseases (**Figure 5**) [16].

During endogenous metabolic reactions, aerobic cells produce ROS such as superoxide anion (O2 − ), hydrogen peroxide (H2 O2 ), hydroxyl radical (OH•), and organic peroxides as normal products of the biological reduction of molecular oxygen [17]. Under hypoxic conditions, the mitochondrial respiratory chain also produces nitric oxide (NO•<sup>−</sup> ), which can generate other reactive nitrogen species (RNS) [18].

**Figure 5.** Oxidative stress.

#### **4. Antioxidants**

Cells have natural defense systems against ROS that consist of antioxidant enzymes, vitamins, etc., some of these antioxidants are produced inside the human body, mostly falling into the enzymatic category, as they are predominantly protein in nature. These proteins include the superoxide dismutase (SOD) enzymes (which have differential subcellular localization and superoxide dismutation to H2 O2 ), glutathione peroxidase (GPx), and catalase (both of which clear peroxide), thioredoxins (Trxs; reduce oxidized proteins), and glutathione synthetase (GSS; synthesizes glutathione [GSH], an important antioxidant), among others. Vitamins are mostly obtained from nutritional sources and include ascorbic acid (vitamin A), tocopherol. Therefore, a fine balance exists between the levels of ROS and antioxidants within the cell [19]. Increased ROS/RNS can result in a greater number of mutations, oxidation of critical proteins, and other alterations, finally culminating in cell death. Identification of potentially modifiable factors that affect oxidative stress in breast cancer patients is an increasingly important task.

#### **5. Nitric oxide**

Nitric oxide (NO•) is a ubiquitous, short-lived, water soluble and endogenously produced gas that exerts a wide range of biological effects. It is a pleiotropic regulator, critical to numerous biological processes, including vasodilation, neurotransmission, and macrophage-mediated immunity.

The general function of NO• protects against the effects of free radicals but at excessive concentrations, NO• or its derivatives may lead to DNA damage and impair the tumor suppressor function of p53, which may cause cancer development [20].

#### **5.1. Intracellular mechanisms of NO•<sup>−</sup>**

When NO• is synthesized, it has a half-life of only a few seconds. Its bioavailability is reduced due to the high affinity binding of superoxide anion (high reactivity in both molecules is due to the unpaired electrons). NO• has an ability to tightly bind to the heme moiety of both hemoglobin (Hb) and guanylyl cyclase (GC) and found mostly in the vascular smooth muscle cells and other cells. Thus, NO• when produced in endothelium is quickly diffused into the blood circulation, binds to hemoglobin in blood and forms nitrates. NO• may also activate guanylyl cyclase (GC), an enzyme that catalyzes the dephosphorylation of guanosine triphosphate (GTP) to 3′,5′-cyclic guanosine monophosphate (cGMP) and serves as a second messenger for many important cellular functions, particularly for signaling smooth muscle relaxation [21].

#### **5.2. NO•<sup>−</sup> biological functions**

**4. Antioxidants**

**Figure 5.** Oxidative stress.

184 Nitric Oxide Synthase - Simple Enzyme-Complex Roles

**5. Nitric oxide**

ated immunity.

superoxide dismutation to H2

O2

Cells have natural defense systems against ROS that consist of antioxidant enzymes, vitamins, etc., some of these antioxidants are produced inside the human body, mostly falling into the enzymatic category, as they are predominantly protein in nature. These proteins include the superoxide dismutase (SOD) enzymes (which have differential subcellular localization and

clear peroxide), thioredoxins (Trxs; reduce oxidized proteins), and glutathione synthetase (GSS; synthesizes glutathione [GSH], an important antioxidant), among others. Vitamins are mostly obtained from nutritional sources and include ascorbic acid (vitamin A), tocopherol. Therefore, a fine balance exists between the levels of ROS and antioxidants within the cell [19]. Increased ROS/RNS can result in a greater number of mutations, oxidation of critical proteins, and other alterations, finally culminating in cell death. Identification of potentially modifiable factors that affect oxidative stress in breast cancer patients is an increasingly important task.

Nitric oxide (NO•) is a ubiquitous, short-lived, water soluble and endogenously produced gas that exerts a wide range of biological effects. It is a pleiotropic regulator, critical to numerous biological processes, including vasodilation, neurotransmission, and macrophage-medi-

), glutathione peroxidase (GPx), and catalase (both of which

Nitric oxide (NO•<sup>−</sup> ) is known to play important functional roles in a variety of physiological systems [22].


While, NO also inhibits the contractility of the smooth muscle wall of the uterus. During child birth, as the moment of birth approaches the production of NO decreases.

**5.** NO•<sup>−</sup> affects secretion from several endocrine glands—NO regulates the release of Gonadotropin-releasing hormone (GnRH) from the hypothalamus and adrenaline from the adrenal medulla.

In mammals, under normoxic conditions, NO• is generated endogenously "on demand" when the guanidino nitrogen of L-arginine undergoes a five-electron oxidation to yield the gaseous free radical, nitric oxide, and citrulline in equimolar concentrations (**Figure 6**) [23].

The constitutive forms of NOS are endothelial NOS (eNOS; type III) and inducible NOS (iNOS; type II). Cofactors for NOS include oxygen, NADPH, tetrahydrobiopterin, and flavin adenine nucleotides.

The inducible (calcium-independent) isoform (iNOS) produces much larger amounts of NO and is only expressed during inflammation. Whereas iNOS can produce injurious amounts of RNS (check), eNOS and nNOS produce beneficial amounts under physiological conditions. The constitutive (calcium-dependent) isoform and endothelial NOS (eNOS) produce small amounts of NO, which act as a vasodilator. The third form, neural NOS (nNOS; type I) serves as a transmitter in the brain and in different nerves of the peripheral nervous system to produce vasodilation. mtNOS constitutively expressed and membrane-bound nNOS isoform alpha, precluding a novel alternative spliced product. The mitochondrial production of nitric oxide is catalyzed nitric-oxide synthase (mtNOS). This enzyme has the same cofactor and substrate requirements as other constitutive nitric-oxide synthases (**Figure 6**).

NOS1 is the neural (or brain) isoform, also known as *nNOS*. It plays an important role in neural communication via synaptic transmission from nerve to nerve across synapses, and from peripheral nerves to the brain.

NOS2 is also known as *iNOS*. It generates extremely elevated concentrations of NO, to participate in a host defense mechanism. It also takes several hours to be activated in response to an injury or infection. Unlike nNOS, which takes part in normal neural communication, an abnormal stimulus (a wound, tissue damage, hypoxia, bacterial infection, etc.) may induce iNOS.

**Figure 6.** Mechanism of NO production from NOS.

The third isoform is *eNOS* (or NOS3) which stands for "endothelial cell" NOS. This isoform is active at all times and is found in endothelial cells which are the cells that line the inner surface of all blood vessels and lymph ducts. The eNOS is activated by the pulsatile flow of blood through vessels and maintains the diameter of the blood vessels at an optimal level. In addition, it also promotes angiogenesis, a process of new blood vessels formation.

A fourth type, mitochondrial NOS (*mtNOS*), differentiated by its subcellular localization in the mitochondrial inner membrane, regulates various functions of mitochondria in liver, heart, kidney, breast, etc. The mitochondrial utilization of NO involves superoxide anion and H2 O2 , a species freely diffusible outside the mitochondria that participate in the modulation of cell proliferation and apoptosis and in cell transformation leading to cancer [24, 25].

#### **6. Endothelial nitric oxide synthase (eNOS)**

In mammals, under normoxic conditions, NO• is generated endogenously "on demand" when the guanidino nitrogen of L-arginine undergoes a five-electron oxidation to yield the gaseous free radical, nitric oxide, and citrulline in equimolar concentrations (**Figure 6**) [23].

The constitutive forms of NOS are endothelial NOS (eNOS; type III) and inducible NOS (iNOS; type II). Cofactors for NOS include oxygen, NADPH, tetrahydrobiopterin, and flavin

The inducible (calcium-independent) isoform (iNOS) produces much larger amounts of NO and is only expressed during inflammation. Whereas iNOS can produce injurious amounts of RNS (check), eNOS and nNOS produce beneficial amounts under physiological conditions. The constitutive (calcium-dependent) isoform and endothelial NOS (eNOS) produce small amounts of NO, which act as a vasodilator. The third form, neural NOS (nNOS; type I) serves as a transmitter in the brain and in different nerves of the peripheral nervous system to produce vasodilation. mtNOS constitutively expressed and membrane-bound nNOS isoform alpha, precluding a novel alternative spliced product. The mitochondrial production of nitric oxide is catalyzed nitric-oxide synthase (mtNOS). This enzyme has the same cofactor and

NOS1 is the neural (or brain) isoform, also known as *nNOS*. It plays an important role in neural communication via synaptic transmission from nerve to nerve across synapses, and from

NOS2 is also known as *iNOS*. It generates extremely elevated concentrations of NO, to participate in a host defense mechanism. It also takes several hours to be activated in response to an injury or infection. Unlike nNOS, which takes part in normal neural communication, an abnormal stimulus (a wound, tissue damage, hypoxia, bacterial infection, etc.) may induce iNOS.

substrate requirements as other constitutive nitric-oxide synthases (**Figure 6**).

adenine nucleotides.

186 Nitric Oxide Synthase - Simple Enzyme-Complex Roles

peripheral nerves to the brain.

**Figure 6.** Mechanism of NO production from NOS.

Endothelial nitric oxide synthase enzyme, also known as nitric oxide synthase-3 (NOS-3) or constitutive NOS (cNOS), has been shown to be a critical regulator of carcinogenesis. It is a dimeric structure in their active form containing two identical monomers of 134 kD represented by *a reductase domain* containing the binding sites for nicotinamide adenine dinucleotide phosphate (NADPH), flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD). While *an oxidase domain* comprises the binding sites for the heme group, zinc, the cofactor tetrahydrobiopterin (BH4), and the substrate L-arginine [26]. The reductase domain is attached to the oxidase domain via calmodulin-binding sequence [27].

The eNOS expression is regulated by a range of transcriptional and posttranscriptional mechanisms, generates nitric oxide (NO•) in response to a number of stimuli. Constitutively expressed eNOS oxidizes L-arginine to generate L-citrulline and NO•. The essential cofactors for catalysis of this reaction include as calmodulin (CaM), flavin mononucleotide, flavin adenine dinucleotide, tetrahydrobiopterin (H4B) and NADPH.

Typically, the eNOS isoforms can be activated as a result of calmodulin (CaM) binding following a rise in intracellular calcium. They may also be activated and/or inhibited by phosphorylation via various protein kinases. Oxygen levels also regulate NOS levels in a cell type and isoform-specific manner by altering enzyme expression and by limiting the availability of oxygen, a key substrate for NO• synthesis.

Experimental and epidemiological evidence has shown the contributory role of eNOS induced NO• in tumor progression, suggesting a possible implication of endothelium expressed eNOS in tumors. The role of NO• in cancer biology is widespread, including its involvement in cellular transformation, malignant lesions, initiation, progression of the metastatic process, and induction of genotoxicity [28]. NO•-mediated DNA damage may be due to direct modification of DNA, or inhibition of DNA repair mechanisms [29]. Similarly, most of the RNS can cause DNA strand breaks and result in multiple mutations in DNA [30].

Breast carcinogenesis involves the transformation of a normal cell into a tumor cell mediated by a sequence of cellular and molecular events. These events consist of the attainment of precise characteristics, such as uncontrolled proliferation, avoidance of mitotic control, resistance to apoptosis, replicative immortality, escape from immune surveillance, progression by stimulating invasion and metastasis, angiogenesis, genomic instability, and deregulated metabolism [31].

Tumor-derived eNOS has shown to modulate cancer-related events (inflammation, apoptosis, cell cycle, angiogenesis, invasion, and metastasis) (**Figure 7**) and genetic studies showed that eNOS gene polymorphisms are associated with the development of multiple cancers [32].

**Figure 7.** Multiple roles of eNOS in tumor development.

#### **6.1. Inflammation**

Inflammation a localized protective response elicited by injury is known to cause DNA damage [33]. Chronic inflammation due to infection or injury is estimated to contribute to 25% of all cancers in the world [34]. A growing body of laboratory research has shown that inflammation is a key mediator in the promotion of malignant transformation, where pro-inflammatory cytokines can facilitate tumor growth and metastasis by altering tumor cell biology and activating stromal cells in the tumor microenvironment, such as vascular endothelial cells, tumor-associated macrophages, and fibroblasts.

NO• is closely related to inflammatory status and regarded as a critical inflammation mediator. Pro-inflammatory cytokines can modulate the expression of eNOS and can accelerate the growth and development of cancer. eNOS, for example, can regulate the expression of the pro-inflammatory molecules nuclear factor-κB (NF-κB) and cyclooxygenase-2 [32, 35, 36].

#### **6.2. Apoptosis**

precise characteristics, such as uncontrolled proliferation, avoidance of mitotic control, resistance to apoptosis, replicative immortality, escape from immune surveillance, progression by stimulating invasion and metastasis, angiogenesis, genomic instability, and deregulated

Tumor-derived eNOS has shown to modulate cancer-related events (inflammation, apoptosis, cell cycle, angiogenesis, invasion, and metastasis) (**Figure 7**) and genetic studies showed that eNOS gene polymorphisms are associated with the development of multiple

Inflammation a localized protective response elicited by injury is known to cause DNA damage [33]. Chronic inflammation due to infection or injury is estimated to contribute to 25% of all cancers in the world [34]. A growing body of laboratory research has shown that inflammation is a key mediator in the promotion of malignant transformation, where pro-inflammatory cytokines can facilitate tumor growth and metastasis by altering tumor cell biology and activating stromal cells in the tumor microenvironment, such as vascular endothelial cells,

NO• is closely related to inflammatory status and regarded as a critical inflammation mediator. Pro-inflammatory cytokines can modulate the expression of eNOS and can accelerate the growth and development of cancer. eNOS, for example, can regulate the expres-

metabolism [31].

188 Nitric Oxide Synthase - Simple Enzyme-Complex Roles

cancers [32].

**6.1. Inflammation**

tumor-associated macrophages, and fibroblasts.

**Figure 7.** Multiple roles of eNOS in tumor development.

Apoptosis is an ordered and orchestrated cellular process that occurs in physiological and pathological conditions. Impaired apoptosis has been associated with initiation and development of cancer. The mechanism of apoptosis is regulated by an array of factors triggering and activating signaling cascade, subsequently leading to cellular death [33]. eNOS may be a molecular node in growth factor–mediated inhibition of apoptosis [37]. The antiapoptotic mechanism is understood on the basis of gene transcription of protective proteins and direct inhibition of the apoptotic executive effectors (caspase family protease).

The mechanisms of action that lead to the proneoplastic activity of NO• are via apoptosis inhibition by S-nitrosylation-inactivation of caspases- 1, 2, 4, 8 and 3, 6, 7 and disruption of the apoptotic protease activating factor 1/caspase-9 complex (Apaf-1/caspase-9 apoptosome is an essential initiator of caspase activation that initiates an apoptotic protease cascade) [38]. Other antiapoptotic effects of NO• depends on the interaction of NO•/cGMP, which inhibits the release of cytochrome C, stimulation of heat-shock protein (Hsp) 70 and Hsp 32, elevated Bcl-2 expression, repression of ceramide generation [39], and induces cyclooxygenase-2 activity [40]. In the animal model study, the lipopolysaccharide (LPS)-induced hepatic apoptosis was increased by the administration of NOS inhibitors [41]. Thus at high concentration, NO• has a potential role in cancer, i.e., inhibition of eNOS activity specific to tumor cells may be a viable option for the stimulation of apoptosis and treatment of cancer alone or in combination with chemotherapeutic agents.

#### **6.3. Angiogenesis**

Angiogenesis in mammary tumors can be stimulated by inflammation, which induces proliferation and morphogenesis of vascular endothelial cells in response to a large number of cytokines or angiogenic molecules produced by tumor and host cells [42]. Angiogenesis is essential for tumor growth and metastasis and has been considered the most important prognostic indicator for predicting overall survival [43]. eNOS strongly affects tumor growth by promoting angiogenesis [44]. Tumor growth–enhancing effects of vascular endothelial growth factor (VEGF) are associated with increased NOS activity and inhibition of apoptosis in human breast carcinoma xenografts [45].

Endogenous NO• promotes tumor blood flow via dilatation of arteriolar vessels. It decreases leukocyte-endothelial adhesive interactions and increases vascular permeability [46]. Several cancer treatment methods influence eNOS expression and activity. Low-dose irradiationinduced angiogenesis is believed to be mediated by NO• from eNOS [47]. Studies have shown that VEGF released as a purified protein or produced by tumor cells requires a functional NO•/cGMP pathway within the end compartment to promote neovascular growth. NO• also has an invasion-stimulating effect, which is mediated by upregulation of MMP-2 and MMP-9 (matrix metalloproteinases) and downregulation of TIMP-2 and possibly TIMP-3 (tissue inhibitors of MMP) [48].

#### **6.4. Tumor progression/invasion**

NO• has been investigated regarding its possible involvement in the promotion of breast carcinoma. Increased amounts of NO• have been observed in the blood circulation of advanced grade breast cancer patients [49] where the increased levels of NO• have shown to promote tumor angiogenesis. Nitrotyrosine, a marker derived from NO•, was correlated with expression of VEGF-C and has been associated with lymph node metastasis in breast carcinoma patients, implicating the role of NO• in the development and progression of breast cancer [50]. In relation to the link of eNOS with cell proliferation, the eNOS expression has been detected in tumor cells specifically in breast cancer [51].

Several studies have observed NO• released by eNOS, can stimulate cancer cell cycle progression and proliferation. More specific to eNOS, studies have shown that the eNOS/NO• pathway plays a role in cancer cell DNA/RNA synthesis and proliferation apart from promoting angiogenesis [52]. The eNOS gene plays an essential role in endothelial cell proliferation in cell culture models and is a central mediator of several endothelium growth stimulators, such as vascular endothelial growth factor (VEGF) and prostaglandin E2. In human breast cancer, eNOS appears to be expressed in tumor epithelial cells, and its presence is correlated with histological grade and lymph node status. Higher NOS activity has been found in invasive breast tumors when compared with benign or normal breast tissue carcinoma [53].

#### **6.5. Metastasis**

Metastasis is a complex process by which the malignant cancer cells from the breast expand into other regions of the body. Lymphatic metastasis is a critical determinant of cancer prognosis. Recent findings indicate that eNOS mediates VEGF-C-induced lymph-angiogenesis and, consequently, plays a critical role in lymphatic metastasis [54]. Investigational studies on tumors have provided substantial evidence of the contributory role of NO• in tumor development, where series of tests were performed on tumor-bearing mice using NOS inhibitors showed a delayed tumor growth through eNOS inhibition and barred metastasis, signifying the potential role of endothelium-derived eNOS in metastasis [55].

#### **7. Regulation OF** *NOS3* **gene expression**

The most fundamental level of NOS regulation is reflected in the tissue-specific expression of the different isoforms. The amount of NO produced results from the expression level and activity of eNOS. It is regulated by several interlinking mechanism such as transcriptional, posttranscriptional, and posttranslational modifications. The activity of NOS3 is also controlled by avid binding transcription factors namely Ets-1, Elf-1, Sp1, Sp3, and YY1 to the NOS3 promoter region. Posttranscriptionally, NOS3 activity is controlled by modifications of the primary transcript, stability of mRNA, its subcellular localization, and nucleocytoplasmic transport. Posttranslational modifications of NOS3 consist of fatty acid acylation, substrate, and cofactor availability, protein-protein interactions, and amount of phosphorylation. Another significant epigenetic mechanism for NOS3 gene expression is differential promoter methylation [47].

The gene-encoding eNOS located on chromosome 7q35-36 and is composed of 26 exons (coding sequences) and introns (sequences between exons) with an entire length of 21 kb and has more than 168 polymorphisms [56]. Over the last few years, polymorphisms of the gene have been identified, and their association with various diseases has been explored. Genetic comparison studies on healthy people and cancer patients have shown that polymorphisms in eNOS are associated with the development of cancers.

A single nucleotide polymorphism (SNP), T-786C, was identified in the 5′ flanking region involving a substitution of thymine (T) to cytosine (C) at a locus 786 base pairs upstream [57]. Another common variant of eNOS with a G to T transversion at nucleotide position 894 (G894T) leading to a change in amino acid at 298 (Glu298Asp) has been reported [58], and a 27-bp variable number of tandem repeats (VNTR) polymorphism in intron 4 (intron 4b/4a) [59] and high numbers of CA, which have been repeated in intron 13 of eNOS gene, are also known to be associated with complex disorders. These polymorphisms seem to be functional and have been widely investigated for their associations with cancer risk [60, 61]. Molecular studies of eNOS −786T > C, intron 4b/4a, and 894G > T polymorphisms (**Figure 8**) if performed in large and unbiased can provide valuable insights into the association between the eNOS gene and breast cancer risk.

**Figure 8.** Organization of eNOS gene.

#### **8. Conclusion**

**6.4. Tumor progression/invasion**

190 Nitric Oxide Synthase - Simple Enzyme-Complex Roles

carcinoma [53].

**6.5. Metastasis**

detected in tumor cells specifically in breast cancer [51].

NO• has been investigated regarding its possible involvement in the promotion of breast carcinoma. Increased amounts of NO• have been observed in the blood circulation of advanced grade breast cancer patients [49] where the increased levels of NO• have shown to promote tumor angiogenesis. Nitrotyrosine, a marker derived from NO•, was correlated with expression of VEGF-C and has been associated with lymph node metastasis in breast carcinoma patients, implicating the role of NO• in the development and progression of breast cancer [50]. In relation to the link of eNOS with cell proliferation, the eNOS expression has been

Several studies have observed NO• released by eNOS, can stimulate cancer cell cycle progression and proliferation. More specific to eNOS, studies have shown that the eNOS/NO• pathway plays a role in cancer cell DNA/RNA synthesis and proliferation apart from promoting angiogenesis [52]. The eNOS gene plays an essential role in endothelial cell proliferation in cell culture models and is a central mediator of several endothelium growth stimulators, such as vascular endothelial growth factor (VEGF) and prostaglandin E2. In human breast cancer, eNOS appears to be expressed in tumor epithelial cells, and its presence is correlated with histological grade and lymph node status. Higher NOS activity has been found in invasive breast tumors when compared with benign or normal breast tissue

Metastasis is a complex process by which the malignant cancer cells from the breast expand into other regions of the body. Lymphatic metastasis is a critical determinant of cancer prognosis. Recent findings indicate that eNOS mediates VEGF-C-induced lymph-angiogenesis and, consequently, plays a critical role in lymphatic metastasis [54]. Investigational studies on tumors have provided substantial evidence of the contributory role of NO• in tumor development, where series of tests were performed on tumor-bearing mice using NOS inhibitors showed a delayed tumor growth through eNOS inhibition and barred metastasis, signifying

The most fundamental level of NOS regulation is reflected in the tissue-specific expression of the different isoforms. The amount of NO produced results from the expression level and activity of eNOS. It is regulated by several interlinking mechanism such as transcriptional, posttranscriptional, and posttranslational modifications. The activity of NOS3 is also controlled by avid binding transcription factors namely Ets-1, Elf-1, Sp1, Sp3, and YY1 to the NOS3 promoter region. Posttranscriptionally, NOS3 activity is controlled by modifications of the primary transcript, stability of mRNA, its subcellular localization, and nucleocytoplasmic transport. Posttranslational modifications of NOS3 consist of fatty acid acylation, substrate, and cofactor

the potential role of endothelium-derived eNOS in metastasis [55].

**7. Regulation OF** *NOS3* **gene expression**

All these findings suggest that the expression of eNOS in breast cancer may be a critical event in carcinogenesis. Understanding different actions of NO• induced by eNOS in breast cancer at the molecular level can help in providing diagnostic or prognostic markers and also in devising potential strategies for prevention of breast cancer. The ability of many tumors to exploit eNOS/NO for a survival, proliferative, and metastatic advantage suggests that pharmacological use of eNOS inhibitors might attenuate these effects. Therefore, selective targeting of eNOS may prove a useful therapeutic or chemopreventive measure. However, further careful studies are needed to confirm the potential therapeutic role of eNOS as a novel target for breast cancer therapy.

### **Author details**

Tupurani Mohini Aiyengar, Padala Chiranjeevi and Hanumanth Surekha Rani\*

\*Address all correspondence to: surekharanih@gmail.com

Department of Genetics, University College of Science, Osmania University, Hyderabad, Telangana State, India

#### **References**


at the molecular level can help in providing diagnostic or prognostic markers and also in devising potential strategies for prevention of breast cancer. The ability of many tumors to exploit eNOS/NO for a survival, proliferative, and metastatic advantage suggests that pharmacological use of eNOS inhibitors might attenuate these effects. Therefore, selective targeting of eNOS may prove a useful therapeutic or chemopreventive measure. However, further careful studies are needed to confirm the potential therapeutic role of eNOS as a novel target

Tupurani Mohini Aiyengar, Padala Chiranjeevi and Hanumanth Surekha Rani\*

Department of Genetics, University College of Science, Osmania University, Hyderabad,

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## **Nitric Oxide Synthase and Nitric Oxide Involvement in Different Toxicities**

Emine Atakisi and Oguz Merhan

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.68494

#### **Abstract**

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Nitric oxide (NO) is known to have a very short half‐life, and it is oxidized to nitrate (NO3 **−** ) and nitrite (NO2 **−** ). The activity and/or expression of nitric oxide synthases (NOSs) can change in response to toxins or therapeutic medications. For example, in recent studies in our laboratory and others, it has been reported that the amount of NO was increased in the serum of N‐nitroso compounds‐treated animals. N‐nitroso com‐ pounds, which are found in different types of foodstuffs, including meat, salted fish, alcoholic beverages, agricultural drugs, insecticides, cigarettes, and several vegetables, are known to have carcinogenic effects. In addition, it is experimentally used to induce liver carcinoma to study the mechanisms of liver cytotoxic injury. Uncontrolled, pro‐ longed, and/or massive production of NO by inducible NOS may cause liver damage, inflammation, and even tumor development during N‐nitroso compound toxicity. In this chapter, we explain the roles of NOS and NO in various toxicity conditions, such as toxicity in environment pollutant or food additive, and present the evaluation of the toxicity and the importance of NOSs in human health.

**Keywords:** nitric oxide synthase, nitrate, nitrite, nitric oxide, N‐nitroso compounds, toxicity

#### **1. Introduction**

Nitric oxide (NO) is synthesized by nitric oxide synthase (NOS) (EC: 1.14.13.39) through oxidation of L‐arginine to L‐citrulline [1–5]. NO is a biologically significant molecule for many species from bacteria to mammals. Mechanisms for NO synthesis in an organism are extremely limited. Nitric oxide synthase enzyme is the only source of endogenous NO, except NO formed by metabolism of the nitro compounds entering the organism [6]. Three

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

 different types of NOS isoforms have been isolated from different tissues, such as vascular endothelium, brain, macrophage, and urinary system, of mammals, including (a) neuronal NOS (nNOS), (b) inducible NOS (iNOS), and (c) endothelial NOS (eNOS) [5, 7]. Neuronal NOS (nNOS, NOS1) is a Ca+2‐dependent, ~160 kDa enzyme that is found in the central and peripheral nervous system cells and striated muscle [4, 8]. Inducible NOS (iNOS, NOS2) is a calcium‐insensitive, ~130 kDa enzyme that was first isolated from activated macrophages and that can be activated by some cytokines (IL‐1, TNF, IF‐γ) or bacterial endotoxins [4, 9, 10]. Endothelial NOS (eNOS, NOS3) is a Ca+2/calmodulin‐dependent, ~135 kDa enzyme that is localized in vascular endothelial cells, hippocampal neural cells, pulmonary and renal epithe‐ lial cells, and cardiac myocytes [4, 9, 11].

Level of NO can be determined indirectly by measuring the concentration of nitrate (NO3 **−** ) and nitrite (NO2 **−** ) using an acidic Griess reaction. In recent studies in our laboratory and oth‐ ers, it has been reported that the amount of NO can change in response to various toxicity conditions [12–18] which are closely associated with animal and human disease conditions. In this chapter, we mention NO<sup>3</sup> **−** and NO2 **−** , the molecules which are naturally found in foods as NO sources, agricultural activities such as the use of artificial fertilizers, polluted water, curing process to give a natural smell and taste to meat. The conversion of NO3 **−** and NO2 **−** into NO in the gastrointestinal tract, the effect of NOS, and possible mechanisms for how it is converted back into NO3 **−** and NO2 **−** in the bloodstream will also be covered. Studies on N‐nitrosamines, which are formed by reaction of NO3 **−** and NO2 **−** with amines, and which can be seen in cured meats, cigarette smoke, and rubber industry, reveal the carcinogenic effects of these molecules (**Figure 1**).

**Figure 1.** Formation of N‐nitroso compounds from NO3 − , NO2 − , NO, and their effects on human health.

#### **2. NO3 − and NO2 − as environment pollutants and food additives**

 different types of NOS isoforms have been isolated from different tissues, such as vascular endothelium, brain, macrophage, and urinary system, of mammals, including (a) neuronal NOS (nNOS), (b) inducible NOS (iNOS), and (c) endothelial NOS (eNOS) [5, 7]. Neuronal NOS (nNOS, NOS1) is a Ca+2‐dependent, ~160 kDa enzyme that is found in the central and peripheral nervous system cells and striated muscle [4, 8]. Inducible NOS (iNOS, NOS2) is a calcium‐insensitive, ~130 kDa enzyme that was first isolated from activated macrophages and that can be activated by some cytokines (IL‐1, TNF, IF‐γ) or bacterial endotoxins [4, 9, 10]. Endothelial NOS (eNOS, NOS3) is a Ca+2/calmodulin‐dependent, ~135 kDa enzyme that is localized in vascular endothelial cells, hippocampal neural cells, pulmonary and renal epithe‐

Level of NO can be determined indirectly by measuring the concentration of nitrate (NO3

ers, it has been reported that the amount of NO can change in response to various toxicity conditions [12–18] which are closely associated with animal and human disease conditions.

as NO sources, agricultural activities such as the use of artificial fertilizers, polluted water,

into NO in the gastrointestinal tract, the effect of NOS, and possible mechanisms for how

be seen in cured meats, cigarette smoke, and rubber industry, reveal the carcinogenic effects

− , NO2 −

**−**

and NO2

**−**

N‐nitrosamines, which are formed by reaction of NO3

**Figure 1.** Formation of N‐nitroso compounds from NO3

and NO2

**−**

curing process to give a natural smell and taste to meat. The conversion of NO3

**−**

) using an acidic Griess reaction. In recent studies in our laboratory and oth‐

**−**

and NO2

, the molecules which are naturally found in foods

in the bloodstream will also be covered. Studies on

**−**

, NO, and their effects on human health.

**−** )

**−**

**−**

with amines, and which can

and NO2

lial cells, and cardiac myocytes [4, 9, 11].

198 Nitric Oxide Synthase - Simple Enzyme-Complex Roles

**−**

In this chapter, we mention NO<sup>3</sup>

it is converted back into NO3

of these molecules (**Figure 1**).

and nitrite (NO2

Nitrogen is the basic element for essential micromolecules, such as amino acids, proteins, and nucleic acids. Nitrogen is the most abundant gas in the atmosphere; however, it has to be fixated before it is taken by plants and animals. Fixation is an important part of the nitrogen cycle. In this cycle, N<sup>2</sup> is converted into ammonium and various nitrogen oxides. These higher nitrogen oxides are eventually gradually reduced. The nitrogen is then freed into the atmosphere and the cycle is completed. Bacteria play an important role in the cycle as they can catalyze each step, including the interconversion of different nitrogen oxides. NO3 − , NO2 − , and NO are all necessary intermediate products in the denitrification process and are catalyzed by NO3 − , NO2 − , and NO reductases, respectively [19]. Bacteria use these molecules as terminal electron acceptors in the absence of oxygen. The production and metabolism of nitrogen oxides also occur in mammals. NO3 − is easily converted into NO2 − in mammals by the activity of enzymes in both bacteria and mammals. The NO2 − then later can react with different molecules, such as amines, amides, and to form N‐nitroso compounds which can be carcinogenic [20]. Potentially carcinogenic or inert oxidizing molecules, such as NO3 − and NO2 − , occur as a result of endogenous NO metabolism during the food chain (**Figure 1**) [21].

Although nitrogen is found naturally in surface waters, its amount increases in many parts of the world. The reason for this is the pollution caused by commonly used inorganic fertilizers, soil drainage, or contamination of water resources by sewage [22]. The main causes of water pollution are pollution from industrial and agricultural activities. Chemical fertilizers used in agricultural production have an important role. NO3 **−** is applied in increasing amounts in the fertilizers for agricultural production, and they accumulate in the soil. This accumulated NO3 **−** , in varying amounts depending on the conditions, moves toward the deeper parts of the ground especially with rainwater, and some of it reaches underground and some to surface waters. High NO3 **−** concentrations in water resources pose a potential risk to human health, because sunlight and some bacteria can easily convert NO3 **−** into NO2 **−** [23]. NO3 **−** and NO2 **−** can also occur spontaneously in vegetables and fruits consumed by humans and especially in animal feed [24]. Vegetables and fruits usually receive NO3 **−** and NO2 **−** from the soil [25]. As a result of nitrogenous fertilizers being used in excess to increase appearance and yield in the plants, plants store NO3 **−** in excess of their need. When the amount of received NO3 **−** is high, the reduction to ammonia is limited and NO2 **−** accumulates as an intermediate metabolism product [26, 27].

Excess NO3 **−** and NO2 **−** can also be utilized to cure meats. In order to improve the taste, pro‐ tection, appearance, and quality of the meat, NO3 **−** and NO2 **−** are used for curing purposes. NO2 **−** can also be used as a preservative against the proliferation of microorganisms, especially *Clostridium botulinum*. It also inhibits lipid peroxidation and prevents putrefaction [28].

Dietary NO3 **−** and NO2 **−** , which are taken by the organisms, could cause various physiological and pathological outcomes.

#### **3. Conversion of NO3 − and NO2 − into NO in the gastrointestinal system: possible role of NOSs**

In an organism, NO<sup>2</sup> **−** is converted into NO in three ways:


In this section, the conversion of NO<sup>3</sup> **−** and NO2 **−** , taken from foods in the gastrointestinal tract, into NO will be mentioned. At the end of this pathway, NO3 **−** and NO2 **−** are synthesized again from NO. This synthesis in the tissues is catalyzed by NOSs. With the identification of the NO3 **−** ‐NO2 **−** ‐NO pathway, the importance of the diet, rather than the biological significance of systemic NO3 **−** and NO2 **−** , in the physiological regulation of NO has arisen. NO3 **−** , rich in green leafy vegetables such as beetroot, is reduced to NO2 **−** by bacterial NO3 **−** reductase in the com‐ mensal anaerobic microflora in the oral cavity by saliva secretion and is reduced to NO in the stomach [29–31]. The highest NO concentration is obtained from an acidic stomach pH after a NO3 **−** ‐rich meal (**Figure 1**) [29].

The rapid postprandial increase in gastric NO is directly proportional to many actions, such as mucus production in the gastrointestinal tract, increased vascular tone, antimicrobial effect, and immunomodulation. It has also been shown that this increased NO is related to many physiological mechanisms, such as the prevention of ischemia‐reperfusion injury and increased cerebral blood flow [21, 29].

How endogenous NO3 **−** and NO2 **−** can be synthesized in the body if NO3 **−** and NO2 **−** are not taken into the body with nutrients? The inorganic NO3 **−** and NO2 **−** , which cannot be taken up with nutrients in starving mammals, are mainly derived from NOSs. These enzymes form NO by using l‐arginine and oxygen, and then this NO is rapidly degraded to NO3 **−** and NO2 **−** . Endogenous NO3 **−** and NO2 **−** are synthesized in this way [30]. Endogenous NO can also be produced by NOSs using NO3 **−** and NO2 **−** in our daily nutrients [30, 32].

NO is a highly diffusible free radical that participates in various in vivo signal pathways and is involved in critical physiological events, such as regulation of vascular tone and immune response [31]. NO also exhibits antimicrobial activity [33] other than its regulatory role in vascular tone. Numerous intracellular pathogenic parasites [34] and bacteria [35] are susceptible to NO.

NO2 **−** in saliva is converted non‐enzymatically into NO and some other nitrogen oxide species when it enters into the stomach with low pH [30]. Increasing number of studies on cardio‐ vascular, inflammatory, and gastrointestinal diseases reported that the NO‐related effects of dietary NO3 **−** and NO2 **−** are protective and preventive. Recent data suggest that these anions are beneficial to gastrointestinal cancer [36] and cardiovascular diseases rather than having harmful effects. Dietary NO<sup>3</sup> **−** and salivary NO2 **−** have been shown to protect gastric mucus from experimentally induced gastric damage by increasing gastric mucus thickness and mucosal blood flow [30].

NO plays an important role for the intestine. It is produced from arginine by eNOS and iNOS in a reaction catalyzed in the intestine. eNOS is structurally expressed in low levels in intestinal microcapillaries and is responsible for the initial levels of NO. Low NO lev‐ els regulate vascular tone and mucosal blood flow in cyclic guanosine monophosphate and neuron‐dependent manner and are also crucial for mucosal homeostasis. Additionally, NO can protect from oxidative stress by diminishing oxygen radicals. eNOS‐derived NO facilitates leucocyte uptake by supporting endothelial adhesion of leukocytes. iNOS is upregulated during inflammation and increases NO synthesis. NO also allows dilatation of capillary vessels. Excess NO secreted during inflammation has harmful effects on the intestinal barrier [37, 38].

**3. Conversion of NO3**

**possible role of NOSs**

**−**

200 Nitric Oxide Synthase - Simple Enzyme-Complex Roles

(c) NO can be produced by acidification of NO<sup>2</sup>

**−**

leafy vegetables such as beetroot, is reduced to NO2

into NO will be mentioned. At the end of this pathway, NO3

In this section, the conversion of NO<sup>3</sup>

and NO2

‐rich meal (**Figure 1**) [29].

increased cerebral blood flow [21, 29].

**−**

produced by NOSs using NO3

**−**

and NO2

and NO2

**−**

**−**

**−**

taken into the body with nutrients? The inorganic NO3

**−**

and NO2

How endogenous NO3

Endogenous NO3

NO2 **−**

dietary NO3

**−**

mucosal blood flow [30].

harmful effects. Dietary NO<sup>3</sup>

and NO2

**−**

**−**

In an organism, NO<sup>2</sup>

NO3 **−** ‐NO2 **−**

a NO3 **−**

systemic NO3

**−**

 **and NO2**

**−**

 is converted into NO in three ways: (a) It is enzymatically (via NOSs) reduced to NO in the circulation and tissues.

at low pH and hypoxic conditions that occur during intense exercise.

**−**

and NO2

(b) It is non‐enzymatically reduced to NO in acidic stomach environment, capillary beds, and

**−**

from NO. This synthesis in the tissues is catalyzed by NOSs. With the identification of the

mensal anaerobic microflora in the oral cavity by saliva secretion and is reduced to NO in the stomach [29–31]. The highest NO concentration is obtained from an acidic stomach pH after

The rapid postprandial increase in gastric NO is directly proportional to many actions, such as mucus production in the gastrointestinal tract, increased vascular tone, antimicrobial effect, and immunomodulation. It has also been shown that this increased NO is related to many physiological mechanisms, such as the prevention of ischemia‐reperfusion injury and

with nutrients in starving mammals, are mainly derived from NOSs. These enzymes form

NO is a highly diffusible free radical that participates in various in vivo signal pathways and is involved in critical physiological events, such as regulation of vascular tone and immune response [31]. NO also exhibits antimicrobial activity [33] other than its regulatory role in vascular tone.

 in saliva is converted non‐enzymatically into NO and some other nitrogen oxide species when it enters into the stomach with low pH [30]. Increasing number of studies on cardio‐ vascular, inflammatory, and gastrointestinal diseases reported that the NO‐related effects of

are beneficial to gastrointestinal cancer [36] and cardiovascular diseases rather than having

from experimentally induced gastric damage by increasing gastric mucus thickness and

**−**

NO by using l‐arginine and oxygen, and then this NO is rapidly degraded to NO3

**−**

Numerous intracellular pathogenic parasites [34] and bacteria [35] are susceptible to NO.

and salivary NO2

‐NO pathway, the importance of the diet, rather than the biological significance of

**−**

can be synthesized in the body if NO3

**−**

in our daily nutrients [30, 32].

are protective and preventive. Recent data suggest that these anions

and NO2

are synthesized in this way [30]. Endogenous NO can also be

**−**

, in the physiological regulation of NO has arisen. NO3

**−**

in the oral cavity.

**−**

by bacterial NO3

and NO2

 **into NO in the gastrointestinal system:** 

, taken from foods in the gastrointestinal tract,

**−**

**−**

**−**

have been shown to protect gastric mucus

and NO2

, which cannot be taken up

**−**

**−** are not

and NO2

**−** .

are synthesized again

reductase in the com‐

, rich in green

**−**

NO reacts with the superoxide ion to form a reactive oxygen and nitrogen type, peroxynitrite, which can be harmful for epithelial cells. It can induce enterocyte cell apoptosis and inhibit proliferation. iNOS is expressed in intestinal smooth muscle cells, endothelial and epithelial cells [38]. During inflammatory conditions in the intestine, such as necrotizing enterocolitis [39], ulcer [29], and colon cancer [40], expression of iNOS mRNA increases.

Numerous studies have shown that NO3 **−** and NO2 **−** obtained from pharmacologic supple‐ ments or diet have obvious effects on gastrointestinal function [29, 39, 40]. However, it is still unclear whether endogenous NO3 **−** and NO2 **−** derived from NOSs in the endothelium and elsewhere affect gastric function. This situation has been tried to be illuminated in germ‐free and starved animals [30, 41].

The gastric NO levels are very low in germ‐free animals lacking microflora even after dietary NO3 **−** load. A significant amount of NO<sup>2</sup> **−** is produced in the saliva even in the case of fasting, indicating NO3 **−** production due to endogenous NOS production [30]. Petersson et al. [30] reported that three doses of NO2 **−** given to germ‐free rats not only increased stomach mucus thickness by more than fourfold but also unexpectedly had an effect in the non‐NO<sup>2</sup> **−** group. This suggests that endogenous NO3 **−** from NOSs also plays a role in the regulation of gastric physiology. In a similar study in humans, individuals were given a low NO<sup>3</sup> **−** diet with an antibacterial mouthwash containing chlorhexidine to lower the reduction of oral NO3 **−** , and it was determined that the levels of circulating NO2 **−** lowered, and this then increased the blood pressure [42]. These studies show that NO3 **−** and NO2 **−** have a NO3 **−** ‐NO2 **−** ‐NO pathway that starts from the mouth and ends in the mouth through digestive and circulatory system. It is possible that NO3 **−** and NO2 **−** in the circulation and saliva may originate from the endogenous NOS pathway.

In addition to NO<sup>3</sup> **−** , NO2 **−** , NO, sodium nitrite (NaNO2 ) is an inorganic compound taken by endogenous sources. NaNO2 may have some beneficial and undesirable effects. NaNO<sup>2</sup> is a preservative used in processed meats, such as salami and bacon. NaNO2 is synthesized by several chemical reactions, including the reduction of sodium nitrate. NaNO2 is used as an additive in foods. There are some suspects about the safety of use in foods, but it is still being used, and, on the contrary, there is information that NaNO2 may actually be healthy [43]. There are studies on the effects of NaNO<sup>2</sup> on human health from 1945 [44] to present date [45].

NO3 **−** salts are used as a cheap nitrogen source in fertilizers. Therefore, with the widespread use of nitrogenous fertilizers in agriculture and inappropriate disposal of nitrogenous wastes, humans are exposed to high NO3 **−** /NO2 **−** levels at an alarming rate, especially through contam‐ inated food and water [46]. Infants and individuals with deficiency of glucose‐6‐phosphate dehydrogenase are particularly sensitive to high levels of NO3 **−** /NO2 **−** [45]. The digestive sys‐ tem of newborn babies convert NO3 **−** to NO2 **−** , and NO2 **−** reacts with hemoglobin and prevents oxygen transport to the tissues. As a result, "methemoglobinemia" known as "blue baby syn‐ drome" occurs in infants [47].

Prolonged non‐lethal exposure to high levels of NO3 **−** /NO2 **−** may cause respiratory failure, growth failure, diabetes, neurological disorders, and cancer [48]. NaNO2 causes oxidative stress in human erythrocytes in vitro by increasing lipid and protein oxidation, osmotic fra‐ gility, and membrane damage [49].

Despite the fact that NaNO2 has not been reliable in the past and has the potential to cause many cancers, it has recently been reported that it can prevent myocardial ischemia‐reperfu‐ sion injury in diabetic rats by regulating eNOS and iNOS expression and inhibiting lipid peroxidation in the heart [50]. It also prevents hypertension and increases endothelium‐ dependent relaxation and total NO by regulating eNOS activity [51].

Serum malondialdehyde, NO, arginase, and glutathione S‐transferase activities were incre‐ ased, and glutathione and catalase activities were decreased in NaNO2 ‐treated rats [52]. In another study, decrease in GSH and catalase activity was reported in NaNO2 ‐intoxicated rats [53]. In their study investigating the histopathological, biochemical, and genotoxic effects of low dose NaNO2 administration for 8 months, Ozen et al. [54] reported that the liver and kid‐ ney NO levels were decreased in rats. The reduction in NO levels may be explained by the rapid and/or efficient removal of this molecule from these tissues, resulting in an increase in serum levels due to reduced NO by metabolic depletion. Therefore, the investigators reported that the increased serum NO level did not contradict with the decreasing NO level in the tis‐ sues [54]. In addition, these and other investigators indicated that chronic administration of NaNO2 increased iNOS activity in experimental animals [54, 55].

Peroxynitrite can interact with tyrosine residues to form nitrotyrosine. Ozen et al. [54] showed that the expression of iNOS and nitrotyrosine was increased in the liver and kidney tissues of NaNO2 ‐treated mice, and it caused tissue degeneration in both organs. Peroxynitrite can be decomposed to form NO3 **−** and NO2 **−** which can cause DNA damage, as well [56, 57].

The mechanisms of NaNO2 are still not fully understood; this suggests that further work needs to be performed in the future.

#### **4. N‐nitroso compounds**

#### **4.1. The chemical structure and sources of N‐nitroso compounds**

NO2 **−** is the precursor of N‐nitroso compounds that have carcinogenic effect [58, 59]. NO2 **−** is converted into nitrous acid in acidic environment, and nitrous acids react with secondary amines to form nitrosamine compounds (**Figure 2**) [60].

Nitric Oxide Synthase and Nitric Oxide Involvement in Different Toxicities http://dx.doi.org/10.5772/intechopen.68494 203

**Figure 2.** Formation of nitrosamine from NO3 − .

humans are exposed to high NO3

202 Nitric Oxide Synthase - Simple Enzyme-Complex Roles

tem of newborn babies convert NO3

gility, and membrane damage [49].

Despite the fact that NaNO2

low dose NaNO2

NaNO2

NaNO2

NO2 **−**

decomposed to form NO3

The mechanisms of NaNO2

**4. N‐nitroso compounds**

needs to be performed in the future.

drome" occurs in infants [47].

**−** /NO2 **−**

> **−** to NO2 **−**

growth failure, diabetes, neurological disorders, and cancer [48]. NaNO2

dependent relaxation and total NO by regulating eNOS activity [51].

ased, and glutathione and catalase activities were decreased in NaNO2

increased iNOS activity in experimental animals [54, 55].

**−**

**−**

and NO2

**4.1. The chemical structure and sources of N‐nitroso compounds**

amines to form nitrosamine compounds (**Figure 2**) [60].

another study, decrease in GSH and catalase activity was reported in NaNO2

dehydrogenase are particularly sensitive to high levels of NO3

Prolonged non‐lethal exposure to high levels of NO3

inated food and water [46]. Infants and individuals with deficiency of glucose‐6‐phosphate

oxygen transport to the tissues. As a result, "methemoglobinemia" known as "blue baby syn‐

stress in human erythrocytes in vitro by increasing lipid and protein oxidation, osmotic fra‐

many cancers, it has recently been reported that it can prevent myocardial ischemia‐reperfu‐ sion injury in diabetic rats by regulating eNOS and iNOS expression and inhibiting lipid peroxidation in the heart [50]. It also prevents hypertension and increases endothelium‐

Serum malondialdehyde, NO, arginase, and glutathione S‐transferase activities were incre‐

[53]. In their study investigating the histopathological, biochemical, and genotoxic effects of

ney NO levels were decreased in rats. The reduction in NO levels may be explained by the rapid and/or efficient removal of this molecule from these tissues, resulting in an increase in serum levels due to reduced NO by metabolic depletion. Therefore, the investigators reported that the increased serum NO level did not contradict with the decreasing NO level in the tis‐ sues [54]. In addition, these and other investigators indicated that chronic administration of

Peroxynitrite can interact with tyrosine residues to form nitrotyrosine. Ozen et al. [54] showed that the expression of iNOS and nitrotyrosine was increased in the liver and kidney tissues of

is the precursor of N‐nitroso compounds that have carcinogenic effect [58, 59]. NO2

converted into nitrous acid in acidic environment, and nitrous acids react with secondary

‐treated mice, and it caused tissue degeneration in both organs. Peroxynitrite can be

which can cause DNA damage, as well [56, 57].

are still not fully understood; this suggests that further work

administration for 8 months, Ozen et al. [54] reported that the liver and kid‐

, and NO2

**−**

**−** /NO2 **−**

levels at an alarming rate, especially through contam‐

[45]. The digestive sys‐

causes oxidative

‐treated rats [52]. In

‐intoxicated rats

**−** is

reacts with hemoglobin and prevents

may cause respiratory failure,

**−** /NO2 **−**

has not been reliable in the past and has the potential to cause

**Figure 3.** Chemical formula for nitrosamine.

Nitrosamines are chemical compounds with general formulas as shown in (**Figure 3**).

Nitrosamines are used in the manufacture of some cosmetics, pesticides, and most rubber products [61, 62]. Nitrosamines are found in latex products, cereal, tea, many foods, ciga‐ rettes, and cigarette smoke [60]. They are also formed by the reduction of NO3 **−** , which is abundant in nature, into NO2 **−** by bacteria [27].

The most commonly used N‐nitroso compounds for the purpose of toxicity are N‐nitrosodi‐ methylamine, N‐nitrosodiethylamine, N‐nitrosopyrrolidine, and N‐nitrosopiperidine [63, 64].

Dimethylnitrosamine (also called N‐nitrosodimethylamine, DMN, DMNA, NDMA—C2 H6 N2 O) is found in wheat flour, cheese, smoked meat, fish, and other food products (**Figure 4**) [65]. It can be formed by reaction of dimethylamine with NO2 **−** . In addition, it can be formed by nitro‐ zation and decarboxylation of amino acids, such as glycine and alanine [66].

**Figure 4.** Chemical formula for dimethylnitrosamine.

Diethylnitrosamine (also called N‐nitrosodiethylamine, DEN, DENA, NDEA—C4 H10N2 O) is found in chemicals used in agriculture and rubber industry, cigarette smoke, alcoholic bever‐ ages, and processed meat products (**Figure 5**) [67]. It can be formed by reaction of diethyl‐ amine with NO2 **−** . Additionally, it can be formed by nitrozation and decarboxylation of amino acids, such as glycine alanine [66].

**Figure 5.** Chemical formula for diethylnitrosamine.

N‐nitrosopyrrolidine (also called NPYR—C4 H8 N2 O) is found in cigarette smoke, meat and fish products (**Figure 6**) [68]. In meat products, this compound is formed by the nitrozation and decarboxylation of l‐proline [69].

**Figure 6.** Chemical formula for N‐nitrosopyrrolidine.

N‐nitrosopiperidine (also called NPIP—C<sup>5</sup> H10N2 O) formation follows these steps: decarbox‐ ylation of lysine results in cadaverine; cadaverine is converted into piperidine by heat and the reaction of the resulting piperidine with NO2 **−** (**Figure 7**) [69, 70].

**Figure 7.** Chemical formula for N‐nitrosopiperidine.

N‐nitrosamines can be found in food products and might cause serious health problems for humans.

#### **4.2. N‐nitrosamines in meat and dairy products**

N‐nitrosamines taken with food and found in the environment have been found to cause serious health risks above a certain level, even though they have both food processing and protection functions as additives.

Nitrosamines occur mostly in meat and dairy products [71]. Since meat, an important nutri‐ ent, is easily decomposed by different factors, it is necessary to protect it with various meth‐ ods and to increase its durability. For this purpose, some ingredients are added to meat and meat products. Cured meat products, unlike fresh meat or salted meat with only table salt, have a pleasant smell, flavor, and a natural looking but a heat‐resistant color. Today, this process is applied to most of the meat products consumed [72, 73]. It has been reported that the degradation products of NO3 **−** and NO2 **−** result in the formation of carcinogenic nitrosa‐ mines by combining with amino acids, such as putrescine, thiamine, piperidine, pyrrolidine, histamine, cadaverine, trimethylamine, β‐phenylethylamine, n‐propylamine, and isopropyl‐ amine [74–77].

The most important sources of nitrosamines in dairy products, such as cheese and butter, are NO3 **−** , NO2 **−** , and amine compounds [78]. The metabolic activities of some microorganisms in milk and dairy products result in the formation of histamine and tyramines, and nitrosamines are formed by the reaction of the secondary amines, which are the degradation products of these biogenic amines in various ways, with NO2 **−** [65, 69].

The first formation of these nitrosamines in meat and dairy products is seen in the oral cavity [79]. The salivary secretion contains abundant NO3 **−** and this NO3 **−** is reduced to NO2 **−** by nitrate reductase enzyme [72]. This NO2 **−** causes the formation of nitrosamines [80]. These compounds can be taken into the stomach in various ways, such as ingestion or smoking, or can also be formed by the reaction of NO2 **−** and amines in acidic conditions [81]. Some bacteria in the stomach and intestines increase the formation of nitrosamines by facilitating the conversion of NO3 **−** into NO2 **−** . NO2 **−** transforms into nitrous acid in the acidic environment of the stom‐ ach, and nitrous acid reacts with amines in the environment to form nitrosamines [60, 82]. Nitrosamines are usually excreted through urine [83, 84].

### **5. The roles of NOS isoforms and** *N***‐nitrosamine compounds in liver toxicity or carcinogenesis**

NO3 **−** can easily be formed in mammalian systems through bacterial and mammalian enzymes. The resulting NO3 **−** can then react with amines, amides, and amino acids to form N‐nitroso compounds. While NO3 **−** has relatively low toxicity, NO2 **−** and N‐nitroso compounds have higher toxicity in mammals. For this reason, there are many studies investigating the toxic‐ ity of these two molecules, as well as studies investigating ways to decrease the detrimental effects of these two molecules [20]. It has been suggested in long‐term experimental animal studies that nitrosamines cause cancer in many tissues, but the role of nitrosamines in the formation of cancers is still being investigated.

#### **5.1. NOS isoforms in carcinogenesis**

N‐nitrosopyrrolidine (also called NPYR—C4

N‐nitrosopiperidine (also called NPIP—C<sup>5</sup>

**Figure 6.** Chemical formula for N‐nitrosopyrrolidine.

reaction of the resulting piperidine with NO2

**Figure 7.** Chemical formula for N‐nitrosopiperidine.

**4.2. N‐nitrosamines in meat and dairy products**

protection functions as additives.

humans.

and decarboxylation of l‐proline [69].

**Figure 5.** Chemical formula for diethylnitrosamine.

204 Nitric Oxide Synthase - Simple Enzyme-Complex Roles

H8 N2

fish products (**Figure 6**) [68]. In meat products, this compound is formed by the nitrozation

H10N2

**−**

ylation of lysine results in cadaverine; cadaverine is converted into piperidine by heat and the

N‐nitrosamines can be found in food products and might cause serious health problems for

N‐nitrosamines taken with food and found in the environment have been found to cause serious health risks above a certain level, even though they have both food processing and

Nitrosamines occur mostly in meat and dairy products [71]. Since meat, an important nutri‐ ent, is easily decomposed by different factors, it is necessary to protect it with various meth‐ ods and to increase its durability. For this purpose, some ingredients are added to meat and

(**Figure 7**) [69, 70].

O) is found in cigarette smoke, meat and

O) formation follows these steps: decarbox‐

Various studies have indicated that three NOS isoforms both trigger and prevent cancer etiol‐ ogy. Nitric oxide synthase activity has been detected in a variety of tumor cells, and it has been shown to be closely related to tumor grade, proliferation rate, and cancer development. High NOS expression can be cytotoxic for cancer cells. On the other hand, low NOS expression may have an adverse effect and may increase tumor development [85, 86]. For this reason, NOS can be both genotoxic and angiogenic. High NO production leads to angiogenesis by increas‐ ing the VEGF gene, especially in p53 mutant cells. In addition, NO can alter the expressions of DNA repair proteins, such as poly (ADP‐ribose) polymerase and DNA‐protein kinase in tumor cells. NO may exhibit carcinogenic effect by the production of different NO metabo‐ lites. For example, NO may rapidly react with intracellular environment to form N‐nitroso compounds. These metabolites, for example, can cause genotoxic effects by creating DNA damage [86]. In some other studies, N‐nitroso compounds have been reported to alter the activity of creatine kinase, lactate dehydrogenase [87], pyruvate kinase [88], and Na/K‐ATPase [89] enzymes.

#### **5.2.** *N***‐nitrosamine compounds in liver toxicity and cancer**

Certain levels of nitrosamines taken in the body with any food ingredient are less likely to cause cancer in the human body alone. However, different types of nitrosamines, which are continuously taken from different sources, such as air and cigarettes, increase the risk of developing cancer [65, 90]. Although NO3 **−** and NO2 **−** create a toxicological problem, the main problem is that they turn into nitrosamines, which are carcinogenic. Nitrosamines are known to exhibit carcinogenic effect through binding to proteins and nucleic acids [91]. Nitrosamines also have mutagenic and teratogenic effects [61]. Because many organ‐specific nitrosamines are metabolized in the same way in human and animal tissues, humans are very sensitive to the carcinogenic properties of nitrosamines [67]. N‐nitroso compounds are potent alkylat‐ ing agents that can form endogenously and can cause cancer in surrounding animals [64]. Bacterial decarboxylation of amino acids in NO3 **−** taken with nutrients results in amines and amides [58]. There is a relationship between the formation of N‐nitroso compounds by bacte‐ rial catalysis and increased risk for liver, stomach, esophagus, nasopharynx, chronic urinary tract infections, and bladder squamous cell carcinoma [77, 92].

Metabolic activations of the nitrosamines occur primarily in the liver, and this transfor‐ mation can occur in all cells. Dimethylnitrosamine is a potent carcinogen that can induce malignant tumors in various animal species in various tissues, including the liver, lungs, and stomach [79]. Various studies on different species of mice have shown that adenomas and adenocarcinomas in the lungs and hepatocarcinoma in the liver are formed by dimeth‐ ylnitrosamine. The target organs of dimethylnitrosamine are the liver, lungs, and kidneys [65, 79].

It is suggested that diethylnitrosamine metabolism is catalyzed by the enzymes of the multi‐ functional cytochrome P‐450 monooxygenase system and toxic effects are initiated by its meta‐ bolic activation, and that the resulting reactive intermediate products have little affinity for the catalytic domains of the binding enzymes, so that instead of being excreted with urine, they stimulate the onset of mutation, cancer, and necrosis by forming covalent bonds with impor‐ tant cellular components [93, 94]. Low concentrations of diethylnitrosamine cause mutations and cancer which was shown by the Ames assay [94]. Many studies suggest that the harmful effects of diethylnitrosamine may be reduced by various antioxidant molecules. The adminis‐ tration of molecules such as α‐lipoic acid [95], omega‐3 [96], blueberry [97], and beta‐carotene [98] were stated to reduce the carcinogenic effect of nitrosamines in experimental animals.

N‐nitrosopiperidine and N‐nitrosopyrrolidine are structurally cyclic nitrosamines with dif‐ ferent carcinogenic activities. Comparative carcinogenicity studies of these two nitrosamines in rats revealed that N‐nitrosopiperidine caused esophagus, liver, and stomach tumors, and N‐nitrosopyrrolidine caused tumors mainly in the liver [63].

#### **6. Conclusion**

have an adverse effect and may increase tumor development [85, 86]. For this reason, NOS can be both genotoxic and angiogenic. High NO production leads to angiogenesis by increas‐ ing the VEGF gene, especially in p53 mutant cells. In addition, NO can alter the expressions of DNA repair proteins, such as poly (ADP‐ribose) polymerase and DNA‐protein kinase in tumor cells. NO may exhibit carcinogenic effect by the production of different NO metabo‐ lites. For example, NO may rapidly react with intracellular environment to form N‐nitroso compounds. These metabolites, for example, can cause genotoxic effects by creating DNA damage [86]. In some other studies, N‐nitroso compounds have been reported to alter the activity of creatine kinase, lactate dehydrogenase [87], pyruvate kinase [88], and Na/K‐ATPase

Certain levels of nitrosamines taken in the body with any food ingredient are less likely to cause cancer in the human body alone. However, different types of nitrosamines, which are continuously taken from different sources, such as air and cigarettes, increase the risk of

and NO2

problem is that they turn into nitrosamines, which are carcinogenic. Nitrosamines are known to exhibit carcinogenic effect through binding to proteins and nucleic acids [91]. Nitrosamines also have mutagenic and teratogenic effects [61]. Because many organ‐specific nitrosamines are metabolized in the same way in human and animal tissues, humans are very sensitive to the carcinogenic properties of nitrosamines [67]. N‐nitroso compounds are potent alkylat‐ ing agents that can form endogenously and can cause cancer in surrounding animals [64].

**−**

amides [58]. There is a relationship between the formation of N‐nitroso compounds by bacte‐ rial catalysis and increased risk for liver, stomach, esophagus, nasopharynx, chronic urinary

Metabolic activations of the nitrosamines occur primarily in the liver, and this transfor‐ mation can occur in all cells. Dimethylnitrosamine is a potent carcinogen that can induce malignant tumors in various animal species in various tissues, including the liver, lungs, and stomach [79]. Various studies on different species of mice have shown that adenomas and adenocarcinomas in the lungs and hepatocarcinoma in the liver are formed by dimeth‐ ylnitrosamine. The target organs of dimethylnitrosamine are the liver, lungs, and kidneys

It is suggested that diethylnitrosamine metabolism is catalyzed by the enzymes of the multi‐ functional cytochrome P‐450 monooxygenase system and toxic effects are initiated by its meta‐ bolic activation, and that the resulting reactive intermediate products have little affinity for the catalytic domains of the binding enzymes, so that instead of being excreted with urine, they stimulate the onset of mutation, cancer, and necrosis by forming covalent bonds with impor‐ tant cellular components [93, 94]. Low concentrations of diethylnitrosamine cause mutations and cancer which was shown by the Ames assay [94]. Many studies suggest that the harmful effects of diethylnitrosamine may be reduced by various antioxidant molecules. The adminis‐ tration of molecules such as α‐lipoic acid [95], omega‐3 [96], blueberry [97], and beta‐carotene [98] were stated to reduce the carcinogenic effect of nitrosamines in experimental animals.

**−**

create a toxicological problem, the main

taken with nutrients results in amines and

**−**

**5.2.** *N***‐nitrosamine compounds in liver toxicity and cancer**

developing cancer [65, 90]. Although NO3

206 Nitric Oxide Synthase - Simple Enzyme-Complex Roles

Bacterial decarboxylation of amino acids in NO3

tract infections, and bladder squamous cell carcinoma [77, 92].

[89] enzymes.

[65, 79].

NO‐mediated responses are cell specific, and they depend on the existence of different NOS isoforms at different concentrations, and their regulations at pre‐ and post‐transcriptional levels are quite complex. The latest developments on strategies for treating or preventing pathological events in association with the stimulation or inhibition of excessive production of NO and N‐nitroso compounds present a crucial importance in medicine.

#### **Author details**

Emine Atakisi\* and Oguz Merhan

\*Address all correspondence to: et\_tasci@hotmail.com

Department of Biochemistry, Faculty of Veterinary, Kafkas University, Kars, Turkey

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#### **Chapter 12**

### **Nitric Oxide Synthase Inhibitors**

Elizabeth Igne Ferreira and

Ricardo Augusto Massarico Serafim

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/67027

#### **Abstract**

Nitric oxide (NO) is an endogenic product from plants, bacteria, and animal cells that has many important effects in those organisms. It is produced by nitric oxide synthase (NOS), which is found in main three isoforms, namely endothelial NOS (eNOS), inducible NOS (iNOS), and neuronal NOS (nNOS). It has an important role in homeostasis in different physiological systems, such as micro- and macro-vascularization, inhibition of platelet aggregation, and neurotransmission regulation in the central nervous, gastrointestinal, respiratory, and genitourinary systems. However, its overproduction has been associated with diseases such as arthritis, asthma, cerebral ischemia, Parkinson's disease, neurodegeneration, and seizures. For this reason, and due to better understanding of the molecular mechanisms by which NO provokes those diseases, the interest on the design of NOS inhibitors with therapeutic purposes has highly increased. Based on the foregoing considerations, the proposal of this chapter is to show an overview about the design strategies, mechanism of action at the molecular level, and the main advances toward the search for selective NOS inhibitors available in the literature.

**Keywords:** nitric oxide synthase isoforms, structure-based drug design, enzymatic inhibition, selectivity, heterocyclic compounds

#### **1. Introduction**

Nitric oxide (NO) is a diatomic neutral molecule, produced by bacteria, plants, and animals. Having one unpaired electron, its effect in biological system is related to the stabilization of this electron. It acts as dissolved nonelectrolyte in the organisms, except for the lungs, where it is found in gaseous state [1–3].

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

NO has gain importance mainly in the 1990s, and from then on, it has been studied to obtain interesting pharmacological effects. A review by Serafim and collaborators describes the state of the art of this compound use in drug design [4].

The basal NO production has an important contribution to homeostasis in different physiological systems, such as micro- and macro-vascularization, inhibition of platelet aggregation, and neurotransmission regulation in central nervous, gastrointestinal, respiratory, and genitourinary systems. However, NO overproduction has been strongly associated with some diseases such as arthritis, asthma, cerebral ischemia, Parkinson's disease, neurodegeneration, and seizures [5–9].

Nitric oxide synthase (NOS) is the enzyme responsible for NO biosynthesis, and there are three main kinds of NOS isoforms [endothelial NOS (eNOS), inducible NOS (iNOS), and neuronal NOS (nNOS)]. They are tetramers, constituted of two NOS monomers associated with two calmodulin monomers (CaM), and contain relatively tightly bound cofactors, BH<sup>4</sup> , FAD, FMN, and iron protoporphyrin IX (heme group). Their chemical function is to catalyze the reaction of l-arginine, NADPH, and oxygen to synthesize free radical NO, l-citrulline, and NADP (**Figure 1**) [10].

The substrate l-arginine establishes H-bond networks inside the catalytic site of NOS isoforms with the heme group, mainly due to the guanidine group, which is crucial to bind tightly using a salt-bridge interaction with the conserved carboxylate of Glu597 in human nNOS, Glu377 for iNOS and Glu361 for eNOS. In addition, l-arginine establishes H-bonds with the amide carbonyl from Trp592, in nNOS; with Trp372, in iNOS; and with Trp356, in eNOS. Moreover, α-amino group of l-arginine interacts through H-bond with the heme propionate side chain, and the guanidine *N*-terminal nitrogen of this amino acid coordinates with FeII (**Figure 2**). This ligand-receptor interaction profile is similar to all isoforms, which generates a challenge to selectivity [11].

 **Figure 1.** General reaction of NO formation by NOS. Adapted from [10].

 **Figure 2.** Scheme of nNOS-binding site.

NO has gain importance mainly in the 1990s, and from then on, it has been studied to obtain interesting pharmacological effects. A review by Serafim and collaborators describes the state

The basal NO production has an important contribution to homeostasis in different physiological systems, such as micro- and macro-vascularization, inhibition of platelet aggregation, and neurotransmission regulation in central nervous, gastrointestinal, respiratory, and genitourinary systems. However, NO overproduction has been strongly associated with some diseases such as arthritis, asthma, cerebral ischemia, Parkinson's disease, neurodegeneration,

Nitric oxide synthase (NOS) is the enzyme responsible for NO biosynthesis, and there are three main kinds of NOS isoforms [endothelial NOS (eNOS), inducible NOS (iNOS), and neuronal NOS (nNOS)]. They are tetramers, constituted of two NOS monomers associated with two calmodulin monomers (CaM), and contain relatively tightly bound cofactors, BH<sup>4</sup>

FAD, FMN, and iron protoporphyrin IX (heme group). Their chemical function is to catalyze the reaction of l-arginine, NADPH, and oxygen to synthesize free radical NO, l-citrulline,

The substrate l-arginine establishes H-bond networks inside the catalytic site of NOS isoforms with the heme group, mainly due to the guanidine group, which is crucial to bind tightly using a salt-bridge interaction with the conserved carboxylate of Glu597 in human nNOS, Glu377 for iNOS and Glu361 for eNOS. In addition, l-arginine establishes H-bonds with the amide carbonyl from Trp592, in nNOS; with Trp372, in iNOS; and with Trp356, in eNOS. Moreover, α-amino group of l-arginine interacts through H-bond with the heme propionate side chain, and the guanidine *N*-terminal nitrogen of this amino acid coordinates with FeII (**Figure 2**). This ligand-receptor interaction profile is similar to all isoforms, which

,

of the art of this compound use in drug design [4].

218 Nitric Oxide Synthase - Simple Enzyme-Complex Roles

and seizures [5–9].

and NADP (**Figure 1**) [10].

generates a challenge to selectivity [11].

 **Figure 1.** General reaction of NO formation by NOS. Adapted from [10].

Exacerbated induction of iNOS is associated with septic shock, inflammatory, and noninflammatory impairment processes in different tissues/organs, and, likewise, the nNOS is triggered in neurotoxicity, neurodegeneration process, and proliferation increase of some neoplastic cell lines. Depending on the clinical condition, decreasing NO levels is necessary, and excellent benefits might be achieved using NOS inhibitors. However, it is much important not to inhibit eNOS, because of its central role in smooth muscle relax, controlling vascular tone and blood pressure [12–14].

The first inhibitors designed (during the1980s and early 1990s) were based on l-arginine, the substrate of the enzyme, and this approach led to potent compounds but with poor selectivity level among the isoforms. In the late 1990s, the first crystal structure of iNOS and eNOS was unveiled, showing the high degree of similarity particularly in the active site of both isoforms. The nNOS crystal structure was reported in 2002, allowing the design of selective inhibitors [15, 16]. It is worth noting that changes in some amino acids of the isoforms lead to differences in electronic and steric effects on the binding site region, which can be interesting for designing selective inhibitors [11, 15]. The active collaboration between Richard Silverman and Thomas Poulos' groups has significantly contributed to this field, and some of their papers are discussed in this chapter.

NOS isoforms were validated as target for new drugs soon after their X-ray crystallography was available. From then on the design of effective and selective inhibitors has been an important approach in modern drug discovery involving NO biochemical pathways related to many dysfunctions of the human organism [12, 17–19].

#### **2. Experimental studies**

#### **2.1. Inducible nitric oxide synthase (iNOS) inhibitors**

Garvey and collaborators (1994) were the first to report highly selective iNOS inhibitors. The compounds were isothiourea derivatives (**Figure 3**—**1**) designed as l-arginine-competitive reversible inhibitors of human iNOS, with a *K*<sup>i</sup> = 47 nM and a 190-fold selectivity over eNOS but only ~5-fold over nNOS [10, 20]. Further studies of the group led to the design of compound **1400W** (**Figure 3**), which is highly selective over eNOS and nNOS and able to penetrate into cells and tissues [10, 21].

The selective iNOS inhibition by aminoguanidine (**Figure 3**—**AG**) showed that NO can mediate the disruption of hematopoiesis during acute graft-versus-host disease (GVHD), also decreasing the endogenous bacterial infections in the spleen and liver in mice receiving the inhibitor [22]. The deleterious neuro-inflammation effects of iNOS/NO system stimulated by lipopolysaccharide (LPS) on learning and memory were evaluated in rats. Aminoguanidine decreases TNFα levels, oxidative stress indicators, and NO metabolites [23]. In addition, this compound seems to significantly relieve periapical inflammation in the canine teeth of cats and to reduce histological multiple organ damage in rats [24, 25].

 **Figure 3.** iNOS inhibitors (part 1).

In 2000, Hagmann and collaborators explored the structure-activity relationships of a series of substituted 2-aminopyridines. Compounds 4,6-dialkyl substituted (**Figure 3**—**2** and **3**) were found to be the most potent inhibitors of iNOS, presenting a significant degree of selectivity for this isoform [26]. Exploring the same scaffold, the synthesis of the derivatives *N*-4-piperidinyl-2-aminopyridine led to compound **4** (**Figure 3**), a 4-methoxy substituted derivative, 4-fold more potent in iNOS inhibition compared to the 4-methyl compound. Moreover, 4-cyanobenzamide derivative (**Figure 3**—**AR**-**C133057XX**) presented IC<sup>50</sup> = 0.071 μM, being 1400-fold and around 100-fold selective for eNOS and nNOS, respectively. X-ray crystallography of **AR**-**C133057XX** showed that pyridine moiety binds deeply to the heme pocket, while the exocyclic ring interacts with another binding pocket. This difference in interaction could explain the good selectivity of this molecule [27].

NOS isoforms were validated as target for new drugs soon after their X-ray crystallography was available. From then on the design of effective and selective inhibitors has been an important approach in modern drug discovery involving NO biochemical pathways related

Garvey and collaborators (1994) were the first to report highly selective iNOS inhibitors. The compounds were isothiourea derivatives (**Figure 3**—**1**) designed as l-arginine-competitive reversible inhibitors of human iNOS, with a *K*<sup>i</sup> = 47 nM and a 190-fold selectivity over eNOS but only ~5-fold over nNOS [10, 20]. Further studies of the group led to the design of compound **1400W** (**Figure 3**), which is highly selective over eNOS and nNOS and able to penetrate

The selective iNOS inhibition by aminoguanidine (**Figure 3**—**AG**) showed that NO can mediate the disruption of hematopoiesis during acute graft-versus-host disease (GVHD), also decreasing the endogenous bacterial infections in the spleen and liver in mice receiving the inhibitor [22]. The deleterious neuro-inflammation effects of iNOS/NO system stimulated by lipopolysaccharide (LPS) on learning and memory were evaluated in rats. Aminoguanidine decreases TNFα levels, oxidative stress indicators, and NO metabolites [23]. In addition, this compound seems to significantly relieve periapical inflammation in the canine teeth of cats

to many dysfunctions of the human organism [12, 17–19].

**2.1. Inducible nitric oxide synthase (iNOS) inhibitors**

and to reduce histological multiple organ damage in rats [24, 25].

**2. Experimental studies**

220 Nitric Oxide Synthase - Simple Enzyme-Complex Roles

into cells and tissues [10, 21].

 **Figure 3.** iNOS inhibitors (part 1).

On the other hand, the 1,2-dihydro-4-quinazolinamine compound **AR**-**C102222** (**Figure 3**) showed a dose-dependent inhibition on NO production induced by lipopolysaccharide (LPS). At the highest dose tested (100 μmol/kg) in rats, this compound completely abolished the chronic inflammatory arthritis development all over the 20-day experiment, thus confirming the in vivo efficacy of the class [28].

Structural-based approach using crystal structure and mutagenesis have identified specific induced-fit binding mode, which can generate some conformational changes toward a new specific cavity. Garcin and coworkers showed the *cis*-amidine moiety of quinazoline and aminopyridine chemotypes in the compounds **AR**-**C133057XX** and **AR**-**C102222**, respectively, promoted interactions of hydrogen with Glu (Glu371 and Glu377, mouse and human, respectively) at the binding site and with the heme group (mimicking the l-arginine substrate) (**Figure 4**). Those interactions increased the affinity of the inhibitor-containing bulky groups for iNOS. This occurs when Gln rotates on its own axis, accommodating the rigid bulky moieties of the inhibitors and exposing a new specific pocket to interact. The Gln-open conformation can create a cascade of conformational changes, leading to the generation of this new interaction site and directing the selectivity to the aminopyridine and quinazoline scaffolds [29]. Quantitative structure-activity relationships (QSAR) of quinazoline derivatives have been performed to evaluate the structural features required to interact with the active site of iNOS, allowing the design of more effective inhibitors [30].

 **Figure 4.** (A) iNOS-binding profile of **AR**-**C133057XX**, *PDB code*: 3EAI; (B) iNOS-binding profile of **AR**-**C102222**, *PDB code*: 3E7T.

Other compounds, as acetamidine derivatives (**Figure 5**—**5** and **6**), designed to inhibit iNOS, showed submicromolar activities (IC50 = 0.428 and 0.165 μM, respectively) and excellent selectivity over eNOS (>2300 and 550-fold more selective, respectively). In silico findings revealed that the activity drastically changes when ending amino groups are located instead of carboxylic function in the acceptor H-bond region, which is adjacent to the lipophilic region. This occurs because the charged amino group and its alkyl chain are not able to be stabilized inside the pocket interaction region of the enzyme, decreasing the binding efficiency [31].

 **Figure 5.** iNOS inhibitors (part 2).

A protein called SPSB2 plays an important role in modulating the activity of iNOS through its proteasomal degradation in defense cells. Since this complex is blocked, the NO production from iNOS is prolonged, increasing the killing activity against pathogen microorganisms, making it an interesting anti-infective target [32]. Some cyclic peptidomimetic compounds were designed using this strategy, and the most potent compound **7** (**Figure 5**) showed strong inhibition of SPSB2-iNOS complex in macrophage cell lysates and potent affinity value (*KD* = 29 nM) [33].

High-throughput screening (HTS) strategy has been used to identify new iNOS inhibitors hits such as the compound **8** (**Figure 5**). After structural optimization process, the quinolone amide derivative **9** (**Figure 5**) was generated. This derivative has 2000-fold selectivity over human eNOS, besides being very potent (iNOS IC50 = 11 nM) and presenting oral bioavailability in mouse although its clearance (Clp > 100 mL/min/kg) has shown to be uninteresting [34]. The efforts to overcome this effect was to improve the pharmacokinetic properties, leading to the compound **10** (**Figure 5**), a 4,7-imidazopyrazine derivative. This is a dual iNOS/nNOS inhibitor, showing high potency in human iNOS (IC50 = 0.091 μM) and activity over nNOS (0.30 μM) while maintaining the desired selectivity over eNOS (180-fold). Its mechanism of action at molecular level is based on the inhibition of the iNOS dimerization process. Furthermore, compound **10** was effective using in vivo models of neuropathic pain, presenting desired clearance value (4–9 mL/min/kg), good oral bioavailability, and no tolerance after repeated doses [35].

Phenylpyrroles, pyrazoles, urea kynurenamines, ethynylcyanodienones, and amidine derivatives (**Figure 6**—**11**, **12**, **13**, **14**, and **15**) have also been interesting scaffolds to generate iNOS inhibitors [36–40]. The first derivative reduced significantly the iNOS activity to control  values in MPTP-Parkinson`s disease model, showing a potential to act in central nervous system (CNS) disorders [36].

 **Figure 6.** iNOS inhibitors (part 3).

Other compounds, as acetamidine derivatives (**Figure 5**—**5** and **6**), designed to inhibit iNOS, showed submicromolar activities (IC50 = 0.428 and 0.165 μM, respectively) and excellent selectivity over eNOS (>2300 and 550-fold more selective, respectively). In silico findings revealed that the activity drastically changes when ending amino groups are located instead of carboxylic function in the acceptor H-bond region, which is adjacent to the lipophilic region. This occurs because the charged amino group and its alkyl chain are not able to be stabilized inside

A protein called SPSB2 plays an important role in modulating the activity of iNOS through its proteasomal degradation in defense cells. Since this complex is blocked, the NO production from iNOS is prolonged, increasing the killing activity against pathogen microorganisms, making it an interesting anti-infective target [32]. Some cyclic peptidomimetic compounds were designed using this strategy, and the most potent compound **7** (**Figure 5**) showed strong inhibition of SPSB2-iNOS complex in macrophage cell lysates and potent affinity value

High-throughput screening (HTS) strategy has been used to identify new iNOS inhibitors hits such as the compound **8** (**Figure 5**). After structural optimization process, the quinolone amide derivative **9** (**Figure 5**) was generated. This derivative has 2000-fold selectivity over human eNOS, besides being very potent (iNOS IC50 = 11 nM) and presenting oral bioavailability in mouse although its clearance (Clp > 100 mL/min/kg) has shown to be uninteresting [34]. The efforts to overcome this effect was to improve the pharmacokinetic properties, leading to the compound **10** (**Figure 5**), a 4,7-imidazopyrazine derivative. This is a dual iNOS/nNOS inhibitor, showing high potency in human iNOS (IC50 = 0.091 μM) and activity over nNOS (0.30 μM) while maintaining the desired selectivity over eNOS (180-fold). Its mechanism of action at molecular level is based on the inhibition of the iNOS dimerization process. Furthermore, compound **10** was effective using in vivo models of neuropathic pain, presenting desired clearance value (4–9 mL/min/kg), good oral bioavailability, and no tolerance after repeated doses [35]. Phenylpyrroles, pyrazoles, urea kynurenamines, ethynylcyanodienones, and amidine derivatives (**Figure 6**—**11**, **12**, **13**, **14**, and **15**) have also been interesting scaffolds to generate iNOS inhibitors [36–40]. The first derivative reduced significantly the iNOS activity to control

(*KD* = 29 nM) [33].

 **Figure 5.** iNOS inhibitors (part 2).

222 Nitric Oxide Synthase - Simple Enzyme-Complex Roles

the pocket interaction region of the enzyme, decreasing the binding efficiency [31].

Natural products have been a rich source of new bioactive molecules. Some examples in the NOS inhibition are a sesquiterpenoid, isolated from *Curcuma wenyujin* (**Figure 6**—**16**) and its isomer. They were strong inhibitors of NO production by LPS in iNOS (IC50 = 7.6 and 8.5 μM, respectively). Anmindenols A and B (**Figure 6**—**17** and **18**), from marine-derived bacterium *Streptomyces* sp., also demonstrated a relevant inhibitory activity in macrophage cells NO production (IC50 = 23 and 19 μM, respectively) [41, 42].

#### **2.2. Neuronal nitric oxide synthase (nNOS) inhibitors**

In the beginning of the 1990s, efforts to design selective nNOS inhibitor compounds were addressed, using the substrate l-arginine as the prototype molecule. Series of analogs was synthesized to evaluate which molecular change could interfere in the ligand activity and selectivity over other isoforms. The first selective compound over nNOS was l-nitroarginine (**Figure 7**), producing hypertension in animals due to the lack of selectivity over eNOS. In addition, many peptide analogs were synthesized trying to obtain more promising compounds. After the X-ray crystal complex elucidation, structure-activity relationship findings of several scaffolds have been explored to identify the molecular basis of improving the selectivity toward neuronal isoform [15, 19].

The non-arginine-based compound 7-nitroindazole (**Figure 7**—**NI**) showed little nNOS in vitro selectivity but high in vivo selectivity. Its mechanism of action involves competitive inhibition of H<sup>4</sup> B cofactor, and series of related structures have been designed [11]. This compound has suppressed open-field behavior expressed as distance moved, exploratory rearing and grooming, suggesting that this compound can increase cortical excitability and interfere with some physiological and behavioral parameters [43]. Nevertheless, its anticonvulsant activity should be better understood, since studies in rodents reveal a beneficial activity although proconvulsant effect can be found in kainite-, nicotine- and soman-induced convulsions [44].

Entrena and collaborators, by using kynurenamine scaffold (**Figure 7**—**19**) as a template, carried out the synthesis of a series of new candidates to neuroprotective compounds, showing a pharmacophore model to interact with nNOS catalytic site [45].

 **Figure 7.** nNOS inhibitors (part 1).

*N*-Substituted acetamidines (**Figure 7**—**20** and **21**) showed nNOS inhibition activity (IC50 = 0.2 and 0.3 μM) with good selectivity index (500 and 1166-fold selectivity over eNOS, respectively, and 50 and 100-fold, over iNOS, respectively). In silico studies were useful to understand the fit of these scaffolds inside the catalytic site. nNOS contains a bigger heme group cavity compared with other isoforms, which could explain why bulky groups better accommodate in the neuronal isoform [46].

Aminopyridine is an attractive pharmacophoric group to bind in different regions of nNOS through H-bond. Using this moiety, compound **22** (**Figure 7**) and its optical isomer showed to be selective against nNOS, although unable to penetrate the blood-brain-barrier (BBB) in in vivo studies [15, 47, 48]. Trying to increase the CNS permeability, prodrug design approach was used in primary and secondary amines. However, this strategy did not increase the BBB penetration, even masking the charge by carbamate and azide functions [49]. On the other hand, using these compounds containing basic nitrogen, Xue and coworkers attached electron-withdrawing groups (**Figure 7**—**23**) close to these amine functions, decreasing their p*K*a values and improving the membrane permeability in cell-based assays [50].

Concerning 4,5-dihydro-1-*H*-pyrazole derivatives, they were confirmed as selective nNOS inhibitors. Compound **24** (**Figure 7**), the most active of the derivatives (82% of inhibition), showed that its methoxy electron-donating group is important to improve potency and selectivity. By molecular modeling, it was possible to identify that the phenyl moiety can fit below the heme group, establishing π-π interaction. The methoxy groups adopt a conformation that allows them to interact with Arg481 by H-bonds. Moreover, the amine group interacts by H-bond with one of the carboxylate moieties of the heme group. On the contrary, electronwithdrawing groups are better to generate inhibitors for iNOS [51].

Other interesting structures such as 2-aminoquinolines are effective scaffold to be included in the structure of nNOS inhibitors. Crystallography studies showed that those compounds act as competitive arginine mimics. This scaffold makes important H-bonds with the activesite Glu residue, and the non-coordinating aryl rings are stabilized in a hydrophobic pocket in the extremity of the substrate access channel. Moreover, this structural class showed good pharmacokinetic properties (**Figure 8**—**25**), such as brain penetration and oral bioavailability according to the permeability results in Caco-2 cell assay [52]. Trying to optimize this class of compounds, chlorine was added on the phenyl ether central aryl ring (**Figure 8**—**26**). This substitution was found to be selective and highly potent in the design of nNOS inhibitors while retaining CNS penetration and showing a diminished off-target interaction. In complex with human nNOS, this compound showed a phenyl ring orientation where the alkyl amine makes an H-bond with the H<sup>4</sup> B site [53].

 **Figure 8.** nNOS inhibitors (part 2).

should be better understood, since studies in rodents reveal a beneficial activity although proconvulsant effect can be found in kainite-, nicotine- and soman-induced convulsions [44]. Entrena and collaborators, by using kynurenamine scaffold (**Figure 7**—**19**) as a template, carried out the synthesis of a series of new candidates to neuroprotective compounds, showing

*N*-Substituted acetamidines (**Figure 7**—**20** and **21**) showed nNOS inhibition activity (IC50 = 0.2 and 0.3 μM) with good selectivity index (500 and 1166-fold selectivity over eNOS, respectively, and 50 and 100-fold, over iNOS, respectively). In silico studies were useful to understand the fit of these scaffolds inside the catalytic site. nNOS contains a bigger heme group cavity compared with other isoforms, which could explain why bulky groups better accom-

Aminopyridine is an attractive pharmacophoric group to bind in different regions of nNOS through H-bond. Using this moiety, compound **22** (**Figure 7**) and its optical isomer showed to be selective against nNOS, although unable to penetrate the blood-brain-barrier (BBB) in in vivo studies [15, 47, 48]. Trying to increase the CNS permeability, prodrug design approach was used in primary and secondary amines. However, this strategy did not increase the BBB penetration, even masking the charge by carbamate and azide functions [49]. On the other hand, using these compounds containing basic nitrogen, Xue and coworkers attached electron-withdrawing groups (**Figure 7**—**23**) close to these amine functions, decreasing their p*K*a

Concerning 4,5-dihydro-1-*H*-pyrazole derivatives, they were confirmed as selective nNOS inhibitors. Compound **24** (**Figure 7**), the most active of the derivatives (82% of inhibition),

values and improving the membrane permeability in cell-based assays [50].

modate in the neuronal isoform [46].

 **Figure 7.** nNOS inhibitors (part 1).

a pharmacophore model to interact with nNOS catalytic site [45].

224 Nitric Oxide Synthase - Simple Enzyme-Complex Roles

Exploring the heme-coordinating potential of imidazole group, a series of 2,4-disubtituted pyrimidine compounds (**Figure 8**—**27**) was designed. They presented a nanomolar affinity to both rat and human nNOS (>200-fold and >100-fold selectivity over eNOS and iNOS, respectively), exhibiting a minimal off-target binding to 50 CNS receptors. Crystal structures of the complex (nNOS-**27**) indicate that heme Fe coordinates by the 2-imidazolyl group, and the non-coordinating aryl rings are stabilized in a hydrophobic pocket at the far end of the substrate access channel. The fluorine atom in compound **27** also interacts into the hydrophobic pocket, and the secondary amine of this derivative establishes dual ionic interaction with both heme propionates (**Figure 9**). This molecular interaction profile is important to obtain potency and selectivity. In addition, nitrogen from pyrimidine ring performs an H-bond with the His342 side chain. The imidazole ring of the most active compound acts as a weak CYP3A4 inhibitor, suggesting that modulating hydrophobicity and bulkiness can be useful to attenuate the effects in CYP isoforms [54].

 **Figure 9.** Rat nNOS-binding profile of **27**, *PDB code*: 4V3X.

Studies using aminopyridine-based scaffold with pyridine linker (**Figure 8**—**28**) showed that difference in the position of an amino acid, Asp597 of nNOS versus Asn368 of eNOS, controls the affinity and binding mode of this class of nNOS inhibitors. While the central pyridine is at least partially protonated to points up toward Tyr562 for optimal electrostatic interactions, the Asp597 provides additional and important electrostatic stabilization to the other part of the inhibitor [55–57].

Rational strategy for identifying new nNOS inhibitors using a combination of virtual screening approach based on 3D pharmacophore model and molecular docking was able to identify a hit compound structurally different from the available inhibitors (**Figure 8**—**29**). This strategy can be useful to design novel optimized analogs [58].

#### *2.2.1. Double-headed nNOS inhibitors*

pocket, and the secondary amine of this derivative establishes dual ionic interaction with both heme propionates (**Figure 9**). This molecular interaction profile is important to obtain potency and selectivity. In addition, nitrogen from pyrimidine ring performs an H-bond with the His342 side chain. The imidazole ring of the most active compound acts as a weak CYP3A4 inhibitor, suggesting that modulating hydrophobicity and bulkiness can be useful to

Studies using aminopyridine-based scaffold with pyridine linker (**Figure 8**—**28**) showed that difference in the position of an amino acid, Asp597 of nNOS versus Asn368 of eNOS, controls the affinity and binding mode of this class of nNOS inhibitors. While the central pyridine is at least partially protonated to points up toward Tyr562 for optimal electrostatic interactions, the Asp597 provides additional and important electrostatic stabilization to the other part of

Rational strategy for identifying new nNOS inhibitors using a combination of virtual screening approach based on 3D pharmacophore model and molecular docking was able to identify a hit compound structurally different from the available inhibitors (**Figure 8**—**29**). This strat-

egy can be useful to design novel optimized analogs [58].

 **Figure 9.** Rat nNOS-binding profile of **27**, *PDB code*: 4V3X.

attenuate the effects in CYP isoforms [54].

226 Nitric Oxide Synthase - Simple Enzyme-Complex Roles

the inhibitor [55–57].

Double-headed compounds have been explored by researches with the aim of obtaining high affinity binding in nNOS. Attaching a double-headed aminopyridine moiety in a compound led to a very potent (*Ki* = 25 nM) and selective (107-fold selective over nNOS and eNOS) compound (**Figure 10**—**30**). Both aminopyridine moieties interact in different positions with the enzyme—Glu592 in the active site and the heme group. According to the X-ray crystals, there is a second **30** molecule binding also in the H<sup>4</sup> B site, specifically, and the pyridine moiety coordinates with the Zn atom. This interaction does not take place in eNOS. Cellular permeability studies confirmed compound **30** as an interesting lead [59, 60].

Other symmetric double-headed aminopyridine series without charge groups were designed to contain a tail on the central aromatic ring. The objective is to achieve an interaction in the electronegative region in the catalytic site, since only the neuronal isoform has Asp597 in this region. Derivative **31** (**Figure 10**) was very potent (*Ki* = 56 nM) and highly selective over other isoforms (472-fold selective for eNOS and 239-fold for iNOS). This occurs because an electropositive functional group (Ciano group) is preferred near Asp597 in nNOS, explaining the selectivity of this compound [61].

Furthermore, double-headed inhibitors containing chiral linkers derived from natural amino acids were designed and synthesized. The best compound (**Figure 10**—**32**) showed high potency (*Ki* = 32 nM) against nNOS and a good selectivity profile (475-fold selective and 244-fold over eNOS and iNOS, respectively). The aminomethyl moiety was crucial in this compound, allowing it to bind to the heme propionates in nNOS and leading to a high selectivity level [62].

 **Figure 10.** Double-headed nNOS inhibitors.

2-Amino-4-methylpyridine groups with a chiral linker derived from proline were designed as selective nNOS inhibitors. They showed to be interesting as they can interact in a unique orientation, what led to selectivity toward neuronal isoform. The aminopyridine groups interact with a Glu592 residue and the heme propionate in nNOS active site. In addition, the nitrogen from pyrrolidine linker is important to contribute to additional hydrogen bonds to the heme propionate, resulting in the most potent compound (*Ki* = 9.7 nM) (**Figure 10**—**33**). The finding that the isomer activities are different also reinforces the importance of chirality control of this kind of inhibitors and shows the dynamism of the target [63].

In addition, using chiral double-headed inhibitors, the α-amino-functionalized aminopyridine derivative **34** (**Figure 10**) was more potent than other chiral compounds (*Ki* value of 24 nM for nNOS, with 273-fold and 2822-fold selectivity against iNOS and eNOS, respectively). Structure-activity relationship studies reveal that the α-amino group close to the center phenyl ring is crucial to stabilize the double-headed binding. Those studies also showed that by changing to aminomethyl group the potency is improved. The inhibitor is able to make H-bonds with both the H<sup>4</sup> B binding site and the propionate of the heme A-ring, which is essential to obtain selectivity over other isoforms. It is also important to note that the distinct electrostatic environments in different isoforms resulted in lower binding free energy in nNOS, which also can explain the potency difference [64].

The non-chiral double-headed thiophene-2-carboximidamide compound (**Figure 10**—**35**) exhibited an excellent inhibitory potency and selectivity (*Ki* = 5 nM; 540-fold and 340-fold selective over eNOS and iNOS, respectively). This compound also showed to be active in metastatic melanoma A375 cells, exhibiting EC50 values of 1.3 μM, better that of the drug cisplatin (EC50 = 4.2 μM) [65].

#### **2.3. Bacterial nitric oxide synthase (bNOS) inhibitors**

Bacterial nitric oxide synthase (bNOS) is present in many Gram-positive microorganisms and has been described as part of their defense system against other species and the oxidative stress provoked by antibiotics through NO releasing. Therefore, bNOS inhibition can increase the antibiotic potential and be harmful to bacterial cell [66].

A screening showed that some known nNOS inhibitors can decrease significantly the percent survival of *Bacillus subtilis* WT treated with the antimicrobial acriflavine. This potential is consistent with NO production inhibition, as it decreases the bacterial resistance against that compound [67].

Exploring the potential of bNOS as a drug target, high selectivity levels are necessary to its inhibitors. In this context, the design of compounds that target the active and pterin-binding site has been considered an important strategy (**Figure 11**—**36**). These compounds are able to carry out an unexpected rotameric position of residue Arg247 in the active site, besides interacting with the important residue of Glu243 from the same site [68].

In addition, with the goal to identify the differences among bNOS and other isoforms, crystallography studies were performed using different inhibitor chemotypes. Researchers observed that Tyr706 from nNOS is conserved in bNOS (Tyr 357) and both have the same rotameric behavior, which is very different, compared with eNOS. This molecular feature can be useful to design new selective bNOS over eNOS inhibitor. Since the pharmacokinetic properties are very different between bNOS and nNOS, selectivity over the latter is not a trouble. However, due to steric hindrance in the tail end of thiophenecarboximidamide analogs, this scaffold can bind differently to bNOS comparatively to nNOS [69].

 **Figure 11.** bNOS inhibitor.

interact with a Glu592 residue and the heme propionate in nNOS active site. In addition, the nitrogen from pyrrolidine linker is important to contribute to additional hydro-

(**Figure 10**—**33**). The finding that the isomer activities are different also reinforces the importance of chirality control of this kind of inhibitors and shows the dynamism of the

In addition, using chiral double-headed inhibitors, the α-amino-functionalized aminopyri-

nM for nNOS, with 273-fold and 2822-fold selectivity against iNOS and eNOS, respectively). Structure-activity relationship studies reveal that the α-amino group close to the center phenyl ring is crucial to stabilize the double-headed binding. Those studies also showed that by changing to aminomethyl group the potency is improved. The inhibitor is able to make

essential to obtain selectivity over other isoforms. It is also important to note that the distinct electrostatic environments in different isoforms resulted in lower binding free energy in

The non-chiral double-headed thiophene-2-carboximidamide compound (**Figure 10**—**35**)

selective over eNOS and iNOS, respectively). This compound also showed to be active in metastatic melanoma A375 cells, exhibiting EC50 values of 1.3 μM, better that of the drug cis-

Bacterial nitric oxide synthase (bNOS) is present in many Gram-positive microorganisms and has been described as part of their defense system against other species and the oxidative stress provoked by antibiotics through NO releasing. Therefore, bNOS inhibition can increase

A screening showed that some known nNOS inhibitors can decrease significantly the percent survival of *Bacillus subtilis* WT treated with the antimicrobial acriflavine. This potential is consistent with NO production inhibition, as it decreases the bacterial resistance against that

Exploring the potential of bNOS as a drug target, high selectivity levels are necessary to its inhibitors. In this context, the design of compounds that target the active and pterin-binding site has been considered an important strategy (**Figure 11**—**36**). These compounds are able to carry out an unexpected rotameric position of residue Arg247 in the active site, besides inter-

In addition, with the goal to identify the differences among bNOS and other isoforms, crystallography studies were performed using different inhibitor chemotypes. Researchers observed that Tyr706 from nNOS is conserved in bNOS (Tyr 357) and both have the same rotameric behavior, which is very different, compared with eNOS. This molecular feature can be useful to design new selective bNOS over eNOS inhibitor. Since the pharmacokinetic properties are very different between bNOS and nNOS, selectivity over the latter is not a trouble. However,

B binding site and the propionate of the heme A-ring, which is

= 9.7 nM)

value of 24

= 5 nM; 540-fold and 340-fold

gen bonds to the heme propionate, resulting in the most potent compound (*Ki*

dine derivative **34** (**Figure 10**) was more potent than other chiral compounds (*Ki*

nNOS, which also can explain the potency difference [64].

**2.3. Bacterial nitric oxide synthase (bNOS) inhibitors**

the antibiotic potential and be harmful to bacterial cell [66].

acting with the important residue of Glu243 from the same site [68].

exhibited an excellent inhibitory potency and selectivity (*Ki*

target [63].

H-bonds with both the H<sup>4</sup>

228 Nitric Oxide Synthase - Simple Enzyme-Complex Roles

platin (EC50 = 4.2 μM) [65].

compound [67].

#### **3. Clinical studies**

A nonselective compound l-NMMA (**Figure 12**), also known as tilarginine, was evaluated clinically in Translational Research Investigating Underlying Disparities in Acute Myocardial Infarction Patients' Health Status (TRIUMPH) study in North America and Europe with planned enrollment of 658 patients at 130 centers. The period of study was between January 2005 and August 2006 (the study was terminated early). Using 1 mg/kg bolus and 5 h infusion did not decrease the mortality rates in patients with refractory cardiogenic shock complicating myocardial infarction despite an open infarct artery. Although the good results showed in phase II, it has failed in phase III [70, 71]. In another study l-NMMA resulted in no differences in mean arterial pressure (MAP) after 2 h compared with placebo group [72].

Evaluating another inhibitor, N(G)-nitro-l-arginine methyl ester (**Figure 12**—l-NAME), in the treatment of refractory cardiogenic shock, the death at 1 month was 27% in the l-NAME group versus 67% in the control group [73]. Additional studies have been performed to further examination, concluding that TRIUMPH strongly indicated that nonselective NOS inhibitors are not clinically interesting [74].

 **Figure 12.** Clinically evaluated compounds.

Recent phase I study in advanced solid tumors with the iNOS inhibitor **ASP9853** (**Figure 12**) showed that the efficacy dose predicted in preclinical studies was not achieved due to overall toxicity limitations. In summary all these clinical information showed that the manipulation of the NOS pathway, with or without chemotherapy, appears to be more challenging than expected [75]. While designing new selective NOS inhibitors which should be highlighted, deeply studies to evaluate clinical benefits are also required.

#### **4. Conclusions**

Many scaffolds have been found to inhibit nitric oxide synthases. Some of them were presented in this chapter as promising for important therapeutic activity. It must be emphasized that the research about nitric oxide synthase inhibitors has expressively advanced thanks to the X-ray crystallographic studies of this enzyme. This helps the structure-based design approach toward the search for selective inhibitors of this enzyme and the comprehension of their mechanism of action. Notwithstanding, efforts have been made for imparting them with a drug-like profile.

#### **Ackowledgements**

The authors thank CAPES, for RAM Serafim scholarship, and CNPq, for EI Ferreira fellowship.

#### **Author details**

Elizabeth Igne Ferreira\* and Ricardo Augusto Massarico Serafim

\*Address all correspondence to: elizabeth.igne@gmail.com

LAPEN, Department of Pharmacy, Faculty of Pharmaceutical Sciences, University of Sao Paulo – FCF/USP, Sao Paulo, Brazil

#### **References**


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Recent phase I study in advanced solid tumors with the iNOS inhibitor **ASP9853** (**Figure 12**) showed that the efficacy dose predicted in preclinical studies was not achieved due to overall toxicity limitations. In summary all these clinical information showed that the manipulation of the NOS pathway, with or without chemotherapy, appears to be more challenging than expected [75]. While designing new selective NOS inhibitors which should be highlighted,

Many scaffolds have been found to inhibit nitric oxide synthases. Some of them were presented in this chapter as promising for important therapeutic activity. It must be emphasized that the research about nitric oxide synthase inhibitors has expressively advanced thanks to the X-ray crystallographic studies of this enzyme. This helps the structure-based design approach toward the search for selective inhibitors of this enzyme and the comprehension of their mechanism of action. Notwithstanding, efforts have been made for imparting them with a drug-like profile.

The authors thank CAPES, for RAM Serafim scholarship, and CNPq, for EI Ferreira fellowship.

LAPEN, Department of Pharmacy, Faculty of Pharmaceutical Sciences, University of Sao

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### *Edited by Seyed Soheil Saeedi Saravi*

Nitric Oxide Synthase - Simple Enzyme-Complex Roles provides information on nitric oxide synthase, a biomolecule of key importance for the different biological systems, including central and peripheral nervous, cardiovascular, and reproductive systems. With recent links to the role of nitric oxide in the reactions that can impact cell signaling, and discoveries surrounding the complex role of nitric oxide synthase that have increased research attention across the fields of cell and molecular biology, physiology, pharmacology, toxicology, neuroscience, cardiology, urology, and endocrinology, this book tries to provide a comprehensive overview of biology/ pathobiology of nitric oxide synthases and a perspective from possible therapeutic indication of the enzyme inhibitors.

Photo by defun / iStock

Nitric Oxide Synthase - Simple Enzyme-Complex Roles

Nitric Oxide Synthase

Simple Enzyme-Complex Roles

*Edited by Seyed Soheil Saeedi Saravi*