Nitric Oxide as Vasodilator

রাজশাহী বিশ্ববিদ্যালয়ের ফার্মেসী বিভাগের এম. ফার্ম সিলেবাস অনুযায়ী প্রণীতঃ

Syllabus- Vasodilators: Nitric oxide - Biosynthesis of nitric oxide and its control, Degradation and carriage of nitric oxide, Effects of nitric oxide, Therapeutic use of nitric oxide and nitric oxide donors, Inhibition of nitric oxide, Clinical conditions in which nitric oxide may play a part.

Introduction
Nitric oxide (NO) plays a critical role in various bodily functions, including the vasodilatation of smooth muscle, neurotransmission, regulation of wound healing, and nonspecific immune responses to infection, host defense, and cytotoxicity.
NO is a soluble gas that is produced not only by the endothelial cells, but also by macrophages and specific neurons in the brain. Because the half-life of NO is only a matter of seconds, the gas acts only on cells in close proximity to where it is produced.
NO is synthesized from L-arginine, molecular O2, and nicotinamide adenine dinucleotide phosphate, and other cofactors by the enzyme nitric oxide synthase (NOS). Then, NO induces the guanosine monophosphate (GMP), which initiates a series of intracellular events, leading to response such as vasodilatation.
Discovery of endogenous Nitric OxideIn 1980, Furchgott and Zawadzki discovered that endothelial cells stimulated by acetylcholine released a vasodilator. Initially named endothelium-derived relaxing factor, its real nature was established several years later, and the molecule was identified as nitric oxide. In 1977, Murad’s laboratory has reported that “nitrovasodilators” such as nitroglycerin and nitroprusside caused smooth muscle relaxation via generation of NO that activated soluble guanylyl cyclase and increased the concentration of cyclic guanosine monophosphate (cGMP) in tissues.
Biosynthesis of NO and its controlAfter it was recognized that this diatomic gas performs crucial functions in a wide array of physiological processes, including signal transduction and the immune response, the hunt for its biological source was on, and it was soon discovered that NO was synthesized from the amino acid L-arginine by an enzyme that, predictably, was described nitric-oxide synthase (NOS). This enzyme turned out to be a real gem for structural chemists, enzymologists, and pharmacologists alike. In its active center, NOS contains a heme of the same type as found in cytochrome P450 (P450).
NOS is a modular enzyme that consists of an N-terminal oxygenase and a C-terminal reductase domain. Catalysis takes place at a cysteinyl sulfur-coordinated b-type heme in the oxygenase domain. The heme iron binds O2 as a sixth ligand in the distal pocket, which also serves as the site for substrate binding. In close proximity, (6R)-5,6,7,8-tetrahydro-L-biopterin (BH4) is bound as an additional cofactor. The required electrons are provided by the reductase domain, which shuttles electrons from NADPH to the heme via two flavin cofactors, one FAD moiety that accepts electrons two at a time from NADPH, and one FMN moiety that transfers them one at a time to the heme.
The two domains are separated by a short amino acid sequence that must bind calmodulin to enable interdomain electron transfer. NOS is only active as a homodimer, because electron transfer can only occur from the reductase domain of one subunit to the oxygenase domain of the second subunit.
The stability of the dimer is enhanced by a zinc ion that is coordinated to four cysteinyl sulfurs in the dimer interface. A schematic illustration of NOS structure and cofactor content is presented in Fig.

Fig. Schematic presentation of the NOS homodimer including all cofactors and the electron transfer pathway. Note the presence of calmodulin, BH4, and Zn2+ as additional cofactors in NOS.
Mammalian NOS comes in three isoforms. Neuronal and endothelial NOS (nNOS and eNOS) are constitutively expressed, eNOS being also in cardiac myocytes, renal mesangial cells, osteoblasts and osteocytes and small amounts in platelets, and their activity is under strict regulatory control in keeping with their role in signal transduction. Both constitutive enzymes are sensitive to the calcium ion concentration, because calmodulin binds to these isoforms only in the presence of Ca2+.
In contrast to the constitutive isoforms, the inducible isoform (iNOS) is only expressed in response to cytokines in macrophages and Kupffer cells, neutrophils, fibroblasts, vascular smooth muscle and endothelial cells. The affinity of iNOS for calmodulin is so much higher than that of eNOS and nNOS that it binds calmodulin in the virtual absence of free Ca2+. As a result, iNOS lacks the tight control characteristic of the constitutive isoforms and is able to stir up large quantities of NO for an extended period, in line with its function in the immune response.
Synthesis
Endogenous NOEndogenous NO is synthesized by NO synthase (NOS), which catalyzes NO synthesis by combining O2 with L-arginine in two distinct cycles with N-hydroxy-L-arginine (NHA) as an intermediate product that is processed to citrulline and NO without being released from the enzyme. The reaction requires nicotinamide adenine dinucleotide phosphate, flavin adenine dinucleotide, flavin mononucleotide, and tetrahydrobiopterin as cofactors. Both cycles consume one molecule of O2 and both require the input of exogenous electrons- two in the first and one in the second cycle- that are furnished by NADPH.

In vivo synthesis of NO occurs in the lungs in the vascular endothelium, epithelial cells, nerve cells, smooth muscle cells, and inflammatory cells such as macrophages. In addition, the cells of the upper airways, especially in the nose and paranasal sinuses, generate large amounts of NO, which is inhaled whenever the patient inspires through the nose.
ControlRate of production of NO is determined by the activity of the enzyme rather than by substrate availability. Nevertheless, very high doses of L-arginine are able to restore endothelial NO biosynthesis in some pathological states (eg. Hypercholesterolemia), in which endothelial function is impaired.
The activity of the constitutive isoforms of NOS is controlled by intracellular Ca-calmodulin. The most important stimuli controlling endothelial NO synthesis in resistance vessels under physiological conditions are mechanical and shear stress, Ca++-ionophore, polycations and receptor mediated vasodilator including acetylcholine, bradykinin etc. Occupation of which increases Ca++ ion concentration, thereby stimulating endothelial NO synthesis.
Several drugs with principal actions on other tissues (eg. Propofol, an intravenous anesthetic agent and nebivolol, a beta-adrenoceptor antagonist) also release NO from endothelium.
The activity of iNOS is independent of [Ca++]. Though iNOS contains a binding site for Ca-calmodulin, the very high affinity of their site for its ligand means that iNOS is activated even at the low values of [Ca++] present under resting condition. The enzyme is induced by bacterial lipopolysaccharide (LPS) and/or cytokines synthesized in response to LPS.
Degradation and carriage of Nitric Oxide
a) NO reacts with oxygen to form N2O4, which combines with water to produce a mixture of nitrite and nitrate anions. Nitrite ions are oxidized to nitrate by oxyhemoglobin.

2NO+O2 leads to N2O4
N2O4+H2O leads to NO3-+NO2-+2H+
NO2-+HbO leads to NO3- +Hb

b) NO reacts with O2- to form peroxynitrite (ONOO-), which can further form its acid form, peroxynitrous acid (ONOOH), a very unstable and reactive oxidizing species. ONOO- has a high affinity for sulphydryl groups and thus inactivates several key sulphydryl bearing enzymes.
Carriage
a) NO diffuses freely across cell membranes, accounting adequately for its local actions on vascular smooth muscle or on monocytes or platelets adhering to the endothelium. The possibility that analogous carrier mechanisms (eg., cysteine and/or –SH containing proteins) operate in mammals and allow NO to act at a distance from its site of biosynthesis. When NO diffuses from endothelium into the blood, it reacts rapidly with haem, which has an affinity for NO>10,000 times greater than for O2. In the absence of oxygen, NO bound to hemoglobin is relatively stable but in the presence of oxygen, NO is immediately converted to nitrate and the haem iron oxidized to methaemoglobin.
Glutathione also reacts with NO under physiologic conditions to generate S-nitroglutathione, a more stable form of NO. Nitroglutathione may serve as an endogenous long-lived adduct or carrier of nitric oxide.
Effects of NO- NO has been involved in many physiological and pathophysiological processes.
Vascular effects: NO has a role in vasodilatation, and, because of the rapid combination of NO with hemoglobin contained in red blood cells, it can be rapidly inactivated, thereby limiting vasodilatation to pulmonary vessels.
Shear stress or receptor activation of vascular endothelium by bradykinin or acetylcholine results in an influx of calcium. The consequent increase in intracellular calcium stimulates the constitutive NOS. The NO formed from L-arginine by this enzyme diffuses to nearby smooth-muscle cells, in which it stimulates the soluble guanylate cyclase (sGC), resulting in enhanced synthesis of cyclic GMP (cGMP) from guanosine triphosphate. This increase in cGMP in the smooth-muscle cells leads to their relaxation.

Pulmonary effects: Researchers have postulated that because glyceryl trinitrate and sodium nitroprusside relax the airway smooth muscle in vitro, NO may be expected to act as a bronchodilator, as has been shown in animal models.
NO has several potentially beneficial effects on pulmonary function by maintaining low pulmonary arterial pressures and sustaining normal vascular permeability.
In addition to its effect on the pulmonary vasculature, NO has some antibacterial actions provided through formation of reactive nitrogen oxides like peroxynitrite. NO also modulates ciliary beat frequency and can inhibit or stimulate mucus secretion.
Inhaled NO (iNO) has been considered for a long time to be a selective pulmonary vasodilator that has no clinically significant effect on blood pressure and cardiac output. Its selective action results from the fixation of iNO to the heme moiety of the hemoglobin molecule after passing through the pulmonary vessel wall. NO is then oxidized to NO2 and NO3. Hemoglobin is transformed to methemoglobin, which is secondarily reduced to hemoglobin by methemoglobin reductase. Although iNO has no systemic hemodynamic effects, it does have extra-pulmonary activity.
Effects of NO on coagulation: NO interferes with platelet and leukocyte functions, fibrinolysis, restenosis, and reperfusion injury by inhibiting expression of adhesion molecules at leukocyte surfaces and by activating sGC, which lead to rapid increase in platelet cGMP and inhibition of platelet aggregation. NO also inhibits vascular smooth muscle cell proliferation, leading to decreased neointimal hyperplasia.
Effects on inflammation: NO plays an important role in vascular function during inflammatory responses. NO is a potent vasodilator. In addition to vascular smooth muscle relaxation, NO plays other important roles in inflammation. It reduces platelet aggregation and adhesion, inhibits several features of mast cell-induced inflammation, and serves as a regulator of leukocyte recruitment. Blocking NO production under normal conditions promotes leukocyte rolling and adhesion in postcapillary venules, and delivery of exogenous NO reduces leukocyte recruitment in acute inflammatory processes. Thus, the overproduction of NO from iNOS is a compensatory mechanism that decreases leukocyte recruitment in inflammatory responses.
Effects on infection: NO also acts in the host’s response to infection. NO antimicrobial activity includes the following: (1) reactive species derived from NO synthase possess antimicrobial activity and (2) interactions occur between NO and reactive oxygen species, leading to formation of multiple antimicrobial metabolites (peroxy nitrate, 5-nitrosothiols, and nitrogen dioxide), each able to damage microbial DNA protein and lipids.
High levels of NO production by a variety of cells appear to limit replication of bacteria, helminthes, protozoa, and viruses, at the risk of potential inflammatory damage to host cells and tissues. Experimental evidence is accumulating that indicates NO has antimicrobial activity against a growing list of organisms. In vitro studies have shown that oxides of nitrogen inhibit the growth of or kill a number of fungi, parasites, helminthes, protozoa, yeasts, mycobacteria, and bacteria. NO may play a role in killing tumor cells and in halting viral replication. Strong evidence exists that NO has a static effect on bacterial growth, and recent in vitro studies using NO donor compounds have demonstrated that NO may even be bactericidal.
Role of NO in Immunity: Researchers have shown that resistance to cancer can be enhanced in a nonspecific way by bacterial products. This nonspecific immunity is associated with the induction of NOS. If this is the case, NO-dependent, nonspecific immunity is a general phenomenon involving the reticuloendothelial system, as well as nonreticuloendothelial cells such as vascular smooth muscle, hepatocytes, and the vascular endothelium, in all of which the inducible NOS has been detected. The liver and the lung in NO-dependent nonspecific immunity play crucial roles because both organs are placed in the circulation to serve as immunologic filters. Furthermore, NO has been hypothesized to play a suppressor role in allograft rejection.
Other effects of NO: Inhaled NO increases urine output without changing systemic hemodynamic parameters in pigs, rats, and humans.

Conclusions
NO is an important mediator in host defense mechanisms as well as in homeostatic processes. Future challenges for researchers are to learn more about the beneficial and harmful effects of NO and infection and how to selectively inhibit excessive NO production or to use NO-releasing drugs to treat infection and to avoid toxic effects against nontarget host cells.

Therapeutic use of NO and NO donors
Though inhalation of high concentration of NO causes acute pulmonary oedema and methaemoglobinemia but at 5-300 ppm, NO inhibits bronchospasm in guinea pigs. But main action of inhaled NO is pulmonary vasodilatation. Two distinctive features make this action potentially therapeutically important.
>first, it is limited to the pulmonary circulation
>second, since NO is administered in inspired air, it acts preferentially on ventilated alveoli.
These properties have raised hopes that inhaled NO may be therapeutically useful in disorders such as adult respiratory distress syndrome.
Intrapulmonary shunting (i.e., pulmonary arterial blood entering the pulmonary vein without passing through capillaries in contact with ventilated alveoli) resulting in arterial hypoxemia. Inhaled NO cause vasodilatation especially in ventilated alveoli and thus reduce shunting.
NO donors: There is considerable interest in the potential for selectivity of these agents, for instance glyceryl trinitrate is more potent on vascular smooth muscle than on platelets whereas s-nitroso glutathione selectively inhibits platelet junctions.
Clinical use of glyceryl trinitrates are-
> used sublingually for rapid antianginal effect
> isosorbide mononitrate used orally for prophylaxis and more sustained effect
> organic nitrates are used to reduce cardiac pre-load in patients with heart failure, especially those unable to take angiotensin-converting enzyme inhibitors.

Inhibition of NO
There are many potential mechanisms by which drugs can inhibit NO synthesis or action.
a) The most common useful strategy remains the use of arginine analogues. Several such compounds-
-N-monomethyl-L-arginine, -N-nitro-L-arginine methyl ester
b) Glucocorticoids inhibit biosynthesis of inducible (but not constitutive) NOS.
c) An endogenous protein inhibitor of nNOS (termed PIN) which works by destabilizing the NOS dimer.
d) Assymetric dimethyl arginine has been detected in human urine, raising the possibility that they influence the L-arginine/NO pathway under pathological conditions.
 e) N-iminoethyl-L-ornithine is a potent and irreversible inhibitor of iNOS in activated macrophages, 7-nitroindazole inhibits mouse cerebral NOS.

Clinical conditions in which NO play a part
The wide distribution of NOS and diverse actions of NO have suggested that abnormalities in this pathway could be involved in the pathophysiology of numerous clinical disorders. Either increased or reduced production could play a part in disease states and hypotheses abound.
Evidence is harder to come by but has been sought using various indirect approach including-
a) Analysis of nitrate or cGMP in urine
b) Measurement of vasoconstrictor effects of NOS inhibitors
c) Comparison of vascular responses to endothelium-dependent agonists (e.g.-Ach) with endothelium-independent agonists that work through the same effector mechanism.
d) Measurement of the dilator response to increased blood flow in the brachial artery, which is partly NO-mediated.
e) Study of histochemical appearances and pharmacological responses of tissue in vitro.

Some postulated phalogical roles of excessive or reduced NO production and summarized below- iNOS
a) NO is of benefit in host defence early in the sequence of sepsis by contributing to microbial killing, subsequent excessive NO production can cause harmful hypotension. Chronic low-grade endotoxaemia occurs in patients with cirrhosis of the liver, many of whom are systemically vasodilated. Urinary excretion of cGMP is increased in such patients and the vasodilatation may be caused by induction of NOS leading to increased vascular NO synthesis.
Constitutive NOS isoforms-
eNOS
a) There is suggestive evidence of reduced NO biosynthesis in patients with hypercholesterolaemia and some other disorders that predispose to atheromatous vascular disease including cigarette smoking and diabetes mellitus.
b) An inhibitor of NOS markedly potentiates atherogenesis without increasing blood pressure or influencing plasma lipid concentrations.
nNOS
a) Excessive NMDA receptor activation contributes to several forms of neurological damage. nNOS is absent in pyloric tissue from babies with idiopathic hypertrophic pyloric stenosis.