**Meet the editor**

Seyed Soheil Saeedi Saravi (PharmD, PhD) graduated from the School of Medicine, Tehran University of Medical Sciences, Iran, and has served as an assistant professor of Pharmacology. He has been awarded a postdoctoral fellowship of cardiovascular medicine at Harvard University. Saeedi Saravi has authored and coauthored over 110 publications in the fields of neu-

ro- and cardiovascular pharmacology and written or edited 8 books and chapters for international publishers. He has served as a member of the editorial board and reviewer of over 15 professional journals and of over 10 national scientific associations. He has been first ranked in the National Exam of PhD in Pharmacology and is a recipient of the National Medical Student, National Young Thesis Editor, National Young Researcher, and National Elites Foundation of Iran Awards.

### Contents

#### **Preface XI**


Kinga G. Blecharz-Lang and Malgorzata Burek

#### **X** Contents


#### **Section 5 Nitric Oxide Synthase Affecting Agents 215**

Chapter 12 **Nitric Oxide Synthase Inhibitors 217** Elizabeth Igne Ferreira and Ricardo Augusto Massarico Serafim

### Preface

**Section 3 Nitric Oxide Synthase in Reproductive System 111**

Chapter 8 **Nitric Oxide: Key Features in Spermatozoa 137**

**Section 4 Miscellaneous Roles of Nitric Oxide Synthase 177**

Chapter 11 **Nitric Oxide Synthase and Nitric Oxide Involvement in**

**Gametes and Embryos 155**

Hanumanth Surekha Rani

**Different Toxicities 197** Emine Atakisi and Oguz Merhan

Chapter 12 **Nitric Oxide Synthase Inhibitors 217**

**Section 5 Nitric Oxide Synthase Affecting Agents 215**

**Functions 113**

Mao

**VI** Contents

Chapter 7 **Nitric Oxide Synthase in Male Urological and Andrologic**

Florentin-Daniel Staicu and Carmen Matas Parra

Chapter 9 **From Nitric Oxide Toward S-Nitrosylation: Expanding Roles in**

Chapter 10 **Role of Endothelial Nitric Oxide Synthase in Breast Cancer 179** Tupurani Mohini Aiyengar, Padala Chiranjeevi and

Ješeta Michal, Marketa Sedmikova and Jean-François Bodart

Elizabeth Igne Ferreira and Ricardo Augusto Massarico Serafim

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

The book Nitric Oxide Synthase - Simple Enzyme—Complex Roles is a compilation of 12 chapters focused on the role of nitric oxide synthase and its product, nitric oxide, in neuro‐ nal function and disorders; cardiovascular diseases like hypertension; male and female re‐ production; and cancer and infection.

The first book section (Chapters 1–4) discusses about the isoforms of nitric oxide synthase and addresses the involvement of nitric oxide synthase and nitric oxide pathway in neuro‐ development and prenatal brain injuries; normal brain functions including memory, learn‐ ing, cognition, and hearing/vision senses; as well as pathophysiology of neuropsychiatric and neurodegenerative diseases and brain tumors. Moreover, the role of nitric oxide signal‐ ing system in the function of the central nervous system in healthy and infectious condi‐ tions, along with the pharmacological interactions of this system with other molecular signaling pathways, such as ionotropic and metabotropic glutamate receptors and kinaselinked receptors, is discussed.

The second book section (Chapters 5 and 6) is dedicated to the nitric oxide synthase impacts on cardiovascular system. The role of endothelial nitric oxide synthase in the pathophysiolo‐ gy of hypertension under increased glucocorticoid level is experimentally studied. In addi‐ tion, an experimental method for monitoring nitric oxide in myocardial tissue is established and fully discussed.

The third book section (Chapters 7–9) comments on the presence and involvement of nitric oxide in normal male sexual and reproductive function, as well as urological disorders such as erectile dysfunction, impotence, infertility, benign prostatic hyperplasia, and prostate car‐ cinoma. Furthermore, this section's attempt is to discuss the key features of nitric oxide syn‐ thase in spermatogenesis and gametogenesis.

The fourth book section (Chapters 10 and 11) tries to notice the miscellaneous roles of nitric oxide synthase in the pathophysiology of breast cancer, as well as mechanisms of drug toxicity.

The fifth book section (Chapter 12) presents a picture of therapeutic indication of nitric oxide synthase inhibitors. Although the medical use of these agents has not yet been es‐ tablished, this chapter concludes experimental effects of nitric oxide synthase inhibitors in animal studies.

Since discovery of nitric oxide, as a significant gaseous transmitter, and the isoforms of nitric oxide synthase, many preclinical and clinical investigations have been performed to find the role of the molecule in physiological function of different biological systems and organs, as well as in the pathophysiology of various diseases. The addressed issues in this book associ‐

ated with the involvement of nitric oxide synthase in neurological, cardiovascular, and re‐ productive disorders, along with cancer and drug toxicity, can summarize the important role of nitric oxide in these events. The aim of this book is to present a comprehensive over‐ view of found functions of nitric oxide synthase to scientists, researchers, and students of fields of cell and molecular biology, physiology, pharmacology, toxicology, neuroscience, cardiology, urology, and endocrinology.

The present book was made due to the valuable efforts and expertise of the contributing authors, who are gratefully acknowledged.

> **Dr. Seyed Soheil Saeedi Saravi** School of Medicine, Tehran University of Medical Sciences, Teheran, Iran

**Nitric Oxide Synthase in Central Nervouss System (CNS)**

ated with the involvement of nitric oxide synthase in neurological, cardiovascular, and re‐ productive disorders, along with cancer and drug toxicity, can summarize the important role of nitric oxide in these events. The aim of this book is to present a comprehensive over‐ view of found functions of nitric oxide synthase to scientists, researchers, and students of fields of cell and molecular biology, physiology, pharmacology, toxicology, neuroscience,

The present book was made due to the valuable efforts and expertise of the contributing

**Dr. Seyed Soheil Saeedi Saravi**

Teheran, Iran

School of Medicine, Tehran University of Medical Sciences,

cardiology, urology, and endocrinology.

VIII Preface

authors, who are gratefully acknowledged.

#### **Chapter 1**

### **Neuronal Nitric Oxide Synthase**

Kourosh Masoumeh Arami, Behnam Jameie and Seyed Akbar Moosavi

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/67494

#### **Abstract**

Nitric oxide synthase (NOS), a flavo‐hemoprotein, regulates nitric oxide (NO) synthesis that has dual biological activities: as an important signalling molecule in vasodilatation and neurotransmission at low concentrations and at higher concentrations as a defensive cytotoxin. In central and peripheral nervous system, neuronal NOS (nNOS) produces NO that has been implicated in modulating physiological functions such as synaptic plasticity, learning, memory and neurogenesis as well as some pathological conditions in which overproduction of NO may lead to the generation of highly reactive species, such as peroxynitrite and stable nitrosothiols, which may cause irreversible cell damage in excitotoxicity, ischaemia, Parkinson, Alzheimer's disease (AD) and depression. NOS‐ derived NO also involves in regulation of blood pressure, smooth muscle relaxation and gut peristalsis via peripheral nitrergic nerves.

**Keywords:** neuronal nitric oxide synthase, nitric oxide

#### **1. Introduction**

After discovery of nitric oxide as a biological mediator many researchers have focused on the importance of nitric oxide in the physiology of the nervous system [1–3].

NO, as the smallest signalling molecule, is produced by three types of NO synthase arising from three different genes referred to as neuronal nitric oxide synthase (also known as nNOS, Type I, NOS‐I and NOS‐1) that is found in neuronal tissues, inducible nitric oxide synthase (also known as iNOS, Type II, NOS‐II and NOS‐2) that is synthesized after formation of pro‐ inflammatory cytokines or endotoxin and endothelial nitric oxide synthase (also known as eNOS, Type III, NOS‐III and NOS‐3) that is found in endothelial cells [4]. The nNOS and eNOS are constitutively expressed and considered to be calcium‐dependent, but when the

activity of iNOS is fully activated at basal intracellular calcium concentration, it would be calcium‐independent.

The main difference between three NOS isoform regarding the reactions achieved lies in the rate of the nicotinamide‐adenine‐dinucleotide phosphate (NADPH) oxidation, termed the uncoupled reaction. Moreover, nNOS carry on transferring electrons to the haem and, hence, oxidase NADPH at a high rate, while in eNOS and iNOS this reaction can happen at a much slower rate [5].

The nNOS constitutes the principal source of NO in distinct populations of neurons and syn‐ aptic spines in the brain and the peripheral nervous system while eNOS can occur in some neurons and iNOS may exist in microglia and astrocytes but usually not in neurons [6].

Interneurons expressing nNOS are involved in physiological procedures like neurovascular coupling to regulate neocortical blood flow [7], the homeostatic control of sleep [8], synaptic integration of adult neurons and balance of excitatory and inhibitory signalling in brain [9].

#### **2. nNOS enzymology**

The nNOS monomer with a molecular weight of 160.8 kDa and 1434 amino acids is inactive and can be activated after dimerization by tetrahydrobiopterin (BH<sup>4</sup> ), haem and l‐arginine (L‐Arg) binding [10]. Each nNOS monomer has two domains including a reductase domain (C‐terminal) and an oxygenase domain (N‐terminal) which can be separated by a calmod‐ ulin‐binding motif [11]. The reductase domain which attaches the substrate NADPH com‐ prises a binding site for flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) [12, 13]. An autoinhibitory loop within the FMN‐binding domain regulates nNOS activity [10]. The oxygenase domain which binds the substrate l‐arginine contains BH<sup>4</sup> binding site, cytochrome P‐450‐type haem active site and Zn binding site for nNOS dimerization facilita‐ tion (**Figure 1**).

**Figure 1.** Schematic demonstration of nNOS structure and the metabolic formation of nitric oxide by nNOS include an oxygenase domain (N‐terminal) and a reductase domain (C‐terminal) which can be separated by a calmodulin‐binding motif. The reductase domain which binds NADPH includes a binding site for FMN, FAD and the oxygenase domain which binds l‐arginine contains a tetrahydrobiopterin (BH<sup>4</sup> ) binding site and a cytochrome P‐450‐type haem active site. NADPH electrons (e‐ ) via FMN and FAD transfer from the reductase domain to the oxygenase domain. nNOS catalyzes the oxidation of l‐arginine to form l‐citrulline and NO (Reproduced with permission from Dong‐Ya Zhu).

All NOS proteins comprise a zinc–thiolate cluster formed by a zinc ion that is tetrahedrally coordinated to two Cys motifs (one donated by each monomer) at the NOS dimer interface. Zinc in NOS has a catalytic activity [10].

#### **3. NOS‐catalyzed reaction**

activity of iNOS is fully activated at basal intracellular calcium concentration, it would be

The main difference between three NOS isoform regarding the reactions achieved lies in the rate of the nicotinamide‐adenine‐dinucleotide phosphate (NADPH) oxidation, termed the uncoupled reaction. Moreover, nNOS carry on transferring electrons to the haem and, hence, oxidase NADPH at a high rate, while in eNOS and iNOS this reaction can happen at a much

The nNOS constitutes the principal source of NO in distinct populations of neurons and syn‐ aptic spines in the brain and the peripheral nervous system while eNOS can occur in some neurons and iNOS may exist in microglia and astrocytes but usually not in neurons [6].

Interneurons expressing nNOS are involved in physiological procedures like neurovascular coupling to regulate neocortical blood flow [7], the homeostatic control of sleep [8], synaptic integration of adult neurons and balance of excitatory and inhibitory signalling in brain [9].

The nNOS monomer with a molecular weight of 160.8 kDa and 1434 amino acids is inactive

(L‐Arg) binding [10]. Each nNOS monomer has two domains including a reductase domain (C‐terminal) and an oxygenase domain (N‐terminal) which can be separated by a calmod‐ ulin‐binding motif [11]. The reductase domain which attaches the substrate NADPH com‐ prises a binding site for flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) [12, 13]. An autoinhibitory loop within the FMN‐binding domain regulates nNOS activity

cytochrome P‐450‐type haem active site and Zn binding site for nNOS dimerization facilita‐

**Figure 1.** Schematic demonstration of nNOS structure and the metabolic formation of nitric oxide by nNOS include an oxygenase domain (N‐terminal) and a reductase domain (C‐terminal) which can be separated by a calmodulin‐binding motif. The reductase domain which binds NADPH includes a binding site for FMN, FAD and the oxygenase domain

the oxidation of l‐arginine to form l‐citrulline and NO (Reproduced with permission from Dong‐Ya Zhu).

) via FMN and FAD transfer from the reductase domain to the oxygenase domain. nNOS catalyzes

) binding site and a cytochrome P‐450‐type haem active site.

), haem and l‐arginine

binding site,

and can be activated after dimerization by tetrahydrobiopterin (BH<sup>4</sup>

[10]. The oxygenase domain which binds the substrate l‐arginine contains BH<sup>4</sup>

calcium‐independent.

4 Nitric Oxide Synthase - Simple Enzyme-Complex Roles

**2. nNOS enzymology**

tion (**Figure 1**).

NADPH electrons (e‐

which binds l‐arginine contains a tetrahydrobiopterin (BH<sup>4</sup>

slower rate [5].

All forms of NOS use l‐arginine and molecular oxygen as the substrate and reduced nicotin‐ amide‐adenine‐dinucleotide phosphate (NADPH) as co‐substrates to produce citrulline NO. FAD and FMN with BH<sup>4</sup> are cofactors of all isozymes. All NOS proteins are homodimers. A functional NOS transfers electrons from NADPH, via FAD and FMN in the carboxy‐terminal reductase domain to the haem in the amino‐terminal oxygenase domain. This flowing of elec‐ tron (NADPH → FAD → FMN → haem) can be facilitated by Ca2+/CaM binding. The oxygen‐ ase domain also binds the cofactor BH<sup>4</sup> , molecular oxygen and l‐arginine. At the haem site, the electrons are used to activate O2 to oxidize l‐arginine to l‐citrulline and NO. Sequences near the cysteine ligand of the haem are also implicated in l‐arginine and BH<sup>4</sup> binding. The NOS enzyme is monooxygenases, generating NO and citrulline from L‐arginine (L‐Arg), NADPH and O2 .

The NOS catalysis has two‐step as follows: In first step, the substrate, L‐Arg, is con‐ verted to N‐hydroxy‐L‐arginine (NOHA), that in turn is converted to NO and citrulline in the second step. The nitrogen atom of NO is derived from the guanidino group of the l‐Arg substrate, and the oxygen atom is derived from dioxygen [10, 14]. NO can acti‐ vate some transduction pathways, such as, activation of guanylyl cyclase and conversion of Guanosine‐5'‐triphosphate (GTP) into Cyclic guanosine monophosphate (cGMP) and

**Figure 2.** Signal transduction of nNOS. nNOS is activated by a calcium‐dependent calmodulin. nNOS produces NO from oxidation of arginine into citrulline. NO diffuses and act on pre‐synaptic or post‐synaptic targets. NO activates guanylyl cyclase (GC) that triggers a protein kinase G (PKG) resulting in Erk activation and the stabilization of TORC1 a CREB co‐activator. PL. M, plasma membrane; CAT, cation and anion transporter. (Adapted from Gallo and Iadecola).

consequent activation of protein kinase G (PKG). PKG activity leads to Erk activation of early genes such as c‐fos, Arc and Brain‐derived neurotrophic factor (BDNF) (**Figure 2**). NO/cGMP pathway is implicated in various neurophysiological processes including neu‐ ronal synaptic modulation, development, learning and memory. Some effects of NO are cGMP‐independent. For instance, several pre‐synaptic targets such as SNAP25, synthaxin Ia, n‐Sec 1, neurogranin as well as the post‐synaptic targets ADP ribosyltransferase and NMDA receptors have been identified for NO [15].

#### **4. Histological distribution**

The nNOS has been found in neurons, astrocytes, the adventitia of brain blood vessels and cardiac myocytes. Besides brain tissue, nNOS has been distinguished by immunohisto‐ chemistry in the spinal cord, sympathetic ganglia and adrenal glands, peripheral nitrergic nerves, skeletal and cardiac myocytes, epithelial cells of different organs, kidney macula densa cells, pancreatic islet cells, parasympathetic ganglia, nonadrenergic noncholinergic peripheral autonomic nerve fibres and the vascular smooth muscle and endothelial cells [16]. In mammalians, the largest origin of nNOS regarding tissue mass is the skeletal mus‐ cle [4, 16, 17].

Since NO cannot be stored in the cells, new synthesis is necessary to have its activities. Thus nNOS should be bonded to the plasma membrane directly or through adapter proteins. Fractionation studies have shown that brain nNOS occurs in particulate and soluble forms in cytosol far from membranes in a patch‐like form. Furthermore, through the early six days in the cultured cerebral cortical astrocytes of rats, nNOS immunoreactivity mostly appeared in the cytoplasm. Nevertheless, at day 7, nNOS immunoreactivity was predominantly expressed in the nucleus, and this nuclear localization continued about 10 h. Then, nNOS immunoreactivity was mainly expressed in the cytoplasm again. Recently, some researchers showed nNOS nuclear localization without cytoplasmic staining of nNOS in some parts of neural and glial cells. Therefore, diverse functions of nNOS in the cell may arise from dif‐ ferential subcellular localization [10].

Adapter proteins are involved in transfer of nNOS to distinct sites. For instance, nNOS is anchored to membranes by binding to syntrophin, PSD95/SAP90 or PSD93. CAPON, another adapter protein for nNOS, comprises a C‐terminal domain that binds to the PDZ domain of nNOS.

In rats and mice, five interneuron expressing nNOS have been found as follows: (1) neuro‐ gliaform cells, (2) Ivy cells (IvC), (3) interneurons expressing the vasoactive intestinal peptide (VIP) and calretinin (CR), (4) interneurons expressing PV and (5) projection cells close to the subiculum. PV cells expressing nNOS mainly exist in the dentate gyrus (DG) of hippocam‐ pus. Though species differences between rat and mouse have been noted that coexpression of nNOS and PV in rat DG is much lower than that in mouse. Somatostatin‐expressing interneu‐ rons that express nNOS are resided in hippocampus.

#### **5. Cofactors and prosthetic groups that impact on nNOS activity**

#### **5.1. Phosphorylation**

consequent activation of protein kinase G (PKG). PKG activity leads to Erk activation of early genes such as c‐fos, Arc and Brain‐derived neurotrophic factor (BDNF) (**Figure 2**). NO/cGMP pathway is implicated in various neurophysiological processes including neu‐ ronal synaptic modulation, development, learning and memory. Some effects of NO are cGMP‐independent. For instance, several pre‐synaptic targets such as SNAP25, synthaxin Ia, n‐Sec 1, neurogranin as well as the post‐synaptic targets ADP ribosyltransferase and

The nNOS has been found in neurons, astrocytes, the adventitia of brain blood vessels and cardiac myocytes. Besides brain tissue, nNOS has been distinguished by immunohisto‐ chemistry in the spinal cord, sympathetic ganglia and adrenal glands, peripheral nitrergic nerves, skeletal and cardiac myocytes, epithelial cells of different organs, kidney macula densa cells, pancreatic islet cells, parasympathetic ganglia, nonadrenergic noncholinergic peripheral autonomic nerve fibres and the vascular smooth muscle and endothelial cells [16]. In mammalians, the largest origin of nNOS regarding tissue mass is the skeletal mus‐

Since NO cannot be stored in the cells, new synthesis is necessary to have its activities. Thus nNOS should be bonded to the plasma membrane directly or through adapter proteins. Fractionation studies have shown that brain nNOS occurs in particulate and soluble forms in cytosol far from membranes in a patch‐like form. Furthermore, through the early six days in the cultured cerebral cortical astrocytes of rats, nNOS immunoreactivity mostly appeared in the cytoplasm. Nevertheless, at day 7, nNOS immunoreactivity was predominantly expressed in the nucleus, and this nuclear localization continued about 10 h. Then, nNOS immunoreactivity was mainly expressed in the cytoplasm again. Recently, some researchers showed nNOS nuclear localization without cytoplasmic staining of nNOS in some parts of neural and glial cells. Therefore, diverse functions of nNOS in the cell may arise from dif‐

Adapter proteins are involved in transfer of nNOS to distinct sites. For instance, nNOS is anchored to membranes by binding to syntrophin, PSD95/SAP90 or PSD93. CAPON, another adapter protein for nNOS, comprises a C‐terminal domain that binds to the PDZ

In rats and mice, five interneuron expressing nNOS have been found as follows: (1) neuro‐ gliaform cells, (2) Ivy cells (IvC), (3) interneurons expressing the vasoactive intestinal peptide (VIP) and calretinin (CR), (4) interneurons expressing PV and (5) projection cells close to the subiculum. PV cells expressing nNOS mainly exist in the dentate gyrus (DG) of hippocam‐ pus. Though species differences between rat and mouse have been noted that coexpression of nNOS and PV in rat DG is much lower than that in mouse. Somatostatin‐expressing interneu‐

NMDA receptors have been identified for NO [15].

**4. Histological distribution**

6 Nitric Oxide Synthase - Simple Enzyme-Complex Roles

ferential subcellular localization [10].

rons that express nNOS are resided in hippocampus.

cle [4, 16, 17].

domain of nNOS.

Kinases increase nNOS activity by phosphorylation, whereas, phosphatases decrease nNOS activity by dephosphorylation [18]. nNOS phosphorylation is important for the enzyme activ‐ ity that is regulated by some kinases and phosphatases, for example, calmodulin‐dependent kinases, protein kinase C (PKC), protein kinase A (PKA) and phosphatase 1, which are in fact modulated by extracellular and intracellular factors [19]. Nonetheless, phosphorylation at different sites of nNOS affects its activity differently. The protein kinase CaMKII can phos‐ phorylate nNOS at Ser847 that diminishes nNOS activity by Ca2+–CaM binding inhibition. Ser847‐PO<sup>4</sup> is found in the autoinhibitory loop which inhibits the movement of the loop even in the occurrence of high concentrations of Ca2+–CaM, thus decreasing nNOS activity [20]. In contrast, the protein phosphatase 1 decreases phosphorylation level of nNOS at Ser847, leading to an increased nNOS activity. Another phosphorylation site of nNOS is at Ser1412 in endothelial nitric oxide synthase [21]. In addition, CaM‐KI deregulates nNOS activity by phosphorylation at Ser741 in transfected cells. Since the expression of CaM‐KI declines with the brain development, it still needs to be demonstrated whether nNOS is phosphorylated at Ser741 by CaM‐KI *in vivo* [10].

#### **5.2. Dimerization**

Despite the ability of the reductase and oxygenase domains that function independently in some circumstances, NOS activity is accomplished by the homodimer [5].

Active nNOS is a dimer with two high‐affinity binding sites for BH<sup>4</sup> and l‐arginine. Two cysteine residues make a disulphide bridge or ligate a zinc ion to connect the two monomers. Furthermore, an 'N‐terminal hook' domain sustains the dimer. BH<sup>4</sup> as well as haem and l‐arginine make nNOS a stable dimer.

High‐affinity binding sites for BH<sup>4</sup> and l‐arginine and facilitating electron flow that occurs in dimerization raise nNOS activity. The electron seems to transfer from one monomer to another, which may be the main reason why the nNOS monomer is inactive. PIN (inhibitor of nNOS) destabilizes the nNOS dimer, thus inhibiting nNOS activity. Dimer stabilization preserves nNOS from proteolysis. Destabilization of dimeric nNOS makes it more vulnerable to be phosphorylated by protein kinase C and hydrolyzed by trypsin [5, 10]. nNOS monomers are able to catalyze the cytochrome c reduction. This shows that the electrons transfer within the reductase domain from NADPH by two flavins is independent of dimeric structure [5]. The haem plays a crucial role in dimerization. In absence of haem, NOS is monomer which is principally normal with respect to secondary structure [5].

#### **5.3. Calcium and calmodulin**

Dependence on Ca2+ is the main characteristic between the constitutive and inducible iso‐ forms. eNOS and nNOS are both triggered by an elevation in intracellular Ca2+, followed by the consequent binding of Ca2+/CaM [5]. Calmodulin acts as an allosteric activator of all forms of NOS that facilitates electron flow transferring from NADPH to the reductase domain fla‐ vins and from the reductase domain to the haem center. Calmodulin binding is brought about by an increase in intracellular Ca2+ [22, 23].

When intracellular Ca2+ concentrations decline to basal levels, calmodulin detaches from nNOS, and it becomes inactive again. Hence, nNOS activity is primarily controlled by intra‐ cellular Ca2+ concentrations and so calmodulin‐binding effect on nNOS activity.

#### **5.4. Proteins binding to nNOS PDZ domain**

PDZ (Post Synaptic Density proteins, discs‐large, ZO‐1) domain of the NH2 terminal involves in dimerization, activation and interaction of nNOS with many other proteins in specific areas of the cell [24]. These interactions determine the sub‐cellular distribution and the function of the enzyme [4, 10].

nNOS PDZ domain contains two separate binding sites, one site binds to PDZ domains of other proteins and another site binds to COOH‐terminal peptide ligands. Proteins contain‐ ing PDZ domains are important in connecting constituents of signal transduction pathways in multiple complexes [25]. NO signalling is modified by nNOS attachment to membrane or cytosolic protein by direct PDZ–PDZ domain or C‐terminal‐PDZ interactions. PSD95 (post‐synaptic density protein‐95), a multivalent synaptic protein and main component of the post‐synaptic density, can connect nNOS to *N*‐methyl‐d‐aspartate receptor (NMDAR), and elucidate the NMDAR stimulation effect on nNOS activation. Post‐synaptic targeting of nNOS is directed by binding to PSD95 (**Figure 3**). nNOS–PSD95 is a typical PDZ–PDZ bind‐ ing, which needs the intact tertiary structure of both domains and a 30‐amino acid extension.

CAPON, another adapter protein, comprises a C‐terminal PDZ domain that binds to the N‐ terminal of nNOS PDZ domain and an N‐terminal phosphotyrosine binding (PTB) domain. CAPON interacts with a component of the Ras family of small G proteins, Dexras1, which is induced by dexamethasone. Interaction of CAPON with nNOS delivers NO to Dexras1, leading to S‐nitrosylation of Dexras1 on cysteine‐11. Dexras1 binds to the peripheral benzodi‐ azepine receptor‐associated protein (PAP7), and PAP7 binds to the divalent metal transporter (DMT1), an iron channel mediating iron uptake in neurons [26]. Abnormally high cellular iron levels may lead to disordered neuronal function [27].

#### **5.5. nNOS inhibitors**

It has been shown that the N‐terminus of nNOS could bind to a protein termed PIN which can inhibit nNOS activity. It is found that PIN destabilizes nNOS dimers and inhibits nNOS activity. Recently, it has been demonstrated that PIN inhibits production of NO and O2 , not nNOS dimerization [28].

Another protein that inhibits NO productions is nitric oxide synthase interacting protein (NOSIP) [29]. NOSIP and nNOS co‐localize in different areas of the central and peripheral nervous systems. NOSIP negatively affects nNOS activity in a neuroepithelioma cell line stably expressing nNOS. In addition, over‐expression of NOSIP in cultured primary neurons

**Figure 3.** PSD95–nNOS uncoupling agents dissociates the PSD95–nNOS interaction. Glutamate produces NMDAR activation, result in NMDAR/PSD95/nNOS complex formation, and thereby recruiting nNOS to the calcium pore of the NMDA receptor, which makes a principal component of excitotoxicity. PSD95–nNOS uncoupling agents may dissociate the PSD95–nNOS complex, thus, having a neuroprotective effect. More, particulary, PSD95–nNOS as uncoupling agent does not influence other pathway of PSD95 or nNOS, such as PSD95–GKAP, PSD95–SynGAP, nNOS–CAPON and nNOS–PFK‐M, therefore, physiological functions of nNOS, for example, learning, memory and neurogenesis, were not affected (Reproduced with permission from Dong‐Ya Zhu).

limits nNOS trafficking to terminal dendrites and direct nNOS to the soma. These findings suggest that NOSIP regulates NO production in the nervous system by regulating the activ‐ ity and localization of nNOS. NOSIP upregulation by neuronal activity may prevent NO production in neurons [10].

### **6. Physiological and pathophysiological functions of nNOS**

#### **6.1. Physiological functions of nNOS**

the consequent binding of Ca2+/CaM [5]. Calmodulin acts as an allosteric activator of all forms of NOS that facilitates electron flow transferring from NADPH to the reductase domain fla‐ vins and from the reductase domain to the haem center. Calmodulin binding is brought about

When intracellular Ca2+ concentrations decline to basal levels, calmodulin detaches from nNOS, and it becomes inactive again. Hence, nNOS activity is primarily controlled by intra‐

in dimerization, activation and interaction of nNOS with many other proteins in specific areas of the cell [24]. These interactions determine the sub‐cellular distribution and the function of

nNOS PDZ domain contains two separate binding sites, one site binds to PDZ domains of other proteins and another site binds to COOH‐terminal peptide ligands. Proteins contain‐ ing PDZ domains are important in connecting constituents of signal transduction pathways in multiple complexes [25]. NO signalling is modified by nNOS attachment to membrane or cytosolic protein by direct PDZ–PDZ domain or C‐terminal‐PDZ interactions. PSD95 (post‐synaptic density protein‐95), a multivalent synaptic protein and main component of the post‐synaptic density, can connect nNOS to *N*‐methyl‐d‐aspartate receptor (NMDAR), and elucidate the NMDAR stimulation effect on nNOS activation. Post‐synaptic targeting of nNOS is directed by binding to PSD95 (**Figure 3**). nNOS–PSD95 is a typical PDZ–PDZ bind‐ ing, which needs the intact tertiary structure of both domains and a 30‐amino acid extension.

CAPON, another adapter protein, comprises a C‐terminal PDZ domain that binds to the N‐ terminal of nNOS PDZ domain and an N‐terminal phosphotyrosine binding (PTB) domain. CAPON interacts with a component of the Ras family of small G proteins, Dexras1, which is induced by dexamethasone. Interaction of CAPON with nNOS delivers NO to Dexras1, leading to S‐nitrosylation of Dexras1 on cysteine‐11. Dexras1 binds to the peripheral benzodi‐ azepine receptor‐associated protein (PAP7), and PAP7 binds to the divalent metal transporter (DMT1), an iron channel mediating iron uptake in neurons [26]. Abnormally high cellular

It has been shown that the N‐terminus of nNOS could bind to a protein termed PIN which can inhibit nNOS activity. It is found that PIN destabilizes nNOS dimers and inhibits nNOS activity. Recently, it has been demonstrated that PIN inhibits production of NO and O2

Another protein that inhibits NO productions is nitric oxide synthase interacting protein (NOSIP) [29]. NOSIP and nNOS co‐localize in different areas of the central and peripheral nervous systems. NOSIP negatively affects nNOS activity in a neuroepithelioma cell line stably expressing nNOS. In addition, over‐expression of NOSIP in cultured primary neurons

terminal involves

, not

cellular Ca2+ concentrations and so calmodulin‐binding effect on nNOS activity.

PDZ (Post Synaptic Density proteins, discs‐large, ZO‐1) domain of the NH2

by an increase in intracellular Ca2+ [22, 23].

8 Nitric Oxide Synthase - Simple Enzyme-Complex Roles

**5.4. Proteins binding to nNOS PDZ domain**

iron levels may lead to disordered neuronal function [27].

the enzyme [4, 10].

**5.5. nNOS inhibitors**

nNOS dimerization [28].

Even though nNOS‐derived NO has many functions in neuronal signalling, it varies from a physiological neuromodulator to a neurotoxic factor when extra amount of NO is gen‐ erated. Thus nNOS may play an important role in many physiological and pathologi‐ cal conditions [10]. Neuronal functions of nNOS are modulating physiological functions such as learning, memory and neurogenesis. In the central nervous system (CNS), nNOS‐ derived NO causes long‐term regulation of synaptic transmission (long‐term potentiation and long‐term inhibition) [30], while in acute neurotransmission there is no involvement. NOS inhibitors diminish learning and produce amnesia in animal models, so it involves in memory formation. Also in the CNS, nNOS‐derived NO is implicated in the central regula‐ tion of blood pressure. Blockade of nNOS activity in the medulla and hypothalamus makes systemic hypertension [31].

The nNOS‐derived NO as an important neurotransmitter is participated in neuronal plastic‐ ity (especially in memory formation), peripheral and central transmission of pain signals, regulation of central nervous system blood flow, neurotransmitter release from cholinergic nerve fibres and the functional regulation of organs with nitrergic innervation [32].

#### *6.1.1. Pancreatic nNOS and insulin regulation*

Pancreatic β‐cells express nNOS that controls insulin secretion through two catalytic activi‐ ties: nitric oxide production and cytochrome c reductase activity [33].

PIN is primarily incorporated with insulin secretory granules and co‐located with nNOS. In addition, PIN overproduction increases glucose‐induced insulin secretion, which is reversed by NO donor, sodium nitroprusside. In contrast, nNOS inhibitor increased insulin secretion induced by glucose. Therefore, PIN insulinotropic effect could be related to its co‐localization with the actin‐based molecular motor myosin and as such be involved in the physiological regulation of insulin secretion at the exocytotic machinery [33].

#### *6.1.2. Cardiovascular regulation*

Sarcoplasmic reticulum and mitochondria functions are regulated by nNOS through Ca2+ maintaining, which are directly related to myocardial injury. nNOS overexpression protects mouse hearts from injury and nNOS deficiency increases ventricular arrhythmia and mortal‐ ity after myocardial infarction [34].

It is accepted that the local regulation of vascular tone in health is mainly regulated by eNOS‐ derived NO [35]. However, some studies have suggested that nNOS‐derived NO may also be implicated in this process and plays an important role in the local regulation of basal micro‐ vascular tone as well as in the vasodilator response to mental stress [35]. nNOS can regu‐ late vascular tone, independent of its effects in the central nervous system [35] and by direct effects on vascular smooth muscle. In the kidney *S*‐methyl‐l‐thiocitrulline (SMTC) as a selec‐ tive nNOS‐inhibitor can decrease basal afferent and efferent arteriolar tone. These studies showed that macula densa (the main source of renal nNOS) removal eliminate the vasocon‐ strictor effects of SMTC.

In cerebral vessels, nNOS may regulate vascular tone reflex especially in response to hypoxia and/or hypotension. For example, Bauser‐Heaton et al. have shown that selective inhibition of nNOS with *N*‐(4*S*)‐(4‐amino‐5‐[aminoethyl]aminopentyl)‐*N*'‐nitroguanidine decreased basal cerebral arterial diameter and eliminated the vasodilator response to hypoxia.

In skeletal muscle nNOS is located at the cell membrane bound to the cytoskeletal protein dystrophin. Dystrophin absence in patients with Duchenne muscular dystrophy (DMD) makes a significant reduction in skeletal muscle nNOS expression and also in blood flow.

In coronary microvascular system, nNOS and eNOS have distinct local roles in the physi‐ ologic regulation. While nNOS may be principal factor in regulation of basal vasomotor tone and blood flow, eNOS‐derived NO facilitates dynamic alterations in blood‐flow distribution and has anti‐atherosclerotic effects at endothelium.

Many smooth muscle tissues in the periphery are innervated by nitrergic nerves, that is, nerves that include nNOS that produce and release NO [4]. For example, relaxation of corpus cavernosum and penile erection is occurred by nitrergic nerves. Phosphodiesterase‐5 inhibitors (sildenafil, vardenafil and tadalafil) need at least a residual nNOS activity for their action [4].

Some studies indicated that nNOS‐derived NO may change vascular tone by perivascular sympathetic nerve inhibition. Hatanaka et al. indicated that selective nNOS inhibitor, vinyl‐ l‐5‐(1‐imino‐3‐butenyl)‐l‐ornithine (l‐VNIO) raises arterial vasoconstriction and local norepi‐ nephrine concentration in perivascular nerve stimulation in isolated rat mesenteric arteries without endothelium. However, l‐VNIO could not alter vasoconstrictor response to exoge‐ nous norepinephrine, showing that nNOS‐derived NO may affect release of neurotransmitter from perivascular sympathetic nerves.

In other studies nNOS‐derived NO in central systems may change peripheral vascular tone. For instance, nitric oxidergic neurons in nucleus tractus solitaries influence on blood pressure in diabetic rats. Unilateral microinjection of sodium nitroprusside (100 mmol/60 nL) into the nucleus raised blood pressure in diabetic rats [36, 37]. In another study, it is revealed that nitric oxide in the nucleus raphe magnus modulates cutaneous blood flow in rats during hypothermia [38, 39].

#### *6.1.3. Neurogenesis*

regulation of central nervous system blood flow, neurotransmitter release from cholinergic

Pancreatic β‐cells express nNOS that controls insulin secretion through two catalytic activi‐

PIN is primarily incorporated with insulin secretory granules and co‐located with nNOS. In addition, PIN overproduction increases glucose‐induced insulin secretion, which is reversed by NO donor, sodium nitroprusside. In contrast, nNOS inhibitor increased insulin secretion induced by glucose. Therefore, PIN insulinotropic effect could be related to its co‐localization with the actin‐based molecular motor myosin and as such be involved in the physiological

Sarcoplasmic reticulum and mitochondria functions are regulated by nNOS through Ca2+ maintaining, which are directly related to myocardial injury. nNOS overexpression protects mouse hearts from injury and nNOS deficiency increases ventricular arrhythmia and mortal‐

It is accepted that the local regulation of vascular tone in health is mainly regulated by eNOS‐ derived NO [35]. However, some studies have suggested that nNOS‐derived NO may also be implicated in this process and plays an important role in the local regulation of basal micro‐ vascular tone as well as in the vasodilator response to mental stress [35]. nNOS can regu‐ late vascular tone, independent of its effects in the central nervous system [35] and by direct effects on vascular smooth muscle. In the kidney *S*‐methyl‐l‐thiocitrulline (SMTC) as a selec‐ tive nNOS‐inhibitor can decrease basal afferent and efferent arteriolar tone. These studies showed that macula densa (the main source of renal nNOS) removal eliminate the vasocon‐

In cerebral vessels, nNOS may regulate vascular tone reflex especially in response to hypoxia and/or hypotension. For example, Bauser‐Heaton et al. have shown that selective inhibition of nNOS with *N*‐(4*S*)‐(4‐amino‐5‐[aminoethyl]aminopentyl)‐*N*'‐nitroguanidine decreased basal

In skeletal muscle nNOS is located at the cell membrane bound to the cytoskeletal protein dystrophin. Dystrophin absence in patients with Duchenne muscular dystrophy (DMD) makes a significant reduction in skeletal muscle nNOS expression and also in blood flow.

In coronary microvascular system, nNOS and eNOS have distinct local roles in the physi‐ ologic regulation. While nNOS may be principal factor in regulation of basal vasomotor tone and blood flow, eNOS‐derived NO facilitates dynamic alterations in blood‐flow distribution

Many smooth muscle tissues in the periphery are innervated by nitrergic nerves, that is, nerves that include nNOS that produce and release NO [4]. For example, relaxation of

cerebral arterial diameter and eliminated the vasodilator response to hypoxia.

and has anti‐atherosclerotic effects at endothelium.

nerve fibres and the functional regulation of organs with nitrergic innervation [32].

ties: nitric oxide production and cytochrome c reductase activity [33].

regulation of insulin secretion at the exocytotic machinery [33].

*6.1.1. Pancreatic nNOS and insulin regulation*

10 Nitric Oxide Synthase - Simple Enzyme-Complex Roles

*6.1.2. Cardiovascular regulation*

ity after myocardial infarction [34].

strictor effects of SMTC.

NO as a paracrine messenger in newly produced neurons regulates the proliferation and dif‐ ferentiation of mouse brain neural progenitor cells. L‐NAME, NO synthase inhibitor, raises cell proliferation and reduces neuronal differentiation [40].

The subventricular zone (SVZ) and the subgranular zone (SGZ) of dentate gyrus are two prin‐ cipal neurogenesis sites in the adult brain. In the adult mouse SVZ and olfactory bulb, NO has a negative control on the size of the undifferentiated precursor pool and enhances neuronal differentiation so acts as a physiological inhibitor of neurogenesis [41].

Mice treated with 7‐NI display a rise in the number of mitotic cells in the SVZ, the olfactory bulb and the rostral migratory stream, but not in the DG. Though, a recent research estab‐ lished that nNOS inhibition elevated progenitor cells proliferation in the DG. Also, the anti‐ proliferative role of nNOS‐derived NO on SVZ and DG has been shown in cerebral ischaemia. Thus, endogenous NO‐derived nNOS can inhibit SVZ neurogenesis. Nevertheless, the role of nNOS in hippocampal neurogenesis is arguable. While nNOS‐derived NO has anti‐prolifera‐ tive effect in adult animals, NO donor administration induces neurogenesis. It may be due to the different experimental protocols. Intravenous or hippocampal administration of a NO donor which can result in raised NO levels enhances cerebral blood flow, indirectly influenc‐ ing neurogenesis [10].

#### *6.1.4. Cerebral maps formation*

NO has been involved in the cerebral map formation. In visual system, NO prompts synap‐ tic refinement of immature synaptic connections at retinothalamic and retinocollicular levels. Normal organization of the somatosensory cortex and barrel field plasticity were found by daily injection of nitroarginine before the period of ocular dominance column formation. However, NO may still contribute in establishing and refining neocortical connectivity. Definitely, when NADPHd activity is reformed in the barrel field, abnormal separation of thalamocortical axons happens. In these animals thalamocortical axons show fewer branch points in layer IV and abnormally expansive thalamocortical arbors. These results propose that NO could promote thalamocortical sprouting and participates in the consolidation of synaptic strength in layer IV of the primary somatosensory cortex [40].

#### *6.1.5. Synchronization and coordination*

Also, regulation of gap junctions is mediated by NO. Rorig and colleagues have shown that sodium nitroprusside (an NO donor) reduced the number of gap‐junction‐coupled neurons. Nonetheless, NO can affect electrical coupling, synchronization of metabolic states and coor‐ dination of transcriptional activity between connected neurons [40].

#### *6.1.6. Neurotransmitter release and plasticity*

Release of several neurotransmitters comprising acetyl choline, catecholamines, glutamate and gamma‐Aminobutyric acid (GABA) are regulated by endogenous NO [40]. Furthermore, NO involves in balancing between GABAergic and glutamatergic synaptic transmission in early post‐natal development. Disruption of this balance precipitates pathological disorders such as epilepsy, autism and schizophrenia [42, 43]. Moreover, NO is involved in fine‐tuning synchronous network activity in the developing hippocampus [40].

NO plays an important role in memory formation in hippocampus [44] and NOS inhibition impedes learning and/or memory [45] while some studies failed to find any effect on learning and/or memory [10]. In mature hippocampus, NO regulates LTP at the Schaffer collateral/CA1 synapses and acts as a retrograde messenger. This occurs via the activation of post‐synaptic NMDA receptors, synthesis of NO by NOS expressed in pyramidal cells and then retrograde activation of guanylate cyclase located in axon terminals. In contrast, in the cerebellum NO serves as an anterograde messenger that is produced in parallel fibre terminals or cerebellar interneu‐ rons and then diffuses to the post‐synaptic Purkinje cell to induce long term depression (LTD) through a cGMP‐dependent mechanism [40]. Additionally, NO involves in experience‐depen‐ dent plasticity in the barrel cortex by reduction of bicuculine‐induced activation of Erk and incre‐ ment of c‐Fos, Egr‐1 and Arc.

In water maze, 8‐arm radial maze, passive‐avoidance and elevated plus‐maze, 7‐NI, at a dose inhibiting nNOS but not affecting blood pressure, induced amnesic effects. Before training in avoidance conditioning in goldfish anterograde amnesia was produced. However, imme‐ diately after training retrograde amnesia was formed. Moreover, genetic inhibition of nNOS indicated spatial performance impairment in the Morris water maze [46]. The hippocampus of nNOS knockout mice showed an abnormal expression of a synaptosomal‐associated protein of the exocytotic machinery, glycolytic enzymes, T‐complex protein 1, the signalling structure guanine nucleotide‐binding protein G and heterogeneous nuclear ribonucleoprotein H of the splicing machinery. Therefore, in nNOS knockout mice spatial memory in the Morris water maze may impair by specific hippocampal protein derangements [10].

#### *6.1.7. Gastrointestinal tract*

injection of nitroarginine before the period of ocular dominance column formation. However, NO may still contribute in establishing and refining neocortical connectivity. Definitely, when NADPHd activity is reformed in the barrel field, abnormal separation of thalamocortical axons happens. In these animals thalamocortical axons show fewer branch points in layer IV and abnormally expansive thalamocortical arbors. These results propose that NO could promote thalamocortical sprouting and participates in the consolidation of synaptic strength in layer IV

Also, regulation of gap junctions is mediated by NO. Rorig and colleagues have shown that sodium nitroprusside (an NO donor) reduced the number of gap‐junction‐coupled neurons. Nonetheless, NO can affect electrical coupling, synchronization of metabolic states and coor‐

Release of several neurotransmitters comprising acetyl choline, catecholamines, glutamate and gamma‐Aminobutyric acid (GABA) are regulated by endogenous NO [40]. Furthermore, NO involves in balancing between GABAergic and glutamatergic synaptic transmission in early post‐natal development. Disruption of this balance precipitates pathological disorders such as epilepsy, autism and schizophrenia [42, 43]. Moreover, NO is involved in fine‐tuning

NO plays an important role in memory formation in hippocampus [44] and NOS inhibition impedes learning and/or memory [45] while some studies failed to find any effect on learning and/or memory [10]. In mature hippocampus, NO regulates LTP at the Schaffer collateral/CA1 synapses and acts as a retrograde messenger. This occurs via the activation of post‐synaptic NMDA receptors, synthesis of NO by NOS expressed in pyramidal cells and then retrograde activation of guanylate cyclase located in axon terminals. In contrast, in the cerebellum NO serves as an anterograde messenger that is produced in parallel fibre terminals or cerebellar interneu‐ rons and then diffuses to the post‐synaptic Purkinje cell to induce long term depression (LTD) through a cGMP‐dependent mechanism [40]. Additionally, NO involves in experience‐depen‐ dent plasticity in the barrel cortex by reduction of bicuculine‐induced activation of Erk and incre‐

In water maze, 8‐arm radial maze, passive‐avoidance and elevated plus‐maze, 7‐NI, at a dose inhibiting nNOS but not affecting blood pressure, induced amnesic effects. Before training in avoidance conditioning in goldfish anterograde amnesia was produced. However, imme‐ diately after training retrograde amnesia was formed. Moreover, genetic inhibition of nNOS indicated spatial performance impairment in the Morris water maze [46]. The hippocampus of nNOS knockout mice showed an abnormal expression of a synaptosomal‐associated protein of the exocytotic machinery, glycolytic enzymes, T‐complex protein 1, the signalling structure guanine nucleotide‐binding protein G and heterogeneous nuclear ribonucleoprotein H of the splicing machinery. Therefore, in nNOS knockout mice spatial memory in the Morris water

dination of transcriptional activity between connected neurons [40].

synchronous network activity in the developing hippocampus [40].

maze may impair by specific hippocampal protein derangements [10].

of the primary somatosensory cortex [40].

12 Nitric Oxide Synthase - Simple Enzyme-Complex Roles

*6.1.6. Neurotransmitter release and plasticity*

ment of c‐Fos, Egr‐1 and Arc.

*6.1.5. Synchronization and coordination*

The most obvious phenotype of nNOS knockout mice is stomach enlargement, several times the normal size, proving a role for nNOS in smooth muscle relaxation of the pyloric sphinc‐ ter. Ablation of exon 6 in another nNOS knockout mice results in severe pyloric stenosis and reproductive endocrine abnormalities.

Citrulline measurements showed that nNOS activity in exon 2‐deficient mice is 0.5% of that in wild‐type, compared to 3% in exon 6‐deficient mice [47].

#### **6.2. Pathophysiological functions of nNOS**

Abnormal NO signalling involved in some neurodegenerative pathologies that include exci‐ totoxicity results in stroke, Parkinson's and Alzheimer's diseases and multiple scleroses. NO can involve in excitotoxicity, probably by peroxynitrite activation of poly‐ADP‐ribose poly‐ merase (PARP) and/or mitochondrial permeability transition. High levels of NO can also pro‐ duce energy reduction, caused by mitochondrial respiration inhibition and glycolysis. Some disorders of smooth muscle tone in the gastrointestinal tract (e.g. gastro‐oesophageal reflux disease) may also drive from an excessive NO production by nNOS in peripheral nitrergic nerves [4].

An important mode of inactivation of NO is its reaction with superoxide anion (O2 ‐ ) to form the potent oxidant peroxynitrite (ONOO‐ ). This can make oxidative damage, nitration and S‐nitrosylation of biomolecules including proteins, lipids and DNA [48]. Nitrosative stress by ONOO‐ has been involved in DNA single‐strand breakage, followed by poly‐ADP‐ribose polymerase (PARP) activation [49].

#### *6.2.1. Role of nNOS in neurodegeneration*

Previous studies have shown that NO implicate in the pathogenesis of some neuroinflam‐ matory/degenerative diseases. The constitutive NOS make the cuprizone‐induced model of demyelination/remyelination [50]. Previous results demonstrate that demyelination was mainly prevented in mice lacking nNOS. In eNOS‐/‐ mice, demyelination increased to the same level as in wild type, but they showed a slight delay in spontaneous remyelination [50].

#### *6.2.1.1. Role of nNOS in the pathophysiology of Parkinson's disease*

A progressive loss of dopaminergic input from the substantia nigra pars compacta leads to overactivity in Parkinson's disease (PD) which creates extrapyramidal motor dysfunction, including bradykinesia, rigidity and tremor. 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) yield Parkinson‐like symptoms and has been used to assess the mechanism of PD pathogenesis.

Recent studies indicate that resistance to MPTP neurotoxicity in the nNOS knockout mice are more than wild‐type ones. This was established by 7‐NI that can protect against this neuro‐ toxicity in experimental animals. Furthermore, a great expression of nNOS was reported in basal ganglia and the respiratory burst of circulating neutrophils of PD patients. However, NO production and protein tyrosine nitration were considerably increased [10].

L‐DOPA is the most used drug in Parkinson's disease treatment. Nitric oxide is a satisfying goal for the decrease of L‐DOPA‐induced dyskinesia in PD [51]. Thus, nNOS in the pathogen‐ esis of Parkinson's disease is important.

#### *6.2.1.2. Role of nNOS in the pathophysiology of Alzheimer's disease*

Disruption of neuronal nitric oxide synthase dimerization contributes to the development of Alzheimer's disease (AD). nNOS‐Ser293, a potential site of cyclin‐dependent kinase‐5 phos‐ phorylation, may be participated in the nNOS dimerization reduction and, hence, the devel‐ opment of AD [52].

Extracellular accumulation of amyloid β‐peptide (Aβ) causing the neuritic plaques and intra‐ cellular neurofibrillary tangles is due to the tau protein hyperphosphorylation considered to be an important feature of AD. Chronic infusion of Aβ1‐40 results in ONOO‐formation and subsequent tyrosine nitration of proteins. Nitrotyrosine found in AD was highly co‐localized with nNOS in cortical pyramidal cells. Moreover, all three isoforms of NOS are raised up in AD; therefore, NOS inhibitors could be useful for AD treatment [10].

#### *6.2.1.3. Role of nNOS in the pathophysiology of multiple sclerosis*

Multiple sclerosis (MS) is characterized by demyelination associated with an infiltration of mononuclear white blood cells within the CNS. The demyelination leads to diminishing con‐ duction of the action potential in neurons.

nNOS can be induced in nerve injury. It has been revealed that nitric oxide deriving from nNOS may be toxic to oligodendrocytes and induce axonal degeneration. Additionally, it is shown that nitrate as NO degradation product is increased in cerebrospinal fluid of MS patients. Also, the oxidised agent, peroxynitrite is found within active MS lesions. Additionally, NO scavengers have been revealed to reduce the severity of an MS‐like disease model [53].

#### *6.2.2. Role of nNOS in the neurodevelopment*

Previous studies indicated that nitric oxide derived from nNOS have been associated in social interaction. They have shown that nNOS deletion decreased anxiety‐like behaviour, aug‐ mented general locomotor activity, reduced spatial learning and memory, and diminished preference for social novelty which are characteristics of autism spectrum disorder [54].

Attention deficit/hyperactivity disorder (ADHD) is a psychiatric disorder with inattention, hyperactivity and impulsivity as main signs, and is frequently associated with learning disabil‐ ity, substance abuse, epilepsy and other psychiatric disorders such as anxiety and disruption of circadian rhythm [55]. In other study, they reported that NOS1 Ex1f‐VNTR is involved in impulsive and empathic personality traits and associated with self‐rated impulsiveness and ven‐ turesomeness [56].

NOS1 KO exhibited higher locomotor activity than wild‐type in a novel environment, as mea‐ sured by open‐field test. NOS1 KO mimics certain ADHD‐like behaviours and could poten‐ tially serve as a novel rodent model for ADHD.

Recent results propose changes in NO‐signalling pathways may be associated with ADHD in humans. Neuronal nitric oxide synthase (NOS1) is a main enzyme responsible for the neu‐ ronal creation of NO. Previous studies show that 28% of adult ADHD patients were found to be homozygous for a risk allele in the NOS1 promoter region (termed ex1f‐VNTR) that diminishes NOS1 expression. This allele is apparently associated with developed prefrontal cortex and ventral striatal roles, which are involved in impulsive and aggressive behaviours associated with ADHD. Moreover, past findings showed that NOS1 knockout mice (NOS1 KO) could be a candidate model for ADHD. Behaviourally, NOS1 KO is apparently hyperac‐ tive and exhibits abnormal social, aggressive and impulsive behaviours as well as deficits in learning and memory [55].

#### *6.2.3. Role of nNOS in excitotoxicity*

basal ganglia and the respiratory burst of circulating neutrophils of PD patients. However,

L‐DOPA is the most used drug in Parkinson's disease treatment. Nitric oxide is a satisfying goal for the decrease of L‐DOPA‐induced dyskinesia in PD [51]. Thus, nNOS in the pathogen‐

Disruption of neuronal nitric oxide synthase dimerization contributes to the development of Alzheimer's disease (AD). nNOS‐Ser293, a potential site of cyclin‐dependent kinase‐5 phos‐ phorylation, may be participated in the nNOS dimerization reduction and, hence, the devel‐

Extracellular accumulation of amyloid β‐peptide (Aβ) causing the neuritic plaques and intra‐ cellular neurofibrillary tangles is due to the tau protein hyperphosphorylation considered to be an important feature of AD. Chronic infusion of Aβ1‐40 results in ONOO‐formation and subsequent tyrosine nitration of proteins. Nitrotyrosine found in AD was highly co‐localized with nNOS in cortical pyramidal cells. Moreover, all three isoforms of NOS are raised up in

Multiple sclerosis (MS) is characterized by demyelination associated with an infiltration of mononuclear white blood cells within the CNS. The demyelination leads to diminishing con‐

nNOS can be induced in nerve injury. It has been revealed that nitric oxide deriving from nNOS may be toxic to oligodendrocytes and induce axonal degeneration. Additionally, it is shown that nitrate as NO degradation product is increased in cerebrospinal fluid of MS patients. Also, the oxidised agent, peroxynitrite is found within active MS lesions. Additionally, NO scavengers have been revealed to reduce the severity of an MS‐like disease model [53].

Previous studies indicated that nitric oxide derived from nNOS have been associated in social interaction. They have shown that nNOS deletion decreased anxiety‐like behaviour, aug‐ mented general locomotor activity, reduced spatial learning and memory, and diminished preference for social novelty which are characteristics of autism spectrum disorder [54].

Attention deficit/hyperactivity disorder (ADHD) is a psychiatric disorder with inattention, hyperactivity and impulsivity as main signs, and is frequently associated with learning disabil‐ ity, substance abuse, epilepsy and other psychiatric disorders such as anxiety and disruption of circadian rhythm [55]. In other study, they reported that NOS1 Ex1f‐VNTR is involved in impulsive and empathic personality traits and associated with self‐rated impulsiveness and ven‐

NO production and protein tyrosine nitration were considerably increased [10].

*6.2.1.2. Role of nNOS in the pathophysiology of Alzheimer's disease*

AD; therefore, NOS inhibitors could be useful for AD treatment [10].

*6.2.1.3. Role of nNOS in the pathophysiology of multiple sclerosis*

duction of the action potential in neurons.

*6.2.2. Role of nNOS in the neurodevelopment*

turesomeness [56].

esis of Parkinson's disease is important.

14 Nitric Oxide Synthase - Simple Enzyme-Complex Roles

opment of AD [52].

Nitric oxide that occurs naturally in the brain without causing overt toxicity, implicated in cell death. One account is that ischaemia creates overproduction of NO, allowing it to react with superoxide to form the potent oxidant peroxynitrite [6].

Brain ischaemia is a great pre‐synaptic glutamate release or post‐synaptic stimulation of its membrane receptors, reproduce neuronal damage or death. Glutamate by binding to four major types of receptors, metabotropic receptors, NMDA receptors, alpha‐amino‐3‐ hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) receptors, and kainate receptors do its action post‐synaptically. Following focal ischaemia, NMDA receptor activation causes more glutamate excitotoxicity and neuronal injury than other receptors owning to their high cal‐ cium permeability. Great stimulation of NMDAR results in Ca2+ overload in the cell, and therefore triggering the Ca2+‐sensitive enzymes. nNOS, as a Ca2+‐sensitive enzyme, indicates a principal role in excitotoxicity. In primary cortical neuronal cultures of nNOS‐/‐ mice, these neurons would be resistant to NMDA neurotoxicity and to oxygen‐glucose deprivation com‐ pared with wild‐type cultures. These *in vitro* studies indicate that nNOS‐derived NO is the principal source of neurotoxicity in neurons [10]. nNOS‐/‐ mice have a reduced infarct size, under focal ischaemia. nNOS knockout mice are resistant to focal and global cerebral isch‐ aemia, consistent with a role for nNOS‐derived NO in cellular injury following ischaemia.

In addition, 7‐nitroindazole and ARL 17477, as selective nNOS inhibitors, can also decrease the infarct size focal ischaemia. Recent studies demonstrate that nNOS expression and enzy‐ matic activity in the hippocampus of mice was decreased under focal cerebral ischaemia. The nNOS reduction following ischaemia stimulated cell proliferation in the DG. Therefore, nNOS inhibition can improve ischemic injury [10].

Even though NMDA activates NOS, the NOS‐containing neurons resist toxic effects of NMDA and form NO that is released to kill adjacent non‐NOS neurons. The unique resistance of NOS neurons to NMDA toxicity seems to be associated with their very high content of manganese superoxide dismutase, which blocks interactions of NO with superoxide to form the per‐ oxynitrite [10].

Since NO measuring and localizing is difficult, citrulline as a marker of NO synthase activ‐ ity localized completely to nNOS‐containing neurons and is eliminated following NOS inhibitors treatment. Additionally, no other enzyme capable of synthesizing citrulline has been found in the brain. Thus, citrulline staining supplies a useful approach to evaluate NO turnover [6].

Ischaemia triggers a pronounced enhancement in citrulline immunoreactivity but more so in a large population of the neuronal isoform of NO synthase (nNOS) in the peri‐infarct than the infarcted tissue. In contrast, 3‐nitrotyrosine (a marker for peroxynitrite formation) is confined to the infarcted tissue and is not present in the peri‐infarct tissue.

#### *6.2.4. Mood disorders*

NO may play an important role in mediating the effect social interactions have on anxiety. Inhibition of nNOS diminishes anxiety‐like responses to pair housing [57]. Also, anxiety‐like behaviours in aged mice are recovered by modifying nNOS expression levels in the hippo‐ campus or cerebellum [58].

Major depressive disorder is a mental disorder characterized by at least two weeks of low mood that is present across most situations. Paroxetine, a typical anti‐depressant inhibited NOS in humans and animals. Moreover, imipramine (IMI, anti‐depressant) significantly diminished NOS activity. IMI withdrawal significantly amplified NOS activity. Furthermore, plasma nitrate concentrations (indicator of NO production) were highly greater in depressed patients. This finding showed likely participation of NO in depression, in line with the observation with those 7‐NI made anti‐depressant–like effects in the Forced Swimming Test. Interestingly, immobilization‐produced stress elevated nNOS mRNA and protein expression in hypothalamic‐pituitary‐adrenal axis in rats. Meanwhile, a recent research showed that chronic mild stress (CMS) augmented nNOS expression in the hippocampus. nNOS inhibi‐ tion prevented CMS‐produced depression. In addition, mice with targeted deletion of the genes encoding nNOS were resistant to the CMS‐induced depression [10].

#### *6.2.5. Pain modulation*

It is revealed that central anti‐nociceptive effect of tapentadolis augmented by 7NI. Neuronal NOS impact on the anti‐nociceptive action of tapentadol at the spinal and supraspinal level [59].

#### **Acknowledgements**

The authors thank Neuroscience Research Center of Iran University of Medical Sciences for the support of this study.

#### **Author details**

superoxide dismutase, which blocks interactions of NO with superoxide to form the per‐

Since NO measuring and localizing is difficult, citrulline as a marker of NO synthase activ‐ ity localized completely to nNOS‐containing neurons and is eliminated following NOS inhibitors treatment. Additionally, no other enzyme capable of synthesizing citrulline has been found in the brain. Thus, citrulline staining supplies a useful approach to evaluate NO

Ischaemia triggers a pronounced enhancement in citrulline immunoreactivity but more so in a large population of the neuronal isoform of NO synthase (nNOS) in the peri‐infarct than the infarcted tissue. In contrast, 3‐nitrotyrosine (a marker for peroxynitrite formation) is confined

NO may play an important role in mediating the effect social interactions have on anxiety. Inhibition of nNOS diminishes anxiety‐like responses to pair housing [57]. Also, anxiety‐like behaviours in aged mice are recovered by modifying nNOS expression levels in the hippo‐

Major depressive disorder is a mental disorder characterized by at least two weeks of low mood that is present across most situations. Paroxetine, a typical anti‐depressant inhibited NOS in humans and animals. Moreover, imipramine (IMI, anti‐depressant) significantly diminished NOS activity. IMI withdrawal significantly amplified NOS activity. Furthermore, plasma nitrate concentrations (indicator of NO production) were highly greater in depressed patients. This finding showed likely participation of NO in depression, in line with the observation with those 7‐NI made anti‐depressant–like effects in the Forced Swimming Test. Interestingly, immobilization‐produced stress elevated nNOS mRNA and protein expression in hypothalamic‐pituitary‐adrenal axis in rats. Meanwhile, a recent research showed that chronic mild stress (CMS) augmented nNOS expression in the hippocampus. nNOS inhibi‐ tion prevented CMS‐produced depression. In addition, mice with targeted deletion of the

It is revealed that central anti‐nociceptive effect of tapentadolis augmented by 7NI. Neuronal NOS impact on the anti‐nociceptive action of tapentadol at the spinal and supraspinal level [59].

The authors thank Neuroscience Research Center of Iran University of Medical Sciences for

to the infarcted tissue and is not present in the peri‐infarct tissue.

genes encoding nNOS were resistant to the CMS‐induced depression [10].

oxynitrite [10].

16 Nitric Oxide Synthase - Simple Enzyme-Complex Roles

turnover [6].

*6.2.4. Mood disorders*

campus or cerebellum [58].

*6.2.5. Pain modulation*

**Acknowledgements**

the support of this study.

Kourosh Masoumeh Arami1,2\*, Behnam Jameie1,2 and Seyed Akbar Moosavi<sup>3</sup>

\*Address all correspondence to: kourosharami.m@iums.ac.ir

1 Neuroscience Research Center, Iran University of Medical Sciences, Tehran, Iran

2 Department of Basic Sciences, Faculty of Allied Medicine, Iran University of Medical Sciences, Tehran, Iran

3 Department of Medical laboratory Sciences, Faculty of Allied Medicine, Iran University of Medical Sciences, Tehran, Iran

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#### **Chapter 2**

## **Endothelial Nitric Oxide Synthase and Neurodevelopmental Disorders**

Saadat Huseynova, Nushaba Panakhova, Safikhan Hasanov and Mehman Guliyev

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/67814

#### **Abstract**

Endothelial activity reflects the balance of endogenous factors regulating vasoconstriction and vasodilation. Among these factors, nitric oxide (NO) is the most important contributor to the acute regulation of vascular tone. Altered nitric oxide synthesis by the vascular endothelium plays several important roles in the pathogenesis of neonatal disease through its effects on vascular homeostasis. However, the role of NO in the pathogenesis of perinatal brain injury has not been fully investigated. The present chapter explores how NO synthesis is regulated under physiological and pathological conditions, the impact of acute and chronic hypoxia on NO synthase activity in the vascular endothelium, and the role of perinatal endothelial dysfunction in the pathogenesis of neurodevelopmental disorders later in life.

**Keywords:** endothelial dysfunction, eNOS, perinatal hypoxia, neuronal injury, neurodevelopmental disorders

#### **1. Introduction**

Endothelial function and the associated production of nitric oxide (NO) play a key role in the pathogenesis of diseases involving the disturbance of vascular homeostasis [1]. Enzymatic generation of NO in mammalian systems is accomplished by the oxidation of l‐arginine to l‐citrulline with the participation of NADPH as a cofactor. Thus, NO is produced by NO synthase isoforms including endothelial NO synthase (eNOS) and neuronal NO synthase (nNOS), with eNOS being the dominant isoform in the vasculature under physiological conditions [2]. eNOS, also known as nitric oxide synthase 3 (NOS3), participates in the regulation

© 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.

of vascular tone and has a wide range of actions that control cerebral blood flow and metabolism. In contrast, the main role of nNOS is the production of NO for retrograde signaling across neuronal synapses.

Altered NO production by the vascular endothelium contributes to the pathogenesis of neonatal disease and may influence developmental growth [3]. The actions of eNOS in endothelial dysfunction lead to vascular and metabolic disorders and are also implicated in hypoxic‐ischemic brain injury [4]. Studies have shown that hypoxic brain injury is characterized by changes in vascular growth and endothelial dysfunction [5–8]. Despite the widespread confirmation a significant role for NO in physiological and pathological vascular homeostasis, the role of NO in the pathogenesis of perinatal brain injury has not been fully investigated. Specifically, the impact of chronic hypoxia on NO synthase isoenzymes in the neonatal brain is unknown. Therefore, the goal of this chapter is to present the results of recent investigations of the pathological role of eNOS in endothelial dysfunction in preterm infants with hypoxic‐ischemic encephalopathy (HIE) and in early‐age neurodevelopmental disorders.

### **2. Specification and function of vascular endothelium in fetoplacental circulation**

The formation of the mammalian vasculature involves many interdependent processes, including the maturation of multiple cell types within tissue compartments, pulsatile blood flow, blood pressure, the activity of smooth muscle cells in vessel walls, and the transmigration of immune cells. Scientific investigations of endothelial remodeling have confirmed its relevance to vascular barrier function, inflammation, and vascular disease [9].

During embryonic development, the first endothelial cells are derived from the extraembryonic mesoderm and appear around embryonic day 7. The placental barrier to the maternal blood is gradually breached between 8 and 12 weeks of gestation owing to invasion of the uteroplacental spiral arteries of the placental bed by the extravillous trophoblast. Accordingly, placental oxygen tension rises and leads to a phase of branching angiogenesis that lasts 24 weeks [10]. The fetoplacental endothelium is continuous with the fetal circulation, such that its function and potential dysfunction have a profound impact on fetal development [11]. To this end, successful pregnancies are highly dependent on effective vasculogenesis.

The regulatory sites and mechanisms responsible for endothelial function in the uteroplacental and fetal circulation remain unclear; however, it is obvious that endothelial activity is regulated through the balanced production and action of local endogenous constricting and dilating factors. Among vasodilatory factors, NO appears to be a chief regulator of acute vascular tone. NO generated by NO synthase expressed in the uterine artery endothelium is a diffusible gas molecule that produces smooth muscle relaxation and therefore vasodilation in a cGMP‐dependent manner [12]. In general, NO is essential for the formation of endothelial function. In pregnancy, NO promotes endovascular invasion by the cytotrophoblast; interstitial trophoblasts produce NO as they invade the maternal spiral arteries in the uterine wall in order to maintain a low‐resistance and high‐caliber uteroplacental unit. If this process fails, endothelial dysfunction associated with increased vascular resistance and reduced fetoplacental blood flow results in placental ischemia, pregnancy complications, and restrictive effects on fetal growth [13, 14]. Moreover, placental hypoperfusion and ischemia lead to the release of antiangiogenic factors that cause oxidative stress and inflammation, further contributing to endothelial dysfunction.

of vascular tone and has a wide range of actions that control cerebral blood flow and metabolism. In contrast, the main role of nNOS is the production of NO for retrograde signaling

Altered NO production by the vascular endothelium contributes to the pathogenesis of neonatal disease and may influence developmental growth [3]. The actions of eNOS in endothelial dysfunction lead to vascular and metabolic disorders and are also implicated in hypoxic‐ischemic brain injury [4]. Studies have shown that hypoxic brain injury is characterized by changes in vascular growth and endothelial dysfunction [5–8]. Despite the widespread confirmation a significant role for NO in physiological and pathological vascular homeostasis, the role of NO in the pathogenesis of perinatal brain injury has not been fully investigated. Specifically, the impact of chronic hypoxia on NO synthase isoenzymes in the neonatal brain is unknown. Therefore, the goal of this chapter is to present the results of recent investigations of the pathological role of eNOS in endothelial dysfunction in preterm infants with hypoxic‐ischemic encephalopathy (HIE) and in early‐age neurodevelopmental

The formation of the mammalian vasculature involves many interdependent processes, including the maturation of multiple cell types within tissue compartments, pulsatile blood flow, blood pressure, the activity of smooth muscle cells in vessel walls, and the transmigration of immune cells. Scientific investigations of endothelial remodeling have confirmed its

During embryonic development, the first endothelial cells are derived from the extraembryonic mesoderm and appear around embryonic day 7. The placental barrier to the maternal blood is gradually breached between 8 and 12 weeks of gestation owing to invasion of the uteroplacental spiral arteries of the placental bed by the extravillous trophoblast. Accordingly, placental oxygen tension rises and leads to a phase of branching angiogenesis that lasts 24 weeks [10]. The fetoplacental endothelium is continuous with the fetal circulation, such that its function and potential dysfunction have a profound impact on fetal development [11]. To

The regulatory sites and mechanisms responsible for endothelial function in the uteroplacental and fetal circulation remain unclear; however, it is obvious that endothelial activity is regulated through the balanced production and action of local endogenous constricting and dilating factors. Among vasodilatory factors, NO appears to be a chief regulator of acute vascular tone. NO generated by NO synthase expressed in the uterine artery endothelium is a diffusible gas molecule that produces smooth muscle relaxation and therefore vasodilation in a cGMP‐dependent manner [12]. In general, NO is essential for the formation of endothelial function. In pregnancy, NO promotes endovascular invasion by the cytotrophoblast; interstitial trophoblasts produce NO as they invade the maternal spiral arteries in the uterine

**2. Specification and function of vascular endothelium** 

relevance to vascular barrier function, inflammation, and vascular disease [9].

this end, successful pregnancies are highly dependent on effective vasculogenesis.

across neuronal synapses.

24 Nitric Oxide Synthase - Simple Enzyme-Complex Roles

**in fetoplacental circulation**

disorders.

eNOS is well established as a primary physiological source of NO. eNOS affects vascular tone, reduces uteroplacental resistance, regulates uterine and fetoplacental blood flow, and is involved in uterine quiescence prior to parturition in normal pregnancy. Several studies have confirmed that eNOS activity is increased in the uterine artery during pregnancy in several species. Yet, investigations of the role of NO modulators in normal and abnormal pregnancies have shown conflicting results. The concentration of NO in the fetoplacental system depends on many factors including l‐arginine availability, the activity levels of NO synthase isoforms, the presence of endogenous NO synthase inhibitors, and species‐dependent variation. While some studies have reported lower eNOS expression in preeclamptic syncytiotrophoblasts than in normal syncytiotrophoblasts [15, 16], a series of clinical studies revealed that increased NO concentration primarily caused altered fetoplacental circulation, endothelial dysfunction, and reduced flow‐mediated vasodilatation in different pregnancy pathologies [17–19]. Moreover, these studies identified increased endothelial permeability and decreased eNOS expression in the peripheral vasculature under pathological conditions [17]. Norris et al. reported increased NO production in the uteroplacental and fetoplacental circulation during preeclampsia compared to normotensive pregnancies and reasoned that this increase was a compensatory mechanism to offset the pathological effects of preeclampsia [18]. In support of this hypothesis, uterine arteries of pregnant rats exposed to plasma from women with preeclampsia were found to have increased eNOS expression and decreased inducible nitric oxide synthase (iNOS) expression [19]. In contrast, Leiva et al. purported that the bioavailability of NO in the fetoplacental system is decreased in pregnancy pathologies such as preeclampsia, gestational diabetes mellitus, and maternal supraphysiological hypercholesterolemia [20]; the authors hypothesized that altered NO synthesis and bioavailability in these cases are owing to the transcriptional and posttranslational modulation of NO synthases during hypoxia and oxidative stress.

One controversial question regards the putative effect of eNOS depression on fetoplacental blood flow in acute and chronic pathological processes. This topic was investigated in a series of experimental animal models; systemic NO synthase inhibitor administration was found to decrease uteroplacental blood flow and increase peripheral vascular resistance in several species [21]. Rosenfeld and Roy argued that the uteroplacental vasculature is less sensitive to prolonged systemic NO synthase inhibition than the peripheral circulation, which might be explained by the activation of compensatory mechanisms such as those reported for NO synthase in ovine uterine artery smooth muscle [22].

Several studies have investigated eNOS gene polymorphisms and their effects in different pregnancy pathologies. Whereas chronic hypoxia selectively augments the pregnancy‐associated upregulation of eNOS gene expression and endothelium‐dependent relaxation of the uterine artery [23], women with eNOS gene mutations were found to be at risk for developing preeclampsia in a study of Egyptian families [24]. However, other studies do not support a major role for eNOS gene variants in preeclampsia [25, 26]. Comparing the results of studies conducted worldwide, Ma et al. concluded that an eNOS gene polymorphism was related to pregnancy‐induced hypertension risk in Asian populations but not in European and American populations [27].

In summary, altered functionality of the fetal endothelium likely contributes to the formation of extrauterine pathologies from the neonatal period onward. However, the mechanisms underlying fetoplacental vascular development and pathologies thereof remain incompletely defined, such that further studies are necessary to understand the exact role of eNOS in pregnancy pathologies and fetal growth problems.

#### **3. The role of endothelial nitric oxide synthase activity in the pathophysiology of perinatal brain injury**

Perinatal hypoxic‐ischemic brain injury is a major cause of neonatal death and long‐term disability. Approximately 15–25% of newborns with hypoxic‐ischemic encephalopathy (HIE) die during the postnatal period, and surviving infants are at risk for the development of severe and permanent neuropsychological sequelae such as cerebral palsy, seizures, visual impairment, mental retardation, and learning and cognitive impairments [28–30]. Decreased cerebral perfusion, hypoxia, hypoglycemia, and severe anemia can cause critical energy shortages in newborn infants, and accordingly severe hypoxia/ischemia can also affect other tissues of the body [31]. Disorders affecting the peripheral organs are often caused by hemodynamic disturbances resulting from the centralization of the bloodstream and/or poor circulation to the internal organs [32].

In the early days of extrauterine life, the vascular endothelium is exposed to high concentrations of inflammatory stimuli and can become dysfunctional if exposed to a hypoxic environment [33]. Cerebral ischemia induces an inflammatory response in the brain parenchyma and systemic circulation [34, 35], resulting in the augmented secretion of proinflammatory cytokines and chemotactic molecules by the vascular endothelium in newborn infants with hypoxic‐ischemic injury. Hence, cytokines are important upstream effector of brain injury after ischemia [36]. Vasoregulatory mechanisms play essential roles in brain injury and tissue reperfusion in critically ill children; endothelial dysfunction results in an imbalance between vasoconstriction and vasodilatation, which causes tissue reperfusion, cytotoxic edema, and brain injury [37]. A previous study determined that hypoxic inflammation was regulated via bioactive mediators synthesized by endothelium, whereas NO and the sources of its synthesis play a special role in the pathophysiology of leukocyte‐endothelial interactions [38]. To this end, studies show that growth‐retarded fetuses and infants with severe and long‐lasting neuronal injuries exhibit decreased vascular growth and endothelial dysfunction [39].

Of note, brain injury after hypoxic‐ischemic injury progresses over many days even after reperfusion has been achieved. For example, oxygenated blood flow is restored to ischemic brain areas after severe perinatal asphyxia; however, while reperfusion temporarily corrects energy failure, excitotoxicity, and the generation of reactive oxygen species during the ischemic period are together responsible for a significant degree of brain damage. Brain damage after hypoxia‐ischemia includes the primary insult and secondary damage such as delayed neuronal death related to cerebral edema [40]. Primary perinatal insults resulting from hypoxia‐ischemia are associated with the failure of ATPase‐dependent ions channels, which can disrupt synaptic function and lead to the accumulation of extracellular glutamate [41]. The increased availability of reactive oxygen metabolites after reperfusion is also directly involved in augmented glutamate release after injury. Increase in extracellular glutamate concentrations and the activation of glutamate receptors lead to excitotoxicity [42], which involves increased intracellular flux of calcium through open NMDA receptor channels and the release of calcium from intracellular stores. Elevations in intracellular calcium activate lipases, proteases, and endonucleases that lead to cellular damage and death [43]. Moreover, the posthypoxic reperfusion process results in oxidative stress; energy failure activates nNOS and increases NO production, increasing the likelihood of its reaction with superoxide anion to form the powerful oxidant peroxynitrite [44]. Together, cellular energy failure, acidosis, glutamate excitotoxicity, and oxidative stress lead to cytotoxic edema and neuronal death after hypoxia‐ischemia injury [45]. Additionally, there is a continuum of necrosis and apoptosis after such injury: often, early (primary) cell death appears necrotic, whereas later (secondary) cell death appears apoptotic**.** Therefore, while severe insult results in cell necrosis, more moderate asphyxia can cause delayed neuronal death through apoptosis [46]. Secondary apoptosis involves multiple pathophysiological processes such as excitatory neurotransmission, altered growth factor production, and changes in protein synthesis [47].

preeclampsia in a study of Egyptian families [24]. However, other studies do not support a major role for eNOS gene variants in preeclampsia [25, 26]. Comparing the results of studies conducted worldwide, Ma et al. concluded that an eNOS gene polymorphism was related to pregnancy‐induced hypertension risk in Asian populations but not in European and

In summary, altered functionality of the fetal endothelium likely contributes to the formation of extrauterine pathologies from the neonatal period onward. However, the mechanisms underlying fetoplacental vascular development and pathologies thereof remain incompletely defined, such that further studies are necessary to understand the exact role of eNOS in preg-

Perinatal hypoxic‐ischemic brain injury is a major cause of neonatal death and long‐term disability. Approximately 15–25% of newborns with hypoxic‐ischemic encephalopathy (HIE) die during the postnatal period, and surviving infants are at risk for the development of severe and permanent neuropsychological sequelae such as cerebral palsy, seizures, visual impairment, mental retardation, and learning and cognitive impairments [28–30]. Decreased cerebral perfusion, hypoxia, hypoglycemia, and severe anemia can cause critical energy shortages in newborn infants, and accordingly severe hypoxia/ischemia can also affect other tissues of the body [31]. Disorders affecting the peripheral organs are often caused by hemodynamic disturbances resulting from the centralization of the bloodstream and/or poor circulation to

In the early days of extrauterine life, the vascular endothelium is exposed to high concentrations of inflammatory stimuli and can become dysfunctional if exposed to a hypoxic environment [33]. Cerebral ischemia induces an inflammatory response in the brain parenchyma and systemic circulation [34, 35], resulting in the augmented secretion of proinflammatory cytokines and chemotactic molecules by the vascular endothelium in newborn infants with hypoxic‐ischemic injury. Hence, cytokines are important upstream effector of brain injury after ischemia [36]. Vasoregulatory mechanisms play essential roles in brain injury and tissue reperfusion in critically ill children; endothelial dysfunction results in an imbalance between vasoconstriction and vasodilatation, which causes tissue reperfusion, cytotoxic edema, and brain injury [37]. A previous study determined that hypoxic inflammation was regulated via bioactive mediators synthesized by endothelium, whereas NO and the sources of its synthesis play a special role in the pathophysiology of leukocyte‐endothelial interactions [38]. To this end, studies show that growth‐retarded fetuses and infants with severe and long‐lasting neu-

ronal injuries exhibit decreased vascular growth and endothelial dysfunction [39].

Of note, brain injury after hypoxic‐ischemic injury progresses over many days even after reperfusion has been achieved. For example, oxygenated blood flow is restored to ischemic brain areas after severe perinatal asphyxia; however, while reperfusion temporarily corrects

American populations [27].

26 Nitric Oxide Synthase - Simple Enzyme-Complex Roles

the internal organs [32].

nancy pathologies and fetal growth problems.

**3. The role of endothelial nitric oxide synthase activity** 

**in the pathophysiology of perinatal brain injury**

NO serves diverse functions in the perinatal brain, including neuronal differentiation and survival and synaptic formation and plasticity [48]. NO also affects these processes in pathological contexts by (in part) mediating neuronal death and neurodegeneration [49]. Previous studies in growth‐restricted infants demonstrated that elevated NO production was associated with decreased endogenous antioxidant activity, increased lipid peroxidation, and impaired neuronal function [50]. NO supplementation was also found to increase uteroplacental circulation and decrease biomarkers of neuronal injury in the cord blood of infants diagnosed with intrauterine growth retardation [51, 52]. Therefore, it is difficult to interpret the role of NO in the pathogenesis of perinatal neuronal injury: is the concentration of NO increased as a defensive mechanism or does it point to a more profound impairment? Clinical and experimental investigations describing the roles of different NO sources in the pathogenesis of brain injury have provided insight on this problem. In the prospective clinical trial conducted by the Azerbaijan Medical University Neonatology group (ACTRN12612000342819), NO, eNOS, and endotelin‐1 were quantified in 240 preterm infants with high risk for perinatal HIE; the results indicated that while eNOS expression was reduced, NO concentrations were increased in accordance with the severity of HIE (**Figure 1**).

This result provided foundation evidence for nonendothelial sources of NO synthesis in tissue hypoperfusion and hypoxia. Thereafter, the balance of NO/eNOS and its effect on neuronal injury in preterm infants was investigated. An important finding was that infants with severe HIE had higher NO/eNOS ratios compared with mild/moderate HIE and control infants, suggesting a relationship between nonendothelial NO production and neuronal injury (**Figure 2**).

**Figure 1.** Mean total eNOS and NO values in preterm infants with HIE. Error bars indicate the standard error of the mean. Black bars show results from days 1 to 3 and grey bars show results from days 5 to 7. \**p* < 0.05 compared with the control group.

**Figure 2.** NO/eNOS ratios in preterm infants with HIE. \**p* < 0.05 compared with the control group.

Significantly higher NO/eNOS ratios in preterm infants with severe HIE suggest that the activation of neuronal and inducible NO synthases is related to long‐term and severe intrauterine and birth distress in infants. Moreover, increased NO in tandem with eNOS activation in infants with low risk for perinatal HIE might represent a compensatory or defensive strategy in the preterm brain. It should be noted that increased NO generation is not necessarily solely derived from areas of neuronal injury in HIE. Under hypoxic conditions, NO is also produced by the activated endothelium in all injured vasculature. Therefore, it might be difficult to accept the idea that NO/eNOS balance is a good predictor of neuronal injury. Yet, consistent with our previous investigation [53], we observed a statistically significant positive correlation between neuron‐specific enolase (NSE) and NO/eNOS ratio, which suggests that decreased synthesis of NO by endothelial sources is related to more severe hypoxic changes and neuronal injury (**Figure 3**).

It was also found that growth‐restricted infants are subject to significant endothelial dysfunction and eNOS depression, implicating NO in the pathogenesis of intrauterine hypoxic injury [53]. Together, these results provide a strong support for NO/eNOS balance as a marker of endothelial inflammation under hypoxic conditions. Previous experimental and clinical investigations have demonstrated that eNOS is responsible for preserving the functional integrity of the neurovascular unit [54, 55] and may have antiinflammatory effects in aging and other pathological contexts [56, 57].

The follow‐up of newborn infants in the aforementioned study identified significant relationships between peripheral endothelial vasoregulatory markers in the perinatal period and the onset of neurodevelopmental disorders at an early age. It was found that, in the presence of high concentrations of NO, early eNOS activity was insufficient in infants diagnosed with cerebral palsy later in life compared to neonates who did not show neurodevelopmental delays associated with HIE (**Figure 4**). These findings suggest that depressed eNOS activity and increased nonendothelial NO synthesis play important roles in the formation of developmental impairments.

**Figure 1.** Mean total eNOS and NO values in preterm infants with HIE. Error bars indicate the standard error of the mean. Black bars show results from days 1 to 3 and grey bars show results from days 5 to 7. \**p* < 0.05 compared with the

**Figure 2.** NO/eNOS ratios in preterm infants with HIE. \**p* < 0.05 compared with the control group.

control group.

28 Nitric Oxide Synthase - Simple Enzyme-Complex Roles

**Figure 3.** Spearman rank‐order correlation between NO/eNOS ratio and NSE in preterm infants with HIE (*r* = 0.67; *p* = 0.001).

**Figure 4.** Peripheral vasoregulatory markers in the early neonatal period in preterm infants with neurodevelopmental disorders (NDs). Error bars indicate the standard error of the mean. Black bars show results from days 1 to 3 and grey bars show results from days 5 to 7. Control 1: data from infants with HIE who did not develop a ND; Control 2: data from healthy infants. \**p* < 0.05 compared with Control 1; ^*p* < 0.05 compared with Control 2.

As shown in **Figure 4**, cases with substantial eNOS and endothelin‐1 depression during the perinatal period exhibited more profound neurodevelopmental delay and cerebral palsy. In contrast, when eNOS was depressed but vasoconstriction was maintained (i.e., increased endothelin‐1 expression), functional impairments were more moderate, including mild motor and cognitive deviations and minimal brain dysfunction later in life. Therefore, insufficient eNOS activation in combination with the absence of a compensatory mechanism (e.g., peripheral vasospasm and/or the centralization of circulation in vital organs during the early stages of pathology) might ultimately drive the more serious and irreversible injury of brain tissue.

The abovementioned findings are consistent with those of several studies of different NO synthases in the pathogenesis of brain injury. In one study, chronic hypoxia decreased eNOS expression in the hippocampus and increased nNOS expression in neuronal and glial cells of the thalamus [5]. Moreover, in addition to elevated glutamate synthesis, long‐term and severe hypoxemic processes have been reported to alter NO synthase enzyme activity in a manner related to DNA structure, resulting in iNOS and nNOS activation [58, 59]. Wei et al. determined that endothelial NO production by eNOS can decrease ischemic injury by inducing vasodilation, while neuronal NO production can exacerbate neuronal injury [6]. Therefore, several researchers have suggested the potential neuroprotective utility of nNOS inhibitors after brain injury [7, 8].

Many specific biochemical markers of neuronal injury are being investigated as indicators of brain damage in neonates [60]. Some neuron‐specific proteins and cytokines show promise for identifying infants who are at risk for perinatal encephalopathy, although the exact value of these markers for predicting severe brain damage and neurodevelopmental disorders remains controversial [61, 62]. The early assessment of acute cerebral lesions in preterm infants may provide useful information regarding appropriate therapeutic intervention strategies and allow the prevention of future neurological complications. One possibility is that the severity of brain injury in newborns can be assessed by measuring the activity of NO synthases. Future studies are required to validate this hypothesis and better elucidate the clinical significance of NO synthesis in perinatal injury.

#### **4. Conclusion**

**Figure 4.** Peripheral vasoregulatory markers in the early neonatal period in preterm infants with neurodevelopmental disorders (NDs). Error bars indicate the standard error of the mean. Black bars show results from days 1 to 3 and grey bars show results from days 5 to 7. Control 1: data from infants with HIE who did not develop a ND; Control 2: data from

healthy infants. \**p* < 0.05 compared with Control 1; ^*p* < 0.05 compared with Control 2.

30 Nitric Oxide Synthase - Simple Enzyme-Complex Roles

Autoregulation in the neonatal brain is tightly coupled with neuronal and endothelial regulatory mechanisms. Clinical and experimental investigations confirm that neuronal injury is in part mediated by the activation of endothelial and nonendothelial sources of NO synthesis. eNOS activity plays a fundamental role in the autoregulation of vascular tone in the perinatal period and is additionally involved in the formation of hypoxic brain damage during this period; however, the various roles of NO in neuroprotection and metabolism in the brain complicate our exact understanding of the relationship between NO and brain injury. Recent investigations suggest that eNOS plays a protective role in perinatal brain injury whereas other endogenous sources of NO (e.g., iNOS and nNOS) may participate in the pathogenesis of perinatal pathologies and neurodevelopmental disorders. Future studies should further delineate the molecular pathways responsible for the roles of NO synthase isoforms in brain injury and neuroprotection. Finally, the ratio of NO/eNOS expression may indicate the severity of neuronal injury and have clinical utility for predicting long‐term outcomes in infants after perinatal brain injury.

#### **Author details**

Saadat Huseynova<sup>1</sup> \*, Nushaba Panakhova<sup>1</sup> , Safikhan Hasanov<sup>2</sup> and Mehman Guliyev3

\*Address all correspondence to: sadi\_0105@mail.ru


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36 Nitric Oxide Synthase - Simple Enzyme-Complex Roles

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/67267

#### Abstract

Nitric oxide synthase has three isoforms; according to their roles and tissues or cells they are involved. Neuronal NOS (nNOS) takes place in neuronal signalling, endothelial NOS (eNOS) takes place in vasodilation and inducible NOS (iNOS) takes place in immune responses. nNOS and eNOS are dominant but all isoforms have various roles in the central nervous system. nNOS and eNOS separately or together works in healthy brain during cognitive processes and in unhealthy brain during the pathology of related diseases. These roles were shown by inhibitor applied or by transgenic animal model studies and also by investigating the diseases at the molecular level. Besides, it is possible to say that iNOS has roles in some neurological pathologies creating immune responses. Three different isoforms mainly serve in different systems so there are lots of researchers from various disciplines working collaterally not knowing the others related works about NOSs. Because of this, a comprehensive chapter will be valuable for neuroscientists working with either healthy or unhealthy brains. The purpose of this chapter is to gather an overview of NOSs duties during the normal processes of the brain like learning and memory formation and abnormal processes such as depression, schizophrenia and brain cancers.

Keywords: NOS, learning, depression, brain cancers

#### 1. Introduction

Nitric oxide (NO) also known as nitrogen oxide or nitrogen monoxide is a small molecule, which is a gaseous secondary messenger in mammalian cells [1]. Since the early 1990s, the importance of that molecule for biological systems has been investigated by various branches of related fields. Robert F. Furchgott, Louis J. Ignarro and Farid Murad earned a Nobel Prize in physiology or medicine in the year 1998 about the findings of the signalling properties of NO in cardiovascular systems [2]. Thus, the importance of that molecule for biological systems was emphasized.

© 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 pathways that create NO in an organism can differ from system to system and tissue to tissue. Inside the mammalian cells NO is produced as a co-product of a biochemical activity catalysed by the nitric oxide synthase (NOS) enzymes. NOS enzymes are flavoenzymes that contain iron-heme, and these enzymes need nicotinamide-adenine-dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN) and (6R-)5, 6, 7, 8 tetrahydro-L-biopterin (BH4) as cofactors to convert substrate L-arginine to L-citrulline. During that reaction NO is formed. As a water-soluble gas, NO can easily diffuse to neighbouring cells; however, the diffusion is limited because of the short half-life of NO [3].

NOS enzyme has three isoforms, two of them are constitutive isoforms: endothelial NOS (eNOS/NOS3) essentially found in the vascular system and neuronal NOS (nNOS/NOS1) essentially found in the nervous system. The third isoform is inducible isoform: inducible NOS (iNOS/NOS2) principally found in immune system cells [4]. NOSs are homodimeric enzymes and for NO producing reactions NOSs transfer electrons from NADPH to heme via FAD and FMN in the amino terminal. The oxygenase domain of the enzyme also binds the BH4, O2 and L-arginine. L-arginine is oxidized to L-citrullin and NO. All three isoforms bind calmodulin, which work as a molecular switch for NOSs [5].

eNOS is one of the constitutive isoforms, which is generally present in the vascular endothelial cells. eNOS is dominantly expressed by endothelial cells and expressed in little amounts by some other cardiovascular system cells such as cardiomyocytes, erythrocytes, leucocytes, platelets and microparticles in the blood. Ca2+ concentration is an important factor for eNOS activation, also haemodynamic forces, hypoxia, catecholamines, exercise, G-protein activation and post-translational modifications activate eNOS. To response these various stimulants, eNOSs sometimes gather in special sections inside cells, these special sections have caveolinbinding activity. These various stimulant response adaptations make eNOS differ from the other isoforms. Because the other two isoforms are regulated by a smaller number of factors [6], NO is very important to keep the vascular homeostasis at a balance. NO synthesized freshly, travels to the neighbouring cells. Then in the vessel walls, inside the cells, NO binds to guanylate cyclase (GC), thus cyclic guanosine monophosphate (cGMP) concentration increase, Ca2+ channels start to open and when calcium inflows the smooth muscle relaxation is triggered [7].

The three isoforms have small differences between each other. nNOS was first identified in brain and have the biggest molecular weight than the other isoforms with 160 kDa, and eNOS and iNOS having 133 and 131 kDa, respectively. The difference is caused by PDZ domain of nNOS, and caveolin-binding site of eNOS [8]. nNOS is mainly expressed in nervous system cells and is very important for neural functions. The primary nNOS expressing cells are neurons in the brain and also neurons of hypothalamus, pineal gland, spinal cord and nerves innervating other organs. Besides nNOS is also expressed in myocytes, epithelial cells, macula densa cells, testis-urethra cells, mast cells and neutrophils of various mammals [9]. nNOS is also Ca2+-calmodulin dependent, and the activation of nNOS is also regulated by the phosphorylation and neurotransmitter activity. N-methyl-D-aspartate (NMDA) receptors have the key role for nNOS activation in neurons. Sometimes nNOS activation is triggered by presynaptic neurons; glutamate released from the presynaptic neuron increases the Ca2+ in the post-synaptic neuron through NMDA receptor, which increased Ca2+ activating the nNOS in the post-synaptic neuron. Then nNOS produce NO, which diffuses back to the presynaptic neuron and triggers soluble GC [4, 10].

The pathways that create NO in an organism can differ from system to system and tissue to tissue. Inside the mammalian cells NO is produced as a co-product of a biochemical activity catalysed by the nitric oxide synthase (NOS) enzymes. NOS enzymes are flavoenzymes that contain iron-heme, and these enzymes need nicotinamide-adenine-dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN) and (6R-)5, 6, 7, 8 tetrahydro-L-biopterin (BH4) as cofactors to convert substrate L-arginine to L-citrulline. During that reaction NO is formed. As a water-soluble gas, NO can easily diffuse to neighbouring cells;

NOS enzyme has three isoforms, two of them are constitutive isoforms: endothelial NOS (eNOS/NOS3) essentially found in the vascular system and neuronal NOS (nNOS/NOS1) essentially found in the nervous system. The third isoform is inducible isoform: inducible NOS (iNOS/NOS2) principally found in immune system cells [4]. NOSs are homodimeric enzymes and for NO producing reactions NOSs transfer electrons from NADPH to heme via FAD and FMN in the amino terminal. The oxygenase domain of the enzyme also binds the BH4, O2 and L-arginine. L-arginine is oxidized to L-citrullin and NO. All three isoforms bind

eNOS is one of the constitutive isoforms, which is generally present in the vascular endothelial cells. eNOS is dominantly expressed by endothelial cells and expressed in little amounts by some other cardiovascular system cells such as cardiomyocytes, erythrocytes, leucocytes, platelets and microparticles in the blood. Ca2+ concentration is an important factor for eNOS activation, also haemodynamic forces, hypoxia, catecholamines, exercise, G-protein activation and post-translational modifications activate eNOS. To response these various stimulants, eNOSs sometimes gather in special sections inside cells, these special sections have caveolinbinding activity. These various stimulant response adaptations make eNOS differ from the other isoforms. Because the other two isoforms are regulated by a smaller number of factors [6], NO is very important to keep the vascular homeostasis at a balance. NO synthesized freshly, travels to the neighbouring cells. Then in the vessel walls, inside the cells, NO binds to guanylate cyclase (GC), thus cyclic guanosine monophosphate (cGMP) concentration increase, Ca2+ channels start to open and when calcium inflows the smooth muscle relaxation

The three isoforms have small differences between each other. nNOS was first identified in brain and have the biggest molecular weight than the other isoforms with 160 kDa, and eNOS and iNOS having 133 and 131 kDa, respectively. The difference is caused by PDZ domain of nNOS, and caveolin-binding site of eNOS [8]. nNOS is mainly expressed in nervous system cells and is very important for neural functions. The primary nNOS expressing cells are neurons in the brain and also neurons of hypothalamus, pineal gland, spinal cord and nerves innervating other organs. Besides nNOS is also expressed in myocytes, epithelial cells, macula densa cells, testis-urethra cells, mast cells and neutrophils of various mammals [9]. nNOS is also Ca2+-calmodulin dependent, and the activation of nNOS is also regulated by the phosphorylation and neurotransmitter activity. N-methyl-D-aspartate (NMDA) receptors have the key role for nNOS activation in neurons. Sometimes nNOS activation is triggered by

however, the diffusion is limited because of the short half-life of NO [3].

38 Nitric Oxide Synthase - Simple Enzyme-Complex Roles

calmodulin, which work as a molecular switch for NOSs [5].

is triggered [7].

There are several answers to the question why NO is important for the brain. That freely diffusible gaseous secondary messenger can be a lifesaver or villain for neurological processes [11]. Thus, nNOS became as important as NO in the brain. nNOS plays an important role in neuronal function, memory formation, sexually different behaviours, neurological regulations and for therapeutics.

nNOS in the whole brain and additionally eNOS in the hippocampus and somatosensory cortex are involved in NO and cGMP formation. These secondary messengers activate synaptic plasticity in the hippocampus, cerebral cortex, amygdala, striatum and cerebellum [12]. In several murine, nNOS inhibition studies and an nNOS knockout mice study revealed that it is crucial for memory formation and recognition of memory [13].

There are lots of studies that try to explain the link between nNOS and behaviour at the molecular level. Sex difference is a very important variable for behavioural studies. There is a significant difference between male and female mice according to their aggressive behaviour if their nNOS gene were knocked-out. Besides in a NOS inhibition study, it was shown that male rats tended to show female-like perceptual style behaviour [14, 15]. It is possible to say that NO and nNOS are important molecules for sex difference dependent behavioural studies.

There are lots of inhibitors for NOSs; arginine-like chemicals bind NOSs instead of L-arginine to inhibit the reaction catalysed by NOSs. Most widely used inhibitors are nitro-L-arginine (L-NA), nitro-L-arginine methyl ester (L-NAME) and N-monomethyl L-arginine (L-NMMA). These inhibitors are not selective for a specific isoform, but there are also selective inhibitors available. The non-selective inhibitors are important as selective ones. These non-selective inhibitors helped to suppress both eNOS and nNOS, because both isoforms take place in regulating vascular activities in the brain. With that inhibitors it was shown that NO/NOS pathway is very important for regulating the cerebral blood flow within the healthy brain, and also very important for the ischaemic brain. The studies done with the ischaemic brain have revealed that NO is also a double-edged sword for that pathology. Scientists trying to find a treatment for the ischaemic brain over NO pathway should consider the risk of manipulating that pathway [16].

There have been some other studies on NO/nNOS pathway for therapeutic concerns in the ischaemic brain. Inhibiting or activating nNOS is a point of view where scientists are dealing with the internal sources of the organism. However, there is another option: to supply NO from outside of the organism. There are some studies that supply NO to the organism by inhalation to treat injured brains. According to the results of these studies, the endogenous sources are generally a better target to manipulate. But for the ischaemic brain, the external NO supply is useful at the ischaemia phase, not in the reperfusion phase. It is possible to interpret that it is not easy to separate the ischaemic and reperfusion phases if it is not a controlled operation. Therefore, external NO inhalation seems not efficient for ischaemic conditions [17].

There are lots of therapeutic strategies to overcome different pathologies involving NOSs. But before developing a technique or a new technology for treatment, the molecular mechanisms behind the normal conditions and/or pathological conditions must be exhibited thoroughly to avoid the devastating side effects of the potential therapy.

The only inducible isoform iNOS is not readily expressed inside the cells. This isoform is mostly expressed in macrophages when there is a pathogenic condition for host defence. There are some differences between constitutive and inducible forms. iNOSs are not Ca2+ dependent, also when induced they produce NO continuously, they are affected by glucocorticoids and less labile. The action of iNOS enzyme starts with binding of cytokines and/or lipopolysaccharides to cell surface receptors instead of calcium influx. Then the similar reaction occurs to produce NO. For stopping the activity, glucocorticoids bind the secondary messengers of cytokine-triggered cascade [18]. The stimuli activating the mice iNOS are not activating the human iNOS [19]. This situation should mislead the researchers while developing a therapeutic strategy. It is more serious if the strategy will be depending on only the animal experiment results.

#### 2. Roles of nitric oxide synthase enzymes in healthy brain

It is important to put forth the molecular mechanism of a molecule for normal conditions. Under this heading, it was aimed to introduce and discuss the roles of NOS enzymes in the healthy brain functions. NOS enzymes have several roles in the healthy brain; especially NO is crucial for learning and memory formation. Besides, NOS enzymes have roles in retinal function, hearing and molecular mechanisms in the cochlea.

#### 2.1. Roles of NOSs in learning and memory formation

One of the main duties of NOS enzymes in the healthy brain is learning and memory formation. Learning is a complex behaviour but the molecular mechanisms of memory formation are mostly enlightened. During the second half of the 1990s, scientists proposed that NO has a role in learning and memory and in neuronal plasticity. Experiments were designed to look for the role of NO/NOSs in learning. NO has a part in both long-term potentiations (LTP) in the hippocampus and long-term depression (LTD) in cerebellum, which are basic mechanisms for memory formation [20].

Neuron-neuron interaction isles called synapses are very plastic; LTP is a form of that plasticity. LTP occur due to the repetitive stimulation of the presynaptic neuron. A consequence of that repetitive stimulation is the influx of calcium to post-synaptic neuron via NMDA receptor, which in turn increases the Ca2+ concentration in the cell. For completing the circuit of LTP, there should be a retrograde messenger. Studies done with knockout mice and inhibitors, revealed that NO is that retrograde messenger for LTP in the hippocampus. But both nNOS and eNOS take place to from LTP [21]. Selective inhibitors for nNOS also showed that there is a deficiency in memory formation in various vertebrates [22]. Not only NO/NOS pathway studies showed the initiation of NO in LTP, but also studies looking for the sGC activity in rat hippocampus support the findings of NO/NOS and LTP correlation [23]. LTD occurs in cerebellar purkinje cells. There are two cells docking each other, climbing fibres and purkinje cells. Also, interneurons are neighbouring that synapse. NO released from interneurons and diffuse to the purkinje cells, then sGC initiates in LTD formation with similar action of LTP but in opposite direction [24, 25].

For memory formation by NOS there is also other signalling molecules in the cascade, like extracellular signal-regulated kinase (ERK). Thus, memory formation processes become more complex [26]. In the stress-exposed rats' hippocampi nNOS activity was diminished. This stress causes a deficit in learning and memory processes in these stressed animals. Same learning and memory problems have seen in hypoxic/ischaemic hippocampi of rats [27].

Within memory-forming circuits between neurons, NO can act as a volume transmitter. Thus, that small molecule can affect the remote parts of the brain at the same time. Also for conceptual learning studies done with invertebrates, it is revealed that indirectly NO/NOS mechanisms take place in learning [28]. It was shown that NOS inhibitors hinder motor learning in adult animals, and the formation of olfactory memories. The motor learning malfunction may arise from the non-selective inhibitors [29, 30]. These are some results explaining the roles of NO/NOS pathway in memory formation.

#### 2.2. Roles of NOSs in seeing and retinal function

that it is not easy to separate the ischaemic and reperfusion phases if it is not a controlled operation. Therefore, external NO inhalation seems not efficient for ischaemic conditions [17]. There are lots of therapeutic strategies to overcome different pathologies involving NOSs. But before developing a technique or a new technology for treatment, the molecular mechanisms behind the normal conditions and/or pathological conditions must be exhibited thoroughly to

The only inducible isoform iNOS is not readily expressed inside the cells. This isoform is mostly expressed in macrophages when there is a pathogenic condition for host defence. There are some differences between constitutive and inducible forms. iNOSs are not Ca2+ dependent, also when induced they produce NO continuously, they are affected by glucocorticoids and less labile. The action of iNOS enzyme starts with binding of cytokines and/or lipopolysaccharides to cell surface receptors instead of calcium influx. Then the similar reaction occurs to produce NO. For stopping the activity, glucocorticoids bind the secondary messengers of cytokine-triggered cascade [18]. The stimuli activating the mice iNOS are not activating the human iNOS [19]. This situation should mislead the researchers while developing a therapeutic strategy. It is more serious if the strategy will be depending on only the animal experiment

It is important to put forth the molecular mechanism of a molecule for normal conditions. Under this heading, it was aimed to introduce and discuss the roles of NOS enzymes in the healthy brain functions. NOS enzymes have several roles in the healthy brain; especially NO is crucial for learning and memory formation. Besides, NOS enzymes have roles in retinal

One of the main duties of NOS enzymes in the healthy brain is learning and memory formation. Learning is a complex behaviour but the molecular mechanisms of memory formation are mostly enlightened. During the second half of the 1990s, scientists proposed that NO has a role in learning and memory and in neuronal plasticity. Experiments were designed to look for the role of NO/NOSs in learning. NO has a part in both long-term potentiations (LTP) in the hippocampus and long-term depression (LTD) in cerebellum, which are basic mechanisms for

Neuron-neuron interaction isles called synapses are very plastic; LTP is a form of that plasticity. LTP occur due to the repetitive stimulation of the presynaptic neuron. A consequence of that repetitive stimulation is the influx of calcium to post-synaptic neuron via NMDA receptor, which in turn increases the Ca2+ concentration in the cell. For completing the circuit of LTP, there should be a retrograde messenger. Studies done with knockout mice and inhibitors, revealed that NO is that retrograde messenger for LTP in the hippocampus. But both nNOS and eNOS take place to from LTP [21]. Selective inhibitors for nNOS also showed that there is a

avoid the devastating side effects of the potential therapy.

40 Nitric Oxide Synthase - Simple Enzyme-Complex Roles

2. Roles of nitric oxide synthase enzymes in healthy brain

function, hearing and molecular mechanisms in the cochlea.

2.1. Roles of NOSs in learning and memory formation

results.

memory formation [20].

Retinal function is very important for developed organisms, and retinal function has various molecular pathways. Also, NO/NOS mechanism is one of the regulators of action of seeing and functioning of the retina. Seeing is a complex process that takes place in visual cortex but the retinal function is essential to carry the visual signals to that processor area of the brain.

In the retina, NOS is found in retinal neurons, pigment epithelium, amacrine and ganglion cells, nerve fibres in the outer and inner plexiform layers and in photoreceptor ellipsoids. But in normal rats' optic nerve, there is not any NOS enzyme present [31]. In photoreceptor cells, ion channels activate the reaction. In the inner segment, Ca2+ concentration increases, then activate the nNOS and thus NO produced. That NO activates the sGC in the same cell not inside the other cells. Then GC pathway will be triggered in on-bipolar cells. Besides in amacrine cell-ganglion cell-bipolar cell synapse; NO produced in amacrine cell, diffuse to the ganglion cell and activate the GC and this cause depolarization [31, 32]. Depolarization in the retina is an essential step for seeing.

There are some studies ranging from developmental biology to animal behaviour that explored the roles of NO/NOS pathway in the retinal function. In the developing human foetal eye nNOS and eNOS are expressed at the same time but in different compartments. Besides, nuclear nNOS was found in progenitor cells, endothelial cells and pericytes. Because of this situation, nNOS may have a transcription regulatory role for some cells during ocular vasculogenesis and angiogenesis [33]. In a study an inhibitor was used which selectively triggered photoreceptor cell death to determine the role of NO in retinal degeneration.

Scientists found that there is a correlation between the increasing NO levels and nNOS activity and mouse retinal cell death [34]. Chick retinas removed during a study revealed the role of NO/NOS mechanism in the brain. The group looked for the expression of NOSs in visual structures of the brain. The NOSs expressions increased after retinal removal. This shows that the NO/NOS mechanism has a role in plasticity processes in visual parts of the chick brain [35].

To unveil the role of NO in optic nerve head blood flow, scientists conducted a NOS inhibitor study in healthy humans. Subjects received L-NMMA and performed isometric exercise during the study. They proposed that NO has an important role in basal optic nerve head blood flow but not in autoregulatory response induced by exercise [36].

In cultured retinal neurons, NO inhibit apoptosis via activating various kinases. In cultured chick embryonic retinal neurons, both endogenous and exogenous NO promoted AKT signalling pathway and probably survival mechanisms [37]. In goldfish optic nerve, scientists showed that NO signalling pathway through nNOS activation has a crucial role in nerve regeneration [38].

In a knockout mice study, roles of all three isoforms were investigated comparatively. It was shown that not having one of the three isoforms did not alter the intraocular pressure or number of neurons in the eye. But eNOS is crucial for endothelium-dependent dilation of murine eye arteries. In conclusion, NOSs are involved in the regulation of ocular vascular tone and blood flow [39].

#### 2.3. Roles of NOSs in hearing and cochlea

Hearing starts at the outer ear and stimuli travels through tympanum and bones and finally arrives at the cochlea, where hair cells and nerve fibres take action. NO and NOSs have roles in hearing function and cochlear activities. And hearing process happens in the auditory cortex.

NO act as a neurotransmitter and/or neuromodulator in the cochlea [40, 41]. In recent years, it was revealed that NO has also a potassium channel modulator role in inner hair cells. Therefore, NO-potassium modulation may be responsible for high-frequency hearing impairment [42].

NO/cGMP pathway was triggered by nerve fibres innervating outer hair cells, NO was produced in these cells and released. But NO affects Deiters' cells and Heusen's cells and not the outer hair cells. Also, nNOS takes place in acoustic overstimulation condition. Inner hair cells release excess glutamate during continuous stimulation. This glutamate increase calcium influx to afferent dendrites where nNOS produce NO. Then overproduction of NO due to acoustic overstimulation kills afferent dendrites because of excitotoxicity [43]. Auditory nerve, lateral wall, vestibule and cochlear neuroepithelium are the areas where NOS activity is the highest in the auditory system. nNOS is the predominant isoform in the cochlea [44].

In a gerbil study, researchers examined the role of NO in cochlelar excitotoxicity. Cochlear compound action potentials thresholds were recorded with NOS inhibitor and glutamate exposed conditions. Overstimulation with glutamate caused NO-mediated excitotoxicity in the cochlea. NOS inhibition should be neuroprotective for cochlea [45].

To trigger iNOS expression in cochlea, bacterial lipopolysaccharides and tumour necrosis factor α was injected to guinea pigs. The iNOS expression was higher than eNOS and nNOS during that experiment. iNOS were localized in the cochlea's blood vessel walls of the spiral ligament and the modiolus, in the organ of Corti, in the limbus, in nerve fibres and in spiral ganglion [46]. This dispersed iNOS is caused by the bacterial lipopolysaccharide-triggered immune response. In another study with immonostaining data, the distribution of NOSs was determined in the auditory system. nNOS was dispersed in the inner and outer hair cells, spiral ganglion cells, cells of the stria vascularis, spiral ligament cells and vessel cells near the modiolus. eNOS was dispersed in vascular endothelial cells, and in spiral ganglion cells. If there were not immune stimulus there would be no iNOS in cochlea [47].

#### 3. Roles of nitric oxide synthase in unhealthy brain

NOS enzymes have important duties during pathophysiology of unhealthy brains. Unlike healthy brains, the roles not only depend on signal transduction, but also on anabolic/catabolic mechanisms. Under that heading various diseases, pathologies, malfunctions and disabilities of brains will be evaluated from the point of view of NOSs.

#### 3.1. Roles of NOSs in neuropsychiatric diseases

Scientists found that there is a correlation between the increasing NO levels and nNOS activity and mouse retinal cell death [34]. Chick retinas removed during a study revealed the role of NO/NOS mechanism in the brain. The group looked for the expression of NOSs in visual structures of the brain. The NOSs expressions increased after retinal removal. This shows that the NO/NOS mechanism has a role in plasticity processes in visual parts of the chick brain [35]. To unveil the role of NO in optic nerve head blood flow, scientists conducted a NOS inhibitor study in healthy humans. Subjects received L-NMMA and performed isometric exercise during the study. They proposed that NO has an important role in basal optic nerve head blood

In cultured retinal neurons, NO inhibit apoptosis via activating various kinases. In cultured chick embryonic retinal neurons, both endogenous and exogenous NO promoted AKT signalling pathway and probably survival mechanisms [37]. In goldfish optic nerve, scientists showed that NO signalling pathway through nNOS activation has a crucial role in nerve

In a knockout mice study, roles of all three isoforms were investigated comparatively. It was shown that not having one of the three isoforms did not alter the intraocular pressure or number of neurons in the eye. But eNOS is crucial for endothelium-dependent dilation of murine eye arteries. In conclusion, NOSs are involved in the regulation of ocular vascular tone

Hearing starts at the outer ear and stimuli travels through tympanum and bones and finally arrives at the cochlea, where hair cells and nerve fibres take action. NO and NOSs have roles in hearing function and cochlear activities. And hearing process happens in the auditory cortex. NO act as a neurotransmitter and/or neuromodulator in the cochlea [40, 41]. In recent years, it was revealed that NO has also a potassium channel modulator role in inner hair cells. Therefore, NO-potassium modulation may be responsible for high-frequency hearing impairment

NO/cGMP pathway was triggered by nerve fibres innervating outer hair cells, NO was produced in these cells and released. But NO affects Deiters' cells and Heusen's cells and not the outer hair cells. Also, nNOS takes place in acoustic overstimulation condition. Inner hair cells release excess glutamate during continuous stimulation. This glutamate increase calcium influx to afferent dendrites where nNOS produce NO. Then overproduction of NO due to acoustic overstimulation kills afferent dendrites because of excitotoxicity [43]. Auditory nerve, lateral wall, vestibule and cochlear neuroepithelium are the areas where NOS activity is the

In a gerbil study, researchers examined the role of NO in cochlelar excitotoxicity. Cochlear compound action potentials thresholds were recorded with NOS inhibitor and glutamate exposed conditions. Overstimulation with glutamate caused NO-mediated excitotoxicity in

highest in the auditory system. nNOS is the predominant isoform in the cochlea [44].

the cochlea. NOS inhibition should be neuroprotective for cochlea [45].

flow but not in autoregulatory response induced by exercise [36].

regeneration [38].

and blood flow [39].

[42].

2.3. Roles of NOSs in hearing and cochlea

42 Nitric Oxide Synthase - Simple Enzyme-Complex Roles

It is very hard to diagnose the neuropsychiatric diseases properly; however, there are globally accepted parameters. Although the criteria for diagnosis is generally evaluated at certain times by prestigious committees and there is still hardships for diagnosis. One of the main goals of the scientists working on neuropsychiatric disorders is to pair a marker molecule with a disease to facilitate the diagnosis. So far there is not any coupling for any NOSs for any neuropsychiatric disorder as a marker but NOSs have various roles for these diseases. Anxiety, depression/major depression and tendency for suicide are important and common neuropsychiatric disorders. NOS has roles for these abnormalities.

In patients with depression it was shown that NO expression altered via eNOS. Besides NO modulates neuropeptides, such as vasopressin, oxytocin and corticotrophin-releasing factor. In patients with depression, these neuropeptides' expression levels were altered. According to post-mortem studies on depression patients, NO signalling was impaired in their hypothalami [48].

Major depression disorder (MDD) will be one of the dominant causes of disability by the year 2020. Antidepressants generally decrease NO levels and/or inhibit NOS activity indirectly. Also NO levels and NOS expressions are higher in MDD patients. Besides, with mice studies, it was shown that there is a correlation between NOS mechanism and depression. NOS inhibitors could be researched as a new target for antidepressant strategies [4, 22].

In a population study done in Taiwan with MDD patients in which the potential genetic variations with healthy and MDD subjects according to their nNOS polymorphisms was researched. There is no difference between subjects; the frequencies are similar for controls and MDD patients [49]. In an autopsy and tissue bank-based study from Holland, there is decrease in nNOS expression in the anterior cingulate cortex of MDD-diagnosed people [50].

In mice along with stress-induced depression, nNOS expression increases in the hippocampi. Due to excitotoxicity neurogenesis in hippocampi is suppressed. To inhibit nNOS signalling may be a novel approach for depression treatment [51]. Also, iNOS is involved in stresstriggered depression. NO derived from iNOS and mRNA levels of iNOS increased in cortices of depression model applied mice [52].

Also in a population study there is no correlation between genetic polymorphisms and MDD. In Japanese population MDD patients were investigated for polymorphisms in their nNOS genes, but found no variation between controls and MDD [53]. From a Czech Republic population genetic study, there is also no correlation between eNOS and MDD [54]. A population study from United Kingdom found a correlation between single nucleotide polymorphisms in nNOS gene and psychosocial stress-triggered depression. The individuals carrying those polymorphisms have a tendency to develop depression if they face financial hardship [55].

There is a link between vascular problems, depressive behaviours and NO metabolism. When vascular dysfunction emerges after depressive symptoms, the characteristics behind it show lack of bioavailable NO. However, H2O2 covers up that deficit [56].

There are studies on ethnopharmacological level to find out if there is a potential drug candidate in botanical material. To investigate NO metabolism is one of the target pathways to detect for antidepressant-like and neuroprotective potential. Aloysia grattissima has the potential to treat depressive disorders depending on the NO metabolism manipulating properties of its extracts [57].

nNOS genes' functional promoter repeat length variant contains sites for transcription factors that has strong relation with hyperactive and aggressive behaviour. Thus, nNOS depending on population genetics studies combined with behaviour is a potential research area [58].

For anxiety-like and depression-like behaviours, there is a strong evidential pathway, hypothalamic-pituitary-adrenal axis (HPA). Also on the ecotoxicological aspect, the nutrients for newborns and expectant mothers are very important because they fall in the risk group. The bisphenol-A supplied pregnant female rats' male littermates revealed anxiety-like and depression-like behaviours according to their HPA experiment results. Those littermates' hippocampal nNOS activity was higher than the control animals [59]. nNOS knockout mice show abnormal social behaviour, hyperactivity and impaired remote spatial memory [60].

It is important to demonstrate how nitric oxide synthases are affected in the brain by psychotropic drugs. Orally treated rats with several psychotropics were sacrificed and iNOS gene expressions in the brain were detected. Psychotropics including antidepressants and anxiolytics modulate the gene expression of iNOS in rat brain [61].

It is a complex and controversial psychological situation: suicide. This behaviour has a strong genetic background. In a study it was shown that a single nucleotide polymorphism of nNOS gene has a correlation between suicides in Japanese population, especially in males [62].

Schizophrenia is a complex illness including biochemical, anatomical and genetic aspects of its pathology. In a post-mortem study, scientists showed that some polymorphic variants of nNOS have overexpression patterns in schizophrenic patients' brains [63].

#### 3.2. Roles of NOSs in neurodegenerative diseases

and MDD patients [49]. In an autopsy and tissue bank-based study from Holland, there is decrease in nNOS expression in the anterior cingulate cortex of MDD-diagnosed people [50].

In mice along with stress-induced depression, nNOS expression increases in the hippocampi. Due to excitotoxicity neurogenesis in hippocampi is suppressed. To inhibit nNOS signalling may be a novel approach for depression treatment [51]. Also, iNOS is involved in stresstriggered depression. NO derived from iNOS and mRNA levels of iNOS increased in cortices

Also in a population study there is no correlation between genetic polymorphisms and MDD. In Japanese population MDD patients were investigated for polymorphisms in their nNOS genes, but found no variation between controls and MDD [53]. From a Czech Republic population genetic study, there is also no correlation between eNOS and MDD [54]. A population study from United Kingdom found a correlation between single nucleotide polymorphisms in nNOS gene and psychosocial stress-triggered depression. The individuals carrying those poly-

There is a link between vascular problems, depressive behaviours and NO metabolism. When vascular dysfunction emerges after depressive symptoms, the characteristics behind it show

There are studies on ethnopharmacological level to find out if there is a potential drug candidate in botanical material. To investigate NO metabolism is one of the target pathways to detect for antidepressant-like and neuroprotective potential. Aloysia grattissima has the potential to treat depressive disorders depending on the NO metabolism manipulating properties of

nNOS genes' functional promoter repeat length variant contains sites for transcription factors that has strong relation with hyperactive and aggressive behaviour. Thus, nNOS depending on

For anxiety-like and depression-like behaviours, there is a strong evidential pathway, hypothalamic-pituitary-adrenal axis (HPA). Also on the ecotoxicological aspect, the nutrients for newborns and expectant mothers are very important because they fall in the risk group. The bisphenol-A supplied pregnant female rats' male littermates revealed anxiety-like and depression-like behaviours according to their HPA experiment results. Those littermates' hippocampal nNOS activity was higher than the control animals [59]. nNOS knockout mice show

It is important to demonstrate how nitric oxide synthases are affected in the brain by psychotropic drugs. Orally treated rats with several psychotropics were sacrificed and iNOS gene expressions in the brain were detected. Psychotropics including antidepressants and anxio-

It is a complex and controversial psychological situation: suicide. This behaviour has a strong genetic background. In a study it was shown that a single nucleotide polymorphism of nNOS gene has a correlation between suicides in Japanese population, especially in males [62].

population genetics studies combined with behaviour is a potential research area [58].

abnormal social behaviour, hyperactivity and impaired remote spatial memory [60].

lytics modulate the gene expression of iNOS in rat brain [61].

morphisms have a tendency to develop depression if they face financial hardship [55].

lack of bioavailable NO. However, H2O2 covers up that deficit [56].

of depression model applied mice [52].

44 Nitric Oxide Synthase - Simple Enzyme-Complex Roles

its extracts [57].

Alois Alzheimer defined the illness during the early 1900s. Alzheimer's disease (AD) is the most common type of dementia. It generally affects the elderly people and is characterized by aggregating senile plaques and/or neurofibrillary tangles in the brain, which leads to progressive neuronal degeneration and death [64]. Deficits in short-term memory formation are characteristic for AD. Short-term memory formation through LTP is dependent on the NO/ NOS pathway. NOSs are very important for creating the new trails between neurons. NOSs take place for activating the presynaptic neurons receptors. However, in the pathology of the AD the harmonization created by NOSs between neurons is blocked by plaques [20–22, 64].

In the brain of an AD patient, β-amyloid peptide aggregates in senile plaques and the arginine within the astrocytes accumulates. These are the classical neuropathology of the disease. Arginine-metabolizing enzymes like NOSs and their association with amyloid peptides are important. The correlation of Aβ-peptide fragments with nNOS has been searched with spectrofluorimetry. The interaction of Aβ-peptide with nNOS causes the molecular movement of two critical tryptophan residues in the structure of the enzyme [65].

Purified nNOS was incubated with Aβ-peptide fragments during 96 hours. The kinetics of the interaction was introduced; nNOS was the amyloidogenic catalyst and all Aβ-peptide fragments were inhibited nNOS [66]. Data from cell culture studies, knockout mice studies and behavioural studies showed that eNOS has a crucial role for decelerating the pathology of AD [67]. If patients with AD start to exercise, they start to increase heart rate, cerebral blood flow and then angiogenesis, which includes NO/eNOS pathway, after which neurogenesis and other self-healing mechanisms are activated [68].

James Parkinson described the disease during the 1810s. Besides the characteristic shakes and tremors, there is a huge molecular mechanism behind Parkinson's disease (PD). The disease is diagnosed generally between 50 and 70 years old people. The mechanism behind PD is still unknown. But the dopamine metabolism decreases significantly in PD; also substantia nigra is one of the potential areas of interest to study the disease.

PD is an illness affecting the dopaminergic pathway in the brain. NO inflict the injuries in dopaminergic neurons. There are several evidences from NOS inhibitor studies about this correlation. However, if a cell goes for the cell death pathway, NO accelerates the process [69]. Some neuroinflammatory responses associated with PD are arousing from NO/iNOS activity. Nitration of α-synuclein triggers the protein aggregation, which worsens the pathology. This mechanism is used to mimic the PD in cell culture [70].

Scientists are trying to create a thorough model of the disease for in vivo or in vitro, however, so far there is not a completely satisfying model. That is caused by the unknown mechanism behind the illness. To mimic PD a group of researchers castrated the male mice. They followed the NO/iNOS mechanisms to test their model. According to the iNOS results, the castration of young male mice induces PD pathologies [71].

During PD pathology, several reasons cause cell death in substantia nigra neurons and/or dopaminergic neurons. Nutrition and false diet should be a cause of the disease. As a strategy to add an antioxidant-rich nutritive to the diet may be beneficial. Rats were supplied with pomegranate juice after a PD model. The change in the diet by adding pomegranate juice enhanced the iNOS expression in the animal brain [72].

#### 3.3. Roles of NOSs in brain cancers

There are lots of cancer types present in the head and neck area. But in this section, only cancers originating from cells inside the cranium will be discussed and not the metastatic pathologies.

There are lots of studies done with cultured cancer cells from various mammals; however, studies done with tumour samples from human are rare. Instead of discussing the cell culture studies, it was important to gather the knowledge, which was hard to reach. Most of cancer cell culture studies are done without the healthy control cell lines or experimental models. But some cancer cell culture studies will be discussed.

A biopsy study done with gliomas, and also with meningiomas, showed that all three NOS isoforms were present in the aforementioned tumours. nNOS was significantly dominant in glial cells of gliomas. However, that NOS dense status becomes sparse in the peritumoral tissues [73]. NOSs of tumour cells, opposite to the healthy cells, synthesize predominantly superoxide and peroxynitrite, which generate oxidative stress [74].

Neuroblastoma cell lines are generally used due to their ability to differentiate neuron-like cells. NOS inhibition in Neuro2a cells blocked that differentiation; it is possible to speculate that nNOS may have important roles for dissolving a neuroblastoma tumour [75].

Astrocytomas/gliomas are the origins of cancers from supporting cells of the nervous system. These types of cancers are dominant and dangerous when compared to other brain cancers. In a human biopsy study, it was shown that iNOS has a role in angiogenesis via vascular endothelial growth factor (VEGF), and there is a correlation with iNOS and VEGF for astrocytomas/gliomas but not for reactive astrogliosis samples [76]. Similar results were reported also from a similar study. iNOS expression was increased in grade I, II and III astrocytic gliomas. However, in the same study it was shown that the iNOS expression was decreased for grade IV astrocytic gliomas [77]. In a study with primary astrocytoma biopsy samples prove that eNOS and VEGF work cooperatively in tumour angiogenesis [78]. Besides, in another study, it was shown that astrocytic tumour vessels have more eNOS expression than normal vessels [79]. Also, it is known that nNOS expression increases in glioma tumours.

Craniopharyngiomas consist of the 2–5% of intracranial tumours. In a study done on rats searched for the immune responses of oily cyst content of human craniopharyngioma. Immunohistochemical studies after injection revealed that eNOS expression increased in a time course manner [80].

Medulloblastomas are highly malignant brain tumours generally affecting children and adolescents. In a study done with medulloblastoma cells revealed that NOS has important roles in medulloblastoma cell death. Scientists applied various chemotherapy agents and PDE5 inhibitors to kill cells. Then, they co-treated cells with L-NAME and found out that NOS inhibition accompanying PDE5 inhibitors suppressed cell killing. Most probably NO/NOS has a role in killing of medulloblastoma cells [81]. A study done in knockout mice exhibited that iNOS has an important role in medulloblastoma formation. Ptch1 heterozygous and iNOS-deficient mice developed medulloblastoma two times higher than Ptch1 heterozygous and iNOS producing mice. This situation may depend on the granule cell precursors' migration [82].

According to studies done with human ptiutary tumour biopsy samples, scientists tried to reveal the roles of NOSs with the disease. In human pituitary adenomas, eNOS expression increased, whereas iNOS and nNOS were stabile [83]. Another human biopsy study showed that highly invasive adenomas have higher upregulated iNOS, whereas noninvasive adenomas did not have upregulated iNOS. Also, eNOS had upregulation with haemorrhagic adenomas [84].

Schwannomas are benign tumours originating from the Schwann cells. It can arise in any peripheral nerve; however, the frequent version arose around the acoustic nerve. Immunohistochemical study done on human biopsy samples revealed that iNOS has a strong expression for this illness. iNOS was stained around the hyalinized vessels' infiltrating leukocytes in Antoni B areas [85].

In conclusion, it is possible to suggest that both clinical and experimental studies are important on the aspect of NOS and brain cancers. It is very hard to mimic pathologies of some brain cancers both in animals and in vitro. Likewise, collecting clinical samples from patients is very hard. NO/NOS pathway is important for brain cancers and more studies needed to reveal the therapeutic potential.

#### Author details

the NO/iNOS mechanisms to test their model. According to the iNOS results, the castration of

During PD pathology, several reasons cause cell death in substantia nigra neurons and/or dopaminergic neurons. Nutrition and false diet should be a cause of the disease. As a strategy to add an antioxidant-rich nutritive to the diet may be beneficial. Rats were supplied with pomegranate juice after a PD model. The change in the diet by adding pomegranate juice

There are lots of cancer types present in the head and neck area. But in this section, only cancers originating from cells inside the cranium will be discussed and not the metastatic

There are lots of studies done with cultured cancer cells from various mammals; however, studies done with tumour samples from human are rare. Instead of discussing the cell culture studies, it was important to gather the knowledge, which was hard to reach. Most of cancer cell culture studies are done without the healthy control cell lines or experimental models. But

A biopsy study done with gliomas, and also with meningiomas, showed that all three NOS isoforms were present in the aforementioned tumours. nNOS was significantly dominant in glial cells of gliomas. However, that NOS dense status becomes sparse in the peritumoral tissues [73]. NOSs of tumour cells, opposite to the healthy cells, synthesize predominantly

Neuroblastoma cell lines are generally used due to their ability to differentiate neuron-like cells. NOS inhibition in Neuro2a cells blocked that differentiation; it is possible to speculate

Astrocytomas/gliomas are the origins of cancers from supporting cells of the nervous system. These types of cancers are dominant and dangerous when compared to other brain cancers. In a human biopsy study, it was shown that iNOS has a role in angiogenesis via vascular endothelial growth factor (VEGF), and there is a correlation with iNOS and VEGF for astrocytomas/gliomas but not for reactive astrogliosis samples [76]. Similar results were reported also from a similar study. iNOS expression was increased in grade I, II and III astrocytic gliomas. However, in the same study it was shown that the iNOS expression was decreased for grade IV astrocytic gliomas [77]. In a study with primary astrocytoma biopsy samples prove that eNOS and VEGF work cooperatively in tumour angiogenesis [78]. Besides, in another study, it was shown that astrocytic tumour vessels have more eNOS expression than normal vessels [79].

Craniopharyngiomas consist of the 2–5% of intracranial tumours. In a study done on rats searched for the immune responses of oily cyst content of human craniopharyngioma. Immunohistochemical studies after injection revealed that eNOS expression increased in a time

that nNOS may have important roles for dissolving a neuroblastoma tumour [75].

young male mice induces PD pathologies [71].

46 Nitric Oxide Synthase - Simple Enzyme-Complex Roles

3.3. Roles of NOSs in brain cancers

pathologies.

course manner [80].

enhanced the iNOS expression in the animal brain [72].

some cancer cell culture studies will be discussed.

superoxide and peroxynitrite, which generate oxidative stress [74].

Also, it is known that nNOS expression increases in glioma tumours.

Melih Dagdeviren

Address all correspondence to: melih.dagdeviren@ege.edu.tr

Department of Biology, Faculty of Science, Ege University, Izmir, Turkey

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54 Nitric Oxide Synthase - Simple Enzyme-Complex Roles

ropathology. 2007 Jun; 27(3):237–44.

Cassiano Felippe Gonçalves de Albuquerque, Tatiana Maron‐Gutierrez, Adriana Ribeiro Silva and Hugo Caire de Castro Faria Neto

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/67816

#### **Abstract**

Nitric oxide (NO) was discovered as an endothelium‐derived relaxing factor more than two decades ago. Since then, it has been shown to participate in many pathways. NO has been described as a key mediator of different pathways in the central nervous system (CNS) in both healthy and diseased processes. The three isoforms of nitric oxide synthase differ in their activity patterns and expression in different cells. Neuronal nitric oxide syn‐ thase (nNOS) is localized in synaptic spines, astrocytes, and the loose connective tissue sur‐ rounding blood vessels in the brain; eNOS is present in both cerebral vascular endothelial cells and motor neurons; and iNOS is induced in astrocytes and microglia under patho‐ logical conditions. During physiological processes, NO produced by eNOS/nNOS, respec‐ tively, controls blood flow activation, and act as a messenger during long‐term potentiation (LTP). However, under pathological conditions, eNOS appears to be impaired, leading to a reduction in blood flow and, consequently, low oxygen/metabolites delivery, efflux of toxicological agents from the brain tissue and disturbance in the blood‐brain barrier. The NO produced by iNOS in glial cells and nNOS, which triggers the NMDA‐excitotoxic pathway, combines with superoxide anion and results in peroxynitrite synthesis, a potent free radical that contributes to tissue damage in the brain. Here, we intend to show the con‐ troversial role of the nitric oxide delivered by the three isoforms of the nitric oxide synthase in the CNS, assess its impact under healthy/pathological conditions and speculate on its possible sequela, particularly in long‐term cognitive decline.

**Keywords:** nitric oxide, nitric oxide synthase, central nervous system, memory, infection

© 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**

Three isoforms of nitric oxide synthase (NOS) have been identified: two constitutive enzymes, neuronal NOS (nNOS) and endothelial NOS (eNOS), and one inducible enzyme (iNOS). These three isoforms of the enzyme nitric oxide synthase (NOS) that are present in the cen‐ tral nervous system (CNS) can produce nitric oxide (NO). eNOS is expressed in the vascular endothelium and choroid plexus; neuronal NOS is mainly expressed in neuronal cell bodies, especially in the cortex, hippocampus, hypothalamus, olfactory bulb, claustrum, amygdala, and thalamus; and inducible NOS is expressed in macrophages, glial cells, infiltrating neu‐ trophils, and, to some extent, neurons [1]. It has also been reported that eNOS can be found in a subset of neurons and astrocytes and that nNOS can be found at low levels in astrocytes [2]. Because these enzymes may have different sites of expression and activation, they have a pivotal role in the divergent functions of NO [3].

Several studies have demonstrated that NO, a freely diffusible gaseous compound, has an important role in a variety of neurobiological processes [4]. Numerous functions of this regu‐ latory molecule have been identified in the CNS, in the process of endothelium‐dependent vasodilatation [5–8], in neurotransmission [9, 10], and in host‐defense mechanisms [11, 12].

NO is produced from the oxidation of the terminal guanidine nitrogen of the amino acid arginine. This reaction is catalyzed by the NADPH‐dependent enzyme, nitric oxide synthase (NOS). After its formation, NO diffuses outside the cell [13]. NO derived from eNOS main‐ tains the CNS microcirculation [14] by inhibiting platelet aggregation and leukocyte adhe‐ sion and migration [15]. NO derived from nNOS is an important neurotransmitter related to neuronal plasticity, memory formation, regulation of CNS blood flow, and neurotransmitter release [16, 17].

#### **2. Implications of nitric oxide synthase in the physiological central nervous system**

Under physiological conditions, the concentration of NO fluctuates within the range of low values [18] and is produced mainly by nNOS and eNOS. Unlike the other two enzymes, iNOS is not expressed unless it is induced by inflammatory mediators, cytokines, and other agents, such as endotoxins [19]. Due to its calcium‐independent activation, iNOS can produce a large amount (100–1000 times greater) of NO in relation to eNOS and nNOS [20]. Until the enzyme is degraded, iNOS constitutively produces NO [21].

NO binds to guanylyl‐cyclase, which is a soluble NO receptor and, through cGMP‐medi‐ ated signaling, acts either as a post‐ or a presynaptic messenger [22]. As a neurotransmitter, NO may activate the cGMP‐dependent protein kinase G (PKG) pathway that phosphorylates synaptophysin, which is critical for fusion of presynaptic vesicles, thereby potentiating and facilitating neurotransmission [22] (**Figure 1**). NO also acts on inhibitory gamma‐aminobu‐ tyric acid (GABA)‐ergic synaptic transmission via cGMP‐dependent pathways as well as on ion channels and exchangers [9].

Role of Nitric Oxide Synthase in the Function of the Central Nervous System under Normal and Infectious Conditions http://dx.doi.org/10.5772/67816 57

**1. Introduction**

56 Nitric Oxide Synthase - Simple Enzyme-Complex Roles

release [16, 17].

**nervous system**

pivotal role in the divergent functions of NO [3].

is degraded, iNOS constitutively produces NO [21].

ion channels and exchangers [9].

Three isoforms of nitric oxide synthase (NOS) have been identified: two constitutive enzymes, neuronal NOS (nNOS) and endothelial NOS (eNOS), and one inducible enzyme (iNOS). These three isoforms of the enzyme nitric oxide synthase (NOS) that are present in the cen‐ tral nervous system (CNS) can produce nitric oxide (NO). eNOS is expressed in the vascular endothelium and choroid plexus; neuronal NOS is mainly expressed in neuronal cell bodies, especially in the cortex, hippocampus, hypothalamus, olfactory bulb, claustrum, amygdala, and thalamus; and inducible NOS is expressed in macrophages, glial cells, infiltrating neu‐ trophils, and, to some extent, neurons [1]. It has also been reported that eNOS can be found in a subset of neurons and astrocytes and that nNOS can be found at low levels in astrocytes [2]. Because these enzymes may have different sites of expression and activation, they have a

Several studies have demonstrated that NO, a freely diffusible gaseous compound, has an important role in a variety of neurobiological processes [4]. Numerous functions of this regu‐ latory molecule have been identified in the CNS, in the process of endothelium‐dependent vasodilatation [5–8], in neurotransmission [9, 10], and in host‐defense mechanisms [11, 12].

NO is produced from the oxidation of the terminal guanidine nitrogen of the amino acid arginine. This reaction is catalyzed by the NADPH‐dependent enzyme, nitric oxide synthase (NOS). After its formation, NO diffuses outside the cell [13]. NO derived from eNOS main‐ tains the CNS microcirculation [14] by inhibiting platelet aggregation and leukocyte adhe‐ sion and migration [15]. NO derived from nNOS is an important neurotransmitter related to neuronal plasticity, memory formation, regulation of CNS blood flow, and neurotransmitter

**2. Implications of nitric oxide synthase in the physiological central** 

Under physiological conditions, the concentration of NO fluctuates within the range of low values [18] and is produced mainly by nNOS and eNOS. Unlike the other two enzymes, iNOS is not expressed unless it is induced by inflammatory mediators, cytokines, and other agents, such as endotoxins [19]. Due to its calcium‐independent activation, iNOS can produce a large amount (100–1000 times greater) of NO in relation to eNOS and nNOS [20]. Until the enzyme

NO binds to guanylyl‐cyclase, which is a soluble NO receptor and, through cGMP‐medi‐ ated signaling, acts either as a post‐ or a presynaptic messenger [22]. As a neurotransmitter, NO may activate the cGMP‐dependent protein kinase G (PKG) pathway that phosphorylates synaptophysin, which is critical for fusion of presynaptic vesicles, thereby potentiating and facilitating neurotransmission [22] (**Figure 1**). NO also acts on inhibitory gamma‐aminobu‐ tyric acid (GABA)‐ergic synaptic transmission via cGMP‐dependent pathways as well as on

**Figure 1.** NO act as an unconventional neurotransmitter that is not stored in synaptic vesicles and not released upon membrane depolarization; it releases as soon it is synthesized and does not bind to any receptors, but diffuses from one neuron to another. NO stimulate soluble guanylyl‐cyclase to form the second messenger molecule, cyclic guanosine monophosphate (cGMP) either as a post‐ or a presynaptic messenger. PKG, protein kinase G; ERK, extracellular signal‐ regulated kinases; LTP, long‐term potentiation. Images: https://mindthegraph.com.

The brain relies on a constant and adequate supply of oxygen and glucose that is provided by blood. Cerebral blood flow is altered in response to both neural activity and humoral *stimuli* (e.g., arterial PO2 and PCO2 ). Thus, augmented neural activation results in locally increased cerebral blood flow via functional hyperemia, whereas humoral *stimuli* produce overall increase in cerebral blood flow [23]. The physiological production of endothelium‐derived NO by eNOS is protective in hypoxic or ischemic brain injury.

All NOS isoforms have phosphorylation sites for different protein kinases, including protein kinase A, B, and C, and calcium‐calmodulin kinase [24]. NOS enzymes are very important for the maintenance of physiological mechanisms within an organism, and the genetic ablation of NOS in mice has been informative for establishing the functional roles of NOS‐generated NO in different systems [25]. For instance, nNOS‐KO mice present with intense gastroparesis due to impaired vagal innervation of stomach muscle cells [26], decreased apoptosis induced by striatal N‐methyl‐D‐aspartate (NMDA) microinjections [27], and early dysfunction of hip‐ pocampal‐dependent spatial memory [28]. Additionally, disrupting the gene that encodes eNOS in mice‐induced spontaneous systemic and pulmonary hypertension [29] and inhibited growth factor‐mediated angiogenesis [30]. Thus, NO acts through numerous mechanisms of different physiological systems and in living cells.

Shortly after its identification, NO emerged as a possible mediator of neurovascular coupling. Neurovascular coupling is an active mechanism with vessel diameter alterations in response to increased metabolic demands from neuronal activity. Under these conditions, NO acts as a potent vasodilator that is released during enhanced neuronal activity and is well suited to mediate the coupling between neuronal activity and cerebral blood flow [31].

The importance of NO as an intermediary in cell communication in the brain is highlighted by the fact that the excitatory amino acid glutamate is an initiator of the reaction that forms NO. NO can act as a "double‐edged sword". Whereas NO supports vascular homeostasis in the endothelium‐ dependent vasodilatation, its over‐ or underproduction is linked to pathological conditions [4].

### **3. Implications of nitric oxide synthase in the pathological central nervous system conditions**

NO is also an important mediator under pathological conditions. For instance, in brain isch‐ emia‐reperfusion injury, NO formation is initially increased and has a protective function by inducing collateral perfusion as a result of its powerful stimulatory effect on vasodilatation and angiogenesis [32]. NO donors induce neuroprotective effects.

NO can exist in distinct oxidation/reduction states and present dual biological actions as either a neuroprotective or a neurotoxic molecule [4]. Under physiological conditions, nNOS produces hydrogen peroxide (H2 O2 ) and superoxide (O2 •−) in addition to NO [33].

The downstream cascade in the breakdown of the BBB appears to be mediated by eNOS activ‐ ity; the systemic administration of a selective eNOS inhibitor abrogates VEGF‐A‐induced BBB disruption and protects against neurologic damage in models of inflammatory disease [34, 35]. Nevertheless, it was suggested that eNOS‐derived NO is a neuromodulator that partici‐ pates in the BBB‐mediated control of the cerebellum in experimental models [36].

Curiously, perivascular macrophage‐derived iNOS‐generated NO, that strategically localizes to leukocytes at brain penetration sites, can serve as a negative feedback regulator that pre‐ vents the unlimited influx of inflammatory cells by restoring BBB integrity [37].

In the brain, NOS regulates cerebral blood flow and neurotransmitter release, and the proper operative eNOS/NO system accounts for the microenvironment homeostasis that is essential for the normal functioning of neurons and glial cells [38]. Therefore, the NO produced by NOS results in vasodilation and controls vascular resistance, platelet adhesion and aggregation, leukocyte‐to‐endothelium interaction, and the maintenance of BBB integrity [36].

Several reports have indicated significant nervous system morbidity due to viral, bacterial, fungal, and parasitic infections (review [39]). During a systemic response to infections, cytokines, chemokines, and damage‐associated soluble mediators of systemic inflammation can gain access to the CNS via blood flow [40]. These mediators can access the brain tissue after the disruption of the BBB. The presence of proinflammatory mediators leads to a disturbance of neuronal and glial homeostasis, with subsequent cognitive and behavioral manifestations that are common during acute infections (anorexia, malaise, depression, and decreased physical activity) and are collec‐ tively known as sickness behavior [40]. Although sickness behavior manifestations are transient and self‐limited, the cognitive and behavioral changes can become permanent or long‐lasting under a persistent systemic inflammatory response. For example, cognitive decline is a common consequence in sepsis and cerebral malaria survivors, as found in both clinical and experimental approaches.

by striatal N‐methyl‐D‐aspartate (NMDA) microinjections [27], and early dysfunction of hip‐ pocampal‐dependent spatial memory [28]. Additionally, disrupting the gene that encodes eNOS in mice‐induced spontaneous systemic and pulmonary hypertension [29] and inhibited growth factor‐mediated angiogenesis [30]. Thus, NO acts through numerous mechanisms of

Shortly after its identification, NO emerged as a possible mediator of neurovascular coupling. Neurovascular coupling is an active mechanism with vessel diameter alterations in response to increased metabolic demands from neuronal activity. Under these conditions, NO acts as a potent vasodilator that is released during enhanced neuronal activity and is well suited to

The importance of NO as an intermediary in cell communication in the brain is highlighted by the fact that the excitatory amino acid glutamate is an initiator of the reaction that forms NO. NO can act as a "double‐edged sword". Whereas NO supports vascular homeostasis in the endothelium‐ dependent vasodilatation, its over‐ or underproduction is linked to pathological conditions [4].

NO is also an important mediator under pathological conditions. For instance, in brain isch‐ emia‐reperfusion injury, NO formation is initially increased and has a protective function by inducing collateral perfusion as a result of its powerful stimulatory effect on vasodilatation

NO can exist in distinct oxidation/reduction states and present dual biological actions as either a neuroprotective or a neurotoxic molecule [4]. Under physiological conditions, nNOS

) and superoxide (O2

The downstream cascade in the breakdown of the BBB appears to be mediated by eNOS activ‐ ity; the systemic administration of a selective eNOS inhibitor abrogates VEGF‐A‐induced BBB disruption and protects against neurologic damage in models of inflammatory disease [34, 35]. Nevertheless, it was suggested that eNOS‐derived NO is a neuromodulator that partici‐

Curiously, perivascular macrophage‐derived iNOS‐generated NO, that strategically localizes to leukocytes at brain penetration sites, can serve as a negative feedback regulator that pre‐

In the brain, NOS regulates cerebral blood flow and neurotransmitter release, and the proper operative eNOS/NO system accounts for the microenvironment homeostasis that is essential for the normal functioning of neurons and glial cells [38]. Therefore, the NO produced by NOS results in vasodilation and controls vascular resistance, platelet adhesion and aggregation,

Several reports have indicated significant nervous system morbidity due to viral, bacterial, fungal, and parasitic infections (review [39]). During a systemic response to infections, cytokines,

pates in the BBB‐mediated control of the cerebellum in experimental models [36].

vents the unlimited influx of inflammatory cells by restoring BBB integrity [37].

leukocyte‐to‐endothelium interaction, and the maintenance of BBB integrity [36].

•−) in addition to NO [33].

mediate the coupling between neuronal activity and cerebral blood flow [31].

**3. Implications of nitric oxide synthase in the pathological central** 

and angiogenesis [32]. NO donors induce neuroprotective effects.

O2

different physiological systems and in living cells.

58 Nitric Oxide Synthase - Simple Enzyme-Complex Roles

**nervous system conditions**

produces hydrogen peroxide (H2

Under healthy conditions, the main cell types that are present in the brain are neurons, oligo‐ dendrocytes, astrocytes, and microglia. Neurons connect to each other through long axonal processes with synapses to transmit electrical/chemical signals, thereby generating memory and emotions that are associated with learning, and to control organ and systemic functions. Oligodendrocytes support axons with myelin sheaths. Astrocytes interact with blood vessels to form the BBB and maintain neuronal synapses. Microglia form long processes to phagocyte apoptotic cells and prune inactive synapses without inducing inflammation while maintaining a type of surveillance of neurons. Systemic inflammatory conditions result in the disruption of the BBB and the efflux of proinflammatory cytokines/chemokines as well as pathogen‐associ‐ ated molecular patterns (PAMPs) and danger‐associated molecular patterns (DAMPS), which together activate glial cells. This results in an inflammatory environment due to the release of cytokines/chemokines and reactive oxygen/nitrogen species that exert direct and indirect neuronal cytotoxic effects [40, 41]. Oligodendroglial myelin sheaths can be affected, thereby leading to axonal degeneration. Astrocytosis leads to reduced BBB and synaptic maintenance. Microgliosis results in a proinflammatory microglial phenotype with reduced tissue mainte‐ nance functions [41, 42]. Neuronal cells can develop an excitotoxicity process due to excessive glutamate in the synaptic cleft and subsequent extra‐synaptic NMDA receptor activation that results in a subsequent increase of Ca2+ efflux in neuronal cells and the activation of proteins calpain 1 and neuronal nitric oxide. This results in mitochondrial dysfunction and oxidative damage by reactive oxygen and nitrogen species [43]. Together, these mechanisms lead to neuronal death, thereby contributing to long‐term cognitive decline, which has been shown to be a consequence of several infectious diseases.

As shown in **Figure 2**, the gaseous signaling molecule NO has a variety of cellular functions, including neurotransmission, regulation of blood‐vessel tone, and immunity. Under patho‐ logical conditions, free radicals may deplete NO produced by eNOS through the formation of ONOO<sup>−</sup> , thus decreasing the vascular bioavailability of NO, which results in BBB dysfunction. This ultimately results in endothelial damage, edema development, and hypoxia. Furthermore, the NO produced by iNOS in glial cells or by nNOS under excitotoxic process can form with free radicals (particularly O2 − ) ONOO<sup>−</sup> and produce several deleterious effects on tissue, such as through tyrosine nitration and cysteine oxidation in various proteins. These free radicals can further decompose into highly toxic‐free radicals, such as NO2 • and •OH (as reviewed by [44]).

Nitric oxide, as described above, is a key molecule in the regulation of physiological brain homeostasis. Nitric oxide is synthetized by neuronal vessels (eNOS), by neurons (nNOS)

**Figure 2.** Different steps in the NO signaling cascade under physiological/pathological conditions in the brain. During long‐term potentiation, NOS1 or neuronal NOS (nNOS) catalyze the NO synthesis after the activation of the NMDA receptor by Ca2+. Under excitotoxic conditions, excessive Ca2+ leads to nNOS hyperactivity, whereas excessive NO production can combine with superoxide to form peroxynitrite, which is responsible for tissue damage due to several biological effects, including blockage of the eNOS pathway and BBB impairment. NO is synthesized following the transcriptional expression of a Ca2+‐independent NOS2 or iNOS isoform in glial cells (astrocytes and microglia) after cytokine exposure, thereby contributing to neuroinflammation and tissue damage in the brain. Intracellular Ca2+ activates NOS3 or eNOS to release NO from brain microvessels. This NO binds to soluble guanylyl‐cyclase (sGC) receptors, which triggers a cGMP‐dependent pathway and interacts with its downstream mediators of the physiological regulation of vasodilation and vascular resistance, platelet adhesion and aggregation, leukocyte‐endothelial interaction, and BBB integrity maintenance. Images: https://mindthegraph.com.

under physiological/pathological *stimuli*, and by iNOS during inflammatory conditions. It is well known that high levels of nitric oxide (NO) released by the inducible NO synthase (iNOS) are critical for defense against parasites and mediate inflammatory tissue damage. However, the suppression or lack of NO production results in the impaired clearance of some types of bacteria by the host [45].

Our group and others have shown that eNOS is impaired during systemic infection, which leads to brain microcirculation dysfunction [46–48]. We showed that during different infec‐ tious diseases, there is a functional capillary rarefaction (**Figure 3**) that can be caused by the impairment of endothelial NO production by a reduction of eNOS activity or by reduced levels of the enzyme cofactor tetrahydrobiopterin—BH4 [49].

Role of Nitric Oxide Synthase in the Function of the Central Nervous System under Normal and Infectious Conditions http://dx.doi.org/10.5772/67816 61

**Figure 3.** Photomicrography from intravital microscopy showing capillary impairment (red arrows) under healthy (A) and infectious conditions (B) malaria, (C) sepsis, and (D) Chagas' disease [47, 48].

Vascular function can be evaluated by the vasodilator response of cerebral arterioles to ace‐ tylcholine (Ach). Our group used intravital microscopy to evaluate the response to Ach in sepsis and Chagas' disease model. The vasodilation‐to‐Ach test is directly related to the avail‐ ability of endothelium‐derived NO generated from l‐arginine by the action of endothelial NO synthase (eNOS). The diffusion of NO to vascular smooth muscle cells and the activation of guanylyl‐cyclase resulted in cGMP‐mediated vasodilation. However, the vasoconstrictive response to Ach results from a direct muscarinic smooth muscle effect [50]. This vasoconstric‐ tive effect can lead to the slow delivery of oxygen to brain tissue, generating hypoxia and increased glycose consume by glycolytic pathway to generate ATP for neuronal functioning. Consequently, mitochondrial function is compromised by low O<sup>2</sup> concentration [51].

In both sepsis and Chagas' disease models, we observed a vasoconstrictive response to Ach. One mechanism of eNOS "uncoupling" from the reduction of NO production and ROS generation involves the oxidative degradation of the cofactor BH4 (tetrahydrobiopterin), especially by per‐ oxynitrite (ONOO<sup>−</sup> ), which is produced in association with superoxide and nitric oxide. Several studies have shown that there is an increase in impaired eNOS‐derived oxidative stress in brain tissue exposed to systemic infection, NO consumption and capillary dysfunction [46, 47, 52, 53].

under physiological/pathological *stimuli*, and by iNOS during inflammatory conditions. It is well known that high levels of nitric oxide (NO) released by the inducible NO synthase (iNOS) are critical for defense against parasites and mediate inflammatory tissue damage. However, the suppression or lack of NO production results in the impaired clearance of some

**Figure 2.** Different steps in the NO signaling cascade under physiological/pathological conditions in the brain. During long‐term potentiation, NOS1 or neuronal NOS (nNOS) catalyze the NO synthesis after the activation of the NMDA receptor by Ca2+. Under excitotoxic conditions, excessive Ca2+ leads to nNOS hyperactivity, whereas excessive NO production can combine with superoxide to form peroxynitrite, which is responsible for tissue damage due to several biological effects, including blockage of the eNOS pathway and BBB impairment. NO is synthesized following the transcriptional expression of a Ca2+‐independent NOS2 or iNOS isoform in glial cells (astrocytes and microglia) after cytokine exposure, thereby contributing to neuroinflammation and tissue damage in the brain. Intracellular Ca2+ activates NOS3 or eNOS to release NO from brain microvessels. This NO binds to soluble guanylyl‐cyclase (sGC) receptors, which triggers a cGMP‐dependent pathway and interacts with its downstream mediators of the physiological regulation of vasodilation and vascular resistance, platelet adhesion and aggregation, leukocyte‐endothelial interaction,

Our group and others have shown that eNOS is impaired during systemic infection, which leads to brain microcirculation dysfunction [46–48]. We showed that during different infec‐ tious diseases, there is a functional capillary rarefaction (**Figure 3**) that can be caused by the impairment of endothelial NO production by a reduction of eNOS activity or by reduced

types of bacteria by the host [45].

60 Nitric Oxide Synthase - Simple Enzyme-Complex Roles

levels of the enzyme cofactor tetrahydrobiopterin—BH4 [49].

and BBB integrity maintenance. Images: https://mindthegraph.com.

Interestingly, in experimental models of malaria [48] and sepsis (Reis et al., submitted) blood flow was recovered by treatment with statins. As reviewed by Giannopoulos et al. [54], statins are shown to enhance eNOS expression and can contribute to the restoration of brain capil‐ lary function.

Resident glial cells in the CNS (i.e., astroglia and microglia) express inducible nitric oxide synthase (iNOS) and produce high levels of NO in response to a wide variety of proinflam‐ matory and degenerative *stimuli*. Excessive NO production by glial cells evoked by inflam‐ matory signals contributes to the pathogenesis of several neurodegenerative diseases, such as multiple sclerosis, HIV dementia, brain ischemia, trauma, Parkinson's disease, and Alzheimer's disease. NO can also be released in glial cells in response to infectious agent. The inducible form of nitric oxide synthase can be strongly associated with tissue damage, despite its activity as an antimicrobial agent. As reviewed by Saha and Pahan [55], astrocytes and microglia can express iNOS and synthetize NO. According to Radi [56], nitric oxide can combine with superoxide (produced mainly by the NADPH oxidase system and partially by mitochondria) under conditions of oxidative stress. It is common under neuroinflamma‐ tory conditions to form peroxynitrite, which binds to tyrosine to produce 3‐nitrotyrosine, and a marker of reactive nitrogen species production. The reactions of peroxynitrite with biomolecules can lead to cytotoxic events and may result in apoptotic or necrotic cell death. Peroxynitrite can act via antioxidant enzyme inhibition; antioxidant depletion; protein aggre‐ gation (e.g., α‐synuclein and microtubule‐associated tau protein modifications in the CNS); activation of specific enzymes (e.g., matrix metalloproteinases (MMPs), cytochrome c, glu‐ tathione‐S‐transferase, protein kinase C‐ε (PKCε), and fibrinogen); impairment of enzyme cofactors (e.g., BH4); modification of mediator pathways, receptors, and cellular signaling molecules; calcium deregulation; DNA injury; and mitochondrial dysfunction, among other processes (reviewed by [57]).

Data from our group show that the absence of the iNOS enzyme (knockout) or treatment with iNOS‐specific inhibitors (e.g., aminoguanidine) has a beneficial effect on the prevention of cognitive impairment (unpublished data) in experimental cerebral malaria. This could be due to the reduced production of the peroxynitrite radical and the subsequent reduction of tissue damage. Other studies are being conducted to clarify the impact of the synthesis of nitric oxide by iNOS in infectious diseases and in the development of cognitive decline. Weberpals et al. also observed that iNOS gene deficiency prevents cognitive decline in addition to pro‐ moting a reduction in gliosis (astrocytes and microglia), proinflammatory cytokines TNF‐α and IL‐1β release, and reduction of synaptic dysfunction in sepsis model [58].

#### **4. NMDA‐nNOS pathway and excitotoxicity development**

The NMDA receptor is a key regulator during glutamatergic long‐term potentiation (LTP) response. During physiological conditions, glutamatergic ionotropic (e.g., kainate, NMDA, and AMPA) and metabotropic receptors (mGlut) are activated, delivering Ca2+ into neuronal cells and resulting in depolarization and the triggering of mitogen‐activated protein kinase (MEK)/extracellular signal‐regulated kinase (ERK) and phosphatidylinositol 3‐kinase (PI3K) signaling pathways. This then activates phosphoinositide‐dependent kinase 1 or 2 (PDK1/2), Akt, and mammalian target of rapamycin (mTOR) downstream signaling [59]. Additionally, calcium‐dependent kinase II (CAMKII) is rapidly activated and all events together lead to the activation of transcription factors (e.g., CREB) and an increase in the synthesis of neurotrophic factors (e.g., BDNF) and proteins associated with the long‐term potentiation (LTP) process.

Resident glial cells in the CNS (i.e., astroglia and microglia) express inducible nitric oxide synthase (iNOS) and produce high levels of NO in response to a wide variety of proinflam‐ matory and degenerative *stimuli*. Excessive NO production by glial cells evoked by inflam‐ matory signals contributes to the pathogenesis of several neurodegenerative diseases, such as multiple sclerosis, HIV dementia, brain ischemia, trauma, Parkinson's disease, and Alzheimer's disease. NO can also be released in glial cells in response to infectious agent. The inducible form of nitric oxide synthase can be strongly associated with tissue damage, despite its activity as an antimicrobial agent. As reviewed by Saha and Pahan [55], astrocytes and microglia can express iNOS and synthetize NO. According to Radi [56], nitric oxide can combine with superoxide (produced mainly by the NADPH oxidase system and partially by mitochondria) under conditions of oxidative stress. It is common under neuroinflamma‐ tory conditions to form peroxynitrite, which binds to tyrosine to produce 3‐nitrotyrosine, and a marker of reactive nitrogen species production. The reactions of peroxynitrite with biomolecules can lead to cytotoxic events and may result in apoptotic or necrotic cell death. Peroxynitrite can act via antioxidant enzyme inhibition; antioxidant depletion; protein aggre‐ gation (e.g., α‐synuclein and microtubule‐associated tau protein modifications in the CNS); activation of specific enzymes (e.g., matrix metalloproteinases (MMPs), cytochrome c, glu‐ tathione‐S‐transferase, protein kinase C‐ε (PKCε), and fibrinogen); impairment of enzyme cofactors (e.g., BH4); modification of mediator pathways, receptors, and cellular signaling molecules; calcium deregulation; DNA injury; and mitochondrial dysfunction, among other

Data from our group show that the absence of the iNOS enzyme (knockout) or treatment with iNOS‐specific inhibitors (e.g., aminoguanidine) has a beneficial effect on the prevention of cognitive impairment (unpublished data) in experimental cerebral malaria. This could be due to the reduced production of the peroxynitrite radical and the subsequent reduction of tissue damage. Other studies are being conducted to clarify the impact of the synthesis of nitric oxide by iNOS in infectious diseases and in the development of cognitive decline. Weberpals et al. also observed that iNOS gene deficiency prevents cognitive decline in addition to pro‐ moting a reduction in gliosis (astrocytes and microglia), proinflammatory cytokines TNF‐α

The NMDA receptor is a key regulator during glutamatergic long‐term potentiation (LTP) response. During physiological conditions, glutamatergic ionotropic (e.g., kainate, NMDA, and AMPA) and metabotropic receptors (mGlut) are activated, delivering Ca2+ into neuronal cells and resulting in depolarization and the triggering of mitogen‐activated protein kinase (MEK)/extracellular signal‐regulated kinase (ERK) and phosphatidylinositol 3‐kinase (PI3K) signaling pathways. This then activates phosphoinositide‐dependent kinase 1 or 2 (PDK1/2), Akt, and mammalian target of rapamycin (mTOR) downstream signaling [59]. Additionally, calcium‐dependent kinase II (CAMKII) is rapidly activated and all events together lead to the

and IL‐1β release, and reduction of synaptic dysfunction in sepsis model [58].

**4. NMDA‐nNOS pathway and excitotoxicity development**

processes (reviewed by [57]).

62 Nitric Oxide Synthase - Simple Enzyme-Complex Roles

Despite the major activity of NMDA receptors in LTP, several reports have related this recep‐ tor with excitotoxicity, which is a neurodegenerative process. This process has been associ‐ ated with a different class of NMDA receptor: the extra‐synaptic receptor. As reviewed by Parsons and Raymond, synaptic NMDA (which expresses the subunit GlutN2A) is associated with LTP, whereas extra‐synaptic receptors (which express the subunit GlutN2B) is associ‐ ated with excitotoxicity and cell death [42]. NMDARs recruit the calcium‐dependent enzyme nNOS via PSD95 (postsynaptic density: membrane‐associated guanylate kinase (MAGUK) scaffolding protein located in neural postsynaptic densities), which is also associated with the LTP process. This is considered a key contributor to excitotoxicity lesions in both stroke and neurodegenerative diseases [60]. nNOS is activated by calcium/calmodulin signaling and is PSD95 protein‐dependent [60]. During excitotoxicity, the activation of NMDA enhances intracellular calcium, leading to nNOS activation and NO production. Additionally, excessive intracellular calcium activates calpain 1, which then disrupts mitochondrial function, thereby triggering the intrinsic apoptotic pathway by releasing cytochrome C and activating apopto‐ somal protein complex [61]. NO can combine with superoxide, which results in peroxynitrite formation and cellular damage. Peroxynitrite can disturb cellular function by the nitration of proteins, which reduces or eliminates protein function, as described earlier, and by DNA damage via the activation of poly (ADP‐ribose) polymerase 1 (PARP‐1). The impact of PARP‐1 on intracellular concentration of its nicotinamideadenine dinucleotide substrate (NAD), cre‐ ates a bioenergetic imbalance that culminates with ATP depletion, thereby triggering necrotic neuronal death [62, 63]. In addition, NO can drive the retraction of the synaptic button via the activation of small GTPase RhoA/ROCK signaling through a paracrine/retrograde‐signal‐ ing pathway [64]. Taken together, these events could contribute to neuronal dysfunction and death associated with cognitive decline.

The roles of NO in neuronal damage following insults, such as hypoxia, traumatic brain injury, and ischemia, have been well established. Recent evidence has implicated an imbalance of ROS and NO signaling in neurodegenerative disorders, such as Alzheimer's disease and Parkinson's disease, and in cognitive impairments associated with normal physiological aging [65–67]. Whereas mild oxidative/nitrosative (nitric oxide (NO)‐related) stress mediates normal neuronal signaling, the accumulation of free radicals is associated with neuronal cell injury or death.

As described above, NO from eNOS modulates blood flow in the brain, and its impairment could be associated with hypoxic events in the brain. Several degenerative and infectious diseases relate hypoxia to neuronal dysfunction and cognitive decline. Using partial eNOS knockout mice model, Tan et al. [68] showed that the development of spontaneous thrombotic cerebral infarction is followed by amyloid protein deposit and cognitive decline. Cognitive decline is a major cause of disability in stroke survivors [69]. We have shown that microvascu‐ lar impairment in malaria, sepsis, and Chagas' diseases may occur in response to other infec‐ tious agents [46–48]. Cerebral metabolism is dependent upon the glucose and oxygen that are delivered by blood, and it is clear that alterations in endothelial function can disrupt neuronal functions. Additionally, activation of endothelial cells by systemic cytokines, PAMP/DAMP and prooxidant molecules can contribute to eNOS dysfunction [70], blood flow disturbance and neuronal dysfunction, which subsequently results in cognitive decline.

The activation of glial cells that may be related with systemic inflammation associated to host response to pathogens can activate inducible isoforms of iNOS and subsequently generate cellular damage via the generation of peroxynitrite by the combination of NO and superox‐ ide radicals. In this way, the induction of iNOS may result in the development of cognitive impairment [71]. Experimental models of sepsis [58] and malaria (unpublished data) have shown that the inhibition of iNOS has a beneficial effect on the central nervous system, par‐ ticularly by abolishing cognitive decline.

The main enzyme target in the central nervous system seems to be nNOS. The NO generated by nNOS controls the release of neurotransmitters and is involved in synaptogenesis, synap‐ tic plasticity, memory function, and neuroendocrine secretion. However, the overproduction of NO during NMDA‐excitotoxic events can lead to neuronal death and directly impact the cognitive function.

Death pathways are activated in mouse brains during experimental model of sepsis and malaria [72, 73]. Additionally, reduced levels of neurotrophic factors, impairment of neurogen‐ esis, and synaptic dysfunction were observed [74–78]. However, the role of excitotoxicity and nNOS delivery during infectious diseases and long‐term cognitive impairment is not yet clear. Neuroinflammation can also induce cell damage/death, and the modulation of the activation of glial cells has been suggested to prevent neuronal damage and cognitive decline. Cognitive impairment is prevented by antioxidants and statin treatment [48, 53, 79], which suggests that the control of oxidative or inflammatory damage is also efficient to avoid cognitive decline.

#### **5. Conclusion**

NO is a key molecule involved in the regulation of CNS function in health and disease (**Table 1**). The impairment of enzymatic activity or the overproduction of NO under inflamma‐ tory/excitotoxic conditions can contribute to neurological sequela during systemic‐infectious diseases (**Figure 3**). The NO synthase complex can be considered a target of pharmacologi‐ cal intervention focusing on the prevention of cognitive sequela; however, this field requires further studies.


**Table 1.** Nitric oxide synthase enzymes functions on health/pathological conditions.

### **Author details**

delivered by blood, and it is clear that alterations in endothelial function can disrupt neuronal functions. Additionally, activation of endothelial cells by systemic cytokines, PAMP/DAMP and prooxidant molecules can contribute to eNOS dysfunction [70], blood flow disturbance

The activation of glial cells that may be related with systemic inflammation associated to host response to pathogens can activate inducible isoforms of iNOS and subsequently generate cellular damage via the generation of peroxynitrite by the combination of NO and superox‐ ide radicals. In this way, the induction of iNOS may result in the development of cognitive impairment [71]. Experimental models of sepsis [58] and malaria (unpublished data) have shown that the inhibition of iNOS has a beneficial effect on the central nervous system, par‐

The main enzyme target in the central nervous system seems to be nNOS. The NO generated by nNOS controls the release of neurotransmitters and is involved in synaptogenesis, synap‐ tic plasticity, memory function, and neuroendocrine secretion. However, the overproduction of NO during NMDA‐excitotoxic events can lead to neuronal death and directly impact the

Death pathways are activated in mouse brains during experimental model of sepsis and malaria [72, 73]. Additionally, reduced levels of neurotrophic factors, impairment of neurogen‐ esis, and synaptic dysfunction were observed [74–78]. However, the role of excitotoxicity and nNOS delivery during infectious diseases and long‐term cognitive impairment is not yet clear. Neuroinflammation can also induce cell damage/death, and the modulation of the activation of glial cells has been suggested to prevent neuronal damage and cognitive decline. Cognitive impairment is prevented by antioxidants and statin treatment [48, 53, 79], which suggests that the control of oxidative or inflammatory damage is also efficient to avoid cognitive decline.

NO is a key molecule involved in the regulation of CNS function in health and disease (**Table 1**). The impairment of enzymatic activity or the overproduction of NO under inflamma‐ tory/excitotoxic conditions can contribute to neurological sequela during systemic‐infectious diseases (**Figure 3**). The NO synthase complex can be considered a target of pharmacologi‐ cal intervention focusing on the prevention of cognitive sequela; however, this field requires

NOS isoform Physiological effect on CNS Pathological effect on CNS

eNOS Vasodilation and increased blood flow ↓—Hypoxia iNOS Sleep [*33*] ↑—Tissue damage

**Table 1.** Nitric oxide synthase enzymes functions on health/pathological conditions.

nNOS Learning and memory ↑—Neuronal death by excitotoxicity

and neuronal dysfunction, which subsequently results in cognitive decline.

ticularly by abolishing cognitive decline.

64 Nitric Oxide Synthase - Simple Enzyme-Complex Roles

cognitive function.

**5. Conclusion**

further studies.

Patricia Alves Reis, Cassiano Felippe Gonçalves de Albuquerque, Tatiana Maron‐Gutierrez, Adriana Ribeiro Silva and Hugo Caire de Castro Faria Neto\*

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

Laboratório de Imunofarmacologia, IOC, Fiocruz, Rio de Janeiro, Brazil

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