**3. NO-related cardiovascular physiology**

#### **3.1 Biosynthesis of NO**

The biochemical history of NO dates back to 1860s when the therapeutic use of nitroglycerin became a prevalent treatment for angina and hypertension [18]. Consequent studies showed that relaxation of blood vessels depended on a molecule present in an intact endothelium lining, which was named as endothelium-derived relaxing factor (EDRF). In 1977, it was demonstrated that NO was the chemical released from nitroglycerin metabolism [19], and EDRF was later identified to be NO [20]. Since then, NO's cardiovascular effect and medical applications have drawn great research interest.

NO biosynthesis primarily relies on the enzyme nitric oxide synthase (NOS). The reaction uses substrate L-arginine and oxygen to generate L-citrulline and NO in the presence of the cofactors, including Ca2+/CaM, reduced nicotinamideadenine-dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), and tetrahydrobiopterin (BH4) [21].

NO is an excellent messenger molecule in the human body because it is a highly reactive free radical and a highly diffusible molecule [22]. NO is able to react with

**7**

3.30 × 10<sup>−</sup><sup>5</sup>

*Nitric Oxide and Oxidative Stress-Mediated Cardiovascular Functionality: From Molecular…*

various species, such as oxygen, superoxide, lipid oxygen radicals, thiols, and metals [23], which is the biochemical base for NO signaling. The ultimate biological effect of NO depends on its concentration, duration of release, and physiological environment [24]. NO concentration varies from subnanomolar (cell survival signaling) to micromolar (cytotoxic effect and apoptotic signaling) in the body [25]. Its small size and lipophilic characteristic allows it to move rapidly across cell membranes. However, NO's effective distance is limited by its extremely high reactivity with species like oxygen, superoxide, and hemoglobin [23]. NO concentration decreases rapidly around the NO source, which makes NO's effect extremely

Three different isoforms of NOS have been identified in the human body. Endothelial NOS (eNOS) is mainly found in vascular endothelial cells (ECs) and is involved in regulating vascular tone and blood clotting. It is also present in cardiomyocytes where it mediates contractility of cardiac muscle [26]. Inducible NOS (iNOS) predominates in immune cells to produce large amounts of NO that aids in the host defense mechanisms. Examples include NO produced by macrophages and neutrophils when induced by lipopolysaccharide, interferon-γ, and tumor necrosis factor alpha [24]. Neuronal NOS (nNOS) is mainly identified in nerve cells where NO assists in nerve transmission. It has also been isolated from cardiomyocytes where NO regulates excitation contraction coupling of cardiac muscles [27]. NO generation by iNOS is mainly regulated at the transcription level, while eNOS and nNOS are constitutive NOS isoforms whose activities are Ca2+- and CaM dependent. Shear forces during blood flow stimulate the opening of Ca2+ channels on ECs to increase [Ca2+]i, resulting in eNOS activation [28]. NO biosynthesis can be inhibited by various chemicals that selectively bind to NOS with high affinities, such as NGmonomethyl L-arginine (L-NMMA) and asymmetric dimethyl L-arginine [9]. S-nitrosothiols and nitrite are alternative endogenous sources of NO. S-nitrosothiols decompose to release NO under physiological pH and are formed through the reaction of NO and thiol [29]. Nitrite can be reduced to NO in the body through pathways involving reducing agents and proteins, such as ascorbic acid, thiols, hemoglobin, and myoglobin [30]. These backup NO generation pathways are emphasized during hypoxia and acidosis when oxygen-dependent NOS-

Endogenous NO has various physiological effects in the body including inhibiting platelet aggregation, regulating SMC proliferation, modulating immune response, participating in neuron signal transmission, and inducing vasodilation [25]. In the vascular system, NO is primarily generated by eNOS in ECs (**Figure 2**). Shear stresses induced by blood flow and chemical stimuli, known as agonists including acetylcholine, bradykinin, adenosine triphosphate, estrogen, and vascular growth factors, are able to activate eNOS [28, 31]. NO is a highly dynamic molecule that diffuses fast in both aqueous and lipid environment (with diffusion coefficient *D* = 2.07–

temperatures). Thus, NO readily enters the SMCs and binds to the heme moiety of soluble guanylyl cyclase (sGC) to activate sGC, probably the most important protein target of NO. This binding causes the formation of a nitrosyl-heme complex, heme conformational change, and breaking-apart of sGC His105 from heme iron [35]. Then, an open central core is formed in porphyrin, which can accept guanosine triphosphate (GTP) to generate cyclic guanosine monophosphate (cGMP).

Cyclic-GMP binds and activates cGMP-dependent protein kinase (protein kinase G, or PKG), which can phosphorylate the downstream targets to trigger vasodilation

/s in water and polymer matrices [32–34] at physiologically relevant

*DOI: http://dx.doi.org/10.5772/intechopen.82556*

localized (within hundreds μm).

mediated NO production is limited.

**3.2 NO-cGMP pathway**

cm2

#### *Nitric Oxide and Oxidative Stress-Mediated Cardiovascular Functionality: From Molecular… DOI: http://dx.doi.org/10.5772/intechopen.82556*

various species, such as oxygen, superoxide, lipid oxygen radicals, thiols, and metals [23], which is the biochemical base for NO signaling. The ultimate biological effect of NO depends on its concentration, duration of release, and physiological environment [24]. NO concentration varies from subnanomolar (cell survival signaling) to micromolar (cytotoxic effect and apoptotic signaling) in the body [25]. Its small size and lipophilic characteristic allows it to move rapidly across cell membranes. However, NO's effective distance is limited by its extremely high reactivity with species like oxygen, superoxide, and hemoglobin [23]. NO concentration decreases rapidly around the NO source, which makes NO's effect extremely localized (within hundreds μm).

Three different isoforms of NOS have been identified in the human body. Endothelial NOS (eNOS) is mainly found in vascular endothelial cells (ECs) and is involved in regulating vascular tone and blood clotting. It is also present in cardiomyocytes where it mediates contractility of cardiac muscle [26]. Inducible NOS (iNOS) predominates in immune cells to produce large amounts of NO that aids in the host defense mechanisms. Examples include NO produced by macrophages and neutrophils when induced by lipopolysaccharide, interferon-γ, and tumor necrosis factor alpha [24]. Neuronal NOS (nNOS) is mainly identified in nerve cells where NO assists in nerve transmission. It has also been isolated from cardiomyocytes where NO regulates excitation contraction coupling of cardiac muscles [27]. NO generation by iNOS is mainly regulated at the transcription level, while eNOS and nNOS are constitutive NOS isoforms whose activities are Ca2+- and CaM dependent. Shear forces during blood flow stimulate the opening of Ca2+ channels on ECs to increase [Ca2+]i, resulting in eNOS activation [28]. NO biosynthesis can be inhibited by various chemicals that selectively bind to NOS with high affinities, such as NGmonomethyl L-arginine (L-NMMA) and asymmetric dimethyl L-arginine [9].

S-nitrosothiols and nitrite are alternative endogenous sources of NO. S-nitrosothiols decompose to release NO under physiological pH and are formed through the reaction of NO and thiol [29]. Nitrite can be reduced to NO in the body through pathways involving reducing agents and proteins, such as ascorbic acid, thiols, hemoglobin, and myoglobin [30]. These backup NO generation pathways are emphasized during hypoxia and acidosis when oxygen-dependent NOSmediated NO production is limited.

#### **3.2 NO-cGMP pathway**

Endogenous NO has various physiological effects in the body including inhibiting platelet aggregation, regulating SMC proliferation, modulating immune response, participating in neuron signal transmission, and inducing vasodilation [25]. In the vascular system, NO is primarily generated by eNOS in ECs (**Figure 2**). Shear stresses induced by blood flow and chemical stimuli, known as agonists including acetylcholine, bradykinin, adenosine triphosphate, estrogen, and vascular growth factors, are able to activate eNOS [28, 31]. NO is a highly dynamic molecule that diffuses fast in both aqueous and lipid environment (with diffusion coefficient *D* = 2.07– 3.30 × 10<sup>−</sup><sup>5</sup> cm2 /s in water and polymer matrices [32–34] at physiologically relevant temperatures). Thus, NO readily enters the SMCs and binds to the heme moiety of soluble guanylyl cyclase (sGC) to activate sGC, probably the most important protein target of NO. This binding causes the formation of a nitrosyl-heme complex, heme conformational change, and breaking-apart of sGC His105 from heme iron [35]. Then, an open central core is formed in porphyrin, which can accept guanosine triphosphate (GTP) to generate cyclic guanosine monophosphate (cGMP).

Cyclic-GMP binds and activates cGMP-dependent protein kinase (protein kinase G, or PKG), which can phosphorylate the downstream targets to trigger vasodilation

*Vascular Biology - Selection of Mechanisms and Clinical Applications*

Intracellular Ca2+ works as a secondary messenger that quickly bonds to troponin, causing a quick conformational change of troponin and tropomyosin [10]. Thus, the myosin-binding site on actin filaments is exposed, and cross-bridge cycling proceeds. Intracellular Ca2+ concentration, or [Ca2+]i, can return to a very low level to cease the contraction and cause relaxation by different mechanisms, such as extruding Ca2+ out of cells or storing cytosolic Ca2+ into sarcoplasmic reticulum (SR) which functions as the Ca2+ reservoir in the cardiomyocyte. Similar mechanisms exist in the vascular tissue. SMC layer lies in between the endothelium layer and adventitia. There is no organized contractile protein fibril or sarcomere structure in SMCs [11]. Instead, the contractile fibrous proteins along with other intermediate filaments form bundles that are immobilized by anchoring proteins onto cell cytoskeletons. These filaments distribute all over the cytoplasm and connect each other through anchoring proteins (dense bodies) to form a threedimensional network (**Figure 1d** and **f**). Unlike in cardiac muscles, actin filament in smooth muscles is associated with caldesmon, tropomyosin, and calmodulin (CaM) [12]. CaM is an important Ca2+ sensing protein, which binds and mediates many enzymes' activities upon Ca2+ signaling. Caldesmon binds to actin, which inhibits the activity and motility of actin-myosin ATPase, and this binding is greatly strengthened by tropomyosin [13]. Ca2+ binds and activates CaM to uncouple the interaction between caldesmon and actin. Thus, actin's myosin-binding sites are exposed to myosins. Different from skeleton or cardiac tissues, the contraction in smooth muscles also depends on phosphorylation level of myosin light chain, which is adjusted by the enzyme activity of CaM-dependent myosin light chain kinase (CaM-dependent MLCK) and myosin light chain phosphatase (MLCP) [14]. MLCK adds the phosphoryl group to the myosin light chain, while MLCP removes it. Thus, increase of [Ca2+]i also facilitates muscle contraction through enhancing myosin

Cardiovascular contractility is crucial for blood pressure homeostasis, thermal exchanging, mass transfer, immune responses, and organ functions [16]. Impaired coronary artery contractility (caused by the block of blood vessel or poor dilation) incurs ischemia heart disease [17]. To maintain cardiomyocyte viability and vascular tones, enhancing vasodilation and restricting oxidative stress are critical.

The biochemical history of NO dates back to 1860s when the therapeutic use of nitroglycerin became a prevalent treatment for angina and hypertension [18]. Consequent studies showed that relaxation of blood vessels depended on a molecule present in an intact endothelium lining, which was named as endothelium-derived relaxing factor (EDRF). In 1977, it was demonstrated that NO was the chemical released from nitroglycerin metabolism [19], and EDRF was later identified to be NO [20]. Since then, NO's cardiovascular effect and medical applications have

NO biosynthesis primarily relies on the enzyme nitric oxide synthase (NOS). The reaction uses substrate L-arginine and oxygen to generate L-citrulline and NO in the presence of the cofactors, including Ca2+/CaM, reduced nicotinamideadenine-dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FAD),

NO is an excellent messenger molecule in the human body because it is a highly reactive free radical and a highly diffusible molecule [22]. NO is able to react with

flavin mononucleotide (FMN), and tetrahydrobiopterin (BH4) [21].

**6**

phosphorylation [15].

**3.1 Biosynthesis of NO**

drawn great research interest.

**3. NO-related cardiovascular physiology**

#### **Figure 2.**

*Biosynthesis of NO by eNOS and the biological effects of NO in the vascular system.*

(**Figure 2**). This signaling pathway can be terminated by removing cGMP through converting cGMP to GMP by various phosphodiesterases (PDE) [35]. There are many types of PDE in the human body, and they play critical roles in regulating cardiovascular function, adrenal steroidogenesis, and phototransduction [35].

Interestingly, protein kinase A (PKA, cAMP-dependent protein kinase) and PKG share very similar nucleotide binding domains. Many studies have shown that cGMP can activate PKA downstream pathways and cAMP can also cross-activate PKG [36]. This cross regulation between cGMP and cAMP pathways sometimes complicates NO signaling, which will be shown later.

Due to NO's vasodilation effect, NO releasing drugs such as organic nitrate, and nitro- and nitroso compounds have been used for treating angina pectoris, congestive heart failure damage from ischemia–reperfusion, and pulmonary hypertension [37, 38]. Potent drugs also include chemicals that target members involved in NO-cGMP pathway. For example, Sildenafil, known as Viagra, is a PDE5 inhibitor. It prohibits cGMP from being hydrolyzed by PDE5 and extends the activation time of vasodilation to widen the blood vessel and increases blood flow into the penis to treat erectile dysfunction [39].
