RECENT TRENDS IN PHARMACOLOGY OF NITRIC OXIDE

OVERVIEW

Nitric oxide(NO)/ Endothelium Derived Relaxing Factor (EDRF)-Properties Synthesis, Mechanism of action and NO Signalling Physiological Role- In Body systems Role in diseases/pathophysiological states, cancer Pharmacology : NO- related drugs NOS modulating drugs activators and inhibitors NO Donors : Classification, clinical potential NO and gene therapy, stem cell-based therapy and nutraceuticals Future prospects and Conclusion

Background Information

Prior to 1990: An air pollutant Named Molecule of the Year by Science magazine in 1992 Robert Furchgott, Louis J Ignore, Ferid Murad: Nobel Prize 1998 Properties of NO: Small water and lipid soluble gas Gaseous free radical Three interchangeable forms: NO: Nitric Oxide NO+: Nitrosonium cation NO- : Nitroxyl Radical

EDRF/NO!

EDRF
1989,

was claimed to be NO by (Ignarro,

Furchgott, 1990 and Skvaril,

2000,.others).

EDFR/NO

system

presented

in

many

tissues, is a regulatory system likely to be

important physiologically.

Nitric Oxide Synthases

NOSs, is a family of related enzymes encoded

by separate genes. It is one of the most

regulated enzymes in biology.

Three

known isoforms, two are constitutive

(cNOS) and the third is inducible (iNOS) (NOS2).

Source: (Majano et al., 1998) (Tylor et al., 1997) (Freid Murad.1998 )

Nitric Oxide Synthases

NOSs

Constitutive

Inducible

Neuornal NOS1 bcNOS

Endothelial NOS3 ecNOS

NOS2 iNOS

Nitric Oxide Biosynthesis

Nitric oxide synthesis involves

arginine, oxygen, and nicotinamide adenine dinucleotide phosphate (NADPH).

Nitric Oxide Biosynthesis

Larginine

NOSs

LCitrulline

Nitrite (NO2-) Nitrate (NO3-)

Synthesis of Nitric Acid

Nitric Oxide Synthesis

Nitric Oxide Synthase oxidizes the quanidine

group of L-arginine in a process that consumes five electrons and results in the formation of NO with stoichiometric formation of L-citrulline.

The process involves the oxidation of NADPH

and the reduction of molecular oxygen.

The transformation occurs at a catalytic site

adjacent to a specific binding site of L-arginine.
Source: (Ignarro, 2001)

Activation of NOS
Glutamate neurotransmitter binds to NMDA receptors Ca++ channels open causing Ca influx into cell Activation of calmodulin, which activates NOS Mechanism for start of synthesis dependent on body system NO synthesis takes place in endothelial cells, lung cells, and neuronal cells

Http://www.kumc.edu/research/medicine/biochemistry/bioc800/sig02-06.h

Types of NOS

NOS I
Central and peripheral neuronal cells Ca+2 dependent, used for neuronal communication

NOS II
Most nucleated cells, particularly macrophages Independent of intracellular Ca+2 Inducible in presence of inflammatory cytokines

NOS III
Vascular endothelial cells Ca+2 dependent Vascular regulation

cNOS/iNOS
Constitutive Cytosolic NADPH dependent Dioxygenase Inhibited by L-arginine analogues Ca2+/calmodulin dependent Picomoles NO released Short-lasting release Unaffected by glucocorticoids Inducible Cytosolic NADPH dependent Dioxygenase Inhibited by L-arginine analogues Ca2+/calmodulin independent Nanomoles NO released Long-lasting release Induction inhibited by glucocorticoids

Source: (Moncada et al., 1991)

eNOS/nNOS/iNOS
Neuronal NOS (nNOS or NOS1) Inducible NOS (iNOS or NOS2) Endothelial NOS (eNOS or NOS3)

Originally cloned from

Chromosome localization

Neuronal Cell
NOS1, Chromosome 12 12q24.2 Ca dependent (Ca-dystrophin)

Macrophage
NOS2, Chromosome 17 17 cen-q11.2 Ca-Calmodulin independent

Endothelial Cell
NOS3, Chromosome 7

7q35-7q36
Ca dependent (Ca-Calmodulin) Ca independent

Ca dependent

Nitric Oxide Signaling

Enzyme Gene No. of exons 29 No. of residues 14291433 11441153 12031205 Subcellular location Mainly soluble (brain); Mainly soluble Mainly particulate Regulation

nNOS

NOS1

Ca2+/Ca M Ca2+ independent Ca2+/CaM

iNOS

NOS2

27

eNOS

NOS3

26

Neuronal Nitric Oxide Synthase (nNOS)

Epithelial Nitric Oxide Synthase (eNOS)

Inducible Nitric Oxide Synthase (iNOS)

Mechanism of Action of NO
Various stimuli 5- HT Acetylcholine Thrombin Calcium ionophore A23187 Arachidonic Acid

Changes in AP &ES

NO Release Prostacyclin Platelet antiaggregation & Vasorelaxation effect

Contd
NO bind to Fe 2+ haem group of Guanylyl Cyclase

NO

Active Guanylate Cyclase

Increased cGMP

Increased intracellular Ca

2+

Relaxes muscle

Dilating the vessel & lowering B.P.

Nitric Oxide Synthesis

Under

normal basal conditions in blood vessels, NO is continually being produced by cNOS but the activity of iNOS is very low.
inflammation the amount of NO produced by iNOS may be a 1,000-fold greater than that produced by cNOS. The activity of iNOS is stimulated during inflammation by bacterial endotoxins (e.g., lipopolysaccharide) and cytokines such as tumor necrosis factor (TNF) and interleukins.
Source: (Archer, 1993) (Davies et al., 1995)

During

Nitric Oxide Synthesis

Nitric oxide synthesis

(Davies et al., 1991)

Nitric Oxide
The chemical effect of NO n biological systems is extensive and complex, it is divided into two major categories: Direct

Indirect

Those reactions fast enough to occur between NO and specific biological molecules. Do not involve NO, but rather are mediated by reactive nitrogen oxide species (RNOS) formed from the reaction of NO either with oxygen or superoxide. RNOS formed from NO can mediate either nitrosative or oxidative stress.
Source: (Wink and Mitchell, 1998)

Mechanism of Action

Oxidation

of iron containing proteins such as

ribonucleotide reductase and aconitase,

Activation of the soluble guanylate cyclase, ADP ribosylation of proteins, Protein sulphhydryl group nitrosylation, and
regulatory factor activation (Shami et al., 1995).

iron

.
(Source: Shami et al., 1995)

Mechanism of Action

NO acts through the stimulation of the

soluble guanylate cyclase which is a heterodimeric enzyme with subsequent formation of cyclic GMP.

Cyclic GMP activates protein kinases

and leads ultimately to the dephosphorylation of the myosine light chain.
Source: (Denninger and Marletta, 1999)

Nitric Oxide
Intracellular calcium Nitric Oxide Synthase Nitric Oxide Guanylate cyclase Cyclic GMP Protein kinase G Protein phosphatase Phosphodiestrase

NO/cGMP signaling processes (Davies et al., 1997)

Nitric Oxide

NO

is an important messenger molecule involved in many physiological and pathological processes within the mammalian body both beneficial and detrimental.

Source: (Kane et al., 1997)

Nitric Oxide: Signaling Molecules

Functions as a signaling molecule that

tells the body to make blood vessels relax and widen. This physiological reaction is important when the body needs more blood.

Works as a signaling molecule in the

cardiovascular system, the nervous system, and in other tissues,. .
Source: (Kuwana, 1998)

Nitric Oxide

Appropriate

levels of NO production are important in protecting an organ such as the liver from ischemic damage. direct tissue toxicity and contribute to the vascular collapse associated with septic shock.

Sustained levels of NO production result in

Chronic

expression of NO is associated with various carcinomas and inflammatory conditions, including juvenile diabetes, multiple sclerosis, arthritis and ulcerative Source: (Tylor et al., 1997) colitis,..

Physiological Role
The synthesis of NO by vascular endothelium is responsible
for the vasodilator tone that is essential for the regulation of blood pressure. NO also contributes to the control of platelet aggregation and the regulation of cardiac contractility. It is now established that NO is the physiological mediator of penile erection. In the central nervous system, NO is a neurotransmitter that underlines several functions including the formation of memory. In the periphery, there is a widespread network of nerves, previously recognized as nonadrenergic and noncholinergic, that operate through a NO dependent mechanism to mediate some forms of neurogenic vasodilatation and regulate various gastrointestinal, respiratory, and genitourinary tract functions.
Source: (Moncada and Higgs, 1993)

Nitric Oxide cGMP Pathway

Physiology of Nitric oxide

NO play important role :

Penile erection
Lung vasodilatation Physiological stimuli for generation of NO are not fully understood, but pulsatile flow and shear forces may be the main determinant. In biological system NO is not stored and diffuses freely to its site of action where it bind covalently to its effectors (t1/2=3-5 second) In coronary artery disease, basal level of NO as well as stimulated release of NO reduced

What is the role of Nitric Oxide in the human body?

Nitric Oxide in the human body has many uses which are best summarized under five categories.
NO in the nervous system NO in the circulatory system NO in the muscular system NO in the immune system NO in the digestive system

Nitric Oxide in the Nervous System

Nitric oxide as a neurotransmitter NO is a signaling molecule, but not necessarily a neurotransmitter NO signals inhibition of smooth muscle contraction, adaptive relaxation, and localized vasodilation

Nitric oxide believed to play a role in long term memory Memory mechanism proposed is a retrograde messenger that facilitates long term potentiation of neurons (memory)

Synthesis mechanism involving Ca/Calmodulin activates NOS-I

NO travels from postsynaptic neuron back to presynaptic neuron which activates guanylyl cyclase, the enzyme that catalyzes cGMP production This starts a cycle of nerve action potentials driven by NO

Is Nitric Oxide a neurotransmitter?

NO serves in the body as a neurotransmitter, but there are definite differences between other neurotransmitters used commonly in the body
NO is synthesized on demand vs. constant synthesis NO diffuses out of the cells making it vs. storage in vesicles and release by exocytosis NO does not bind to surface receptors, but instead exits cytoplasm, enters the target cell, and binds with intracellular guanylyl cyclase

Similarities to normal NTs

Present in presynaptic terminal Natural removal from synaptic junction

Nitric Oxide in the Circulatory System

NO serves as a vasodilator

Released in response to high blood flow rate and signaling molecules (Ach and bradykinin)
Highly localized and effects are brief If NO synthesis is inhibited, blood pressure skyrockets (Diagram of vasodilation mechanism after muscular system)

NO aids in gas exchange between hemoglobin and cells Hemoglobin is a vasoconstrictor, Fe scavenges NO NO is protected by cysteine group when O2 binds to hemoglobin

During O2 delivery, NO locally dilates blood vessels to aid in gas exchange

Excess NO is picked up by HGB with CO2

Nitric Oxide in the Muscular System

NO was orginally called EDRF (endothelium derived relaxation factor) NO signals inhibition of smooth muscle contraction
Ca+2 is released from the vascular lumen activating NOS NO is synthesized from NOS III in vascular endothelial cells This causes guanylyl cyclase to produce cGMP A rise in cGMP causes Ca+2 pumps to be activated, thus reducing Ca+2 concentration in the cell This causes muscle relaxation

Http://www.kumc.edu/research/medicine/biochemistry/bioc800/sig02-11

Nitric Oxide in the Immune System

NOS II catalyzes synthesis of NO used in host defense reactions Activation of NOS II is independent of Ca+2 in the cell Synthesis of NO happens in most nucleated cells, particularly macrophages NO is a potent inhibitor of viral replication NO is a bactericidal agent NO is created from the nitrates extracted from food near the gums This kills bacteria in the mouth that may be harmful to the body

Nitric Oxide in the Digestive System

NO is used in adaptive relaxation

NO promotes the stretching of the stomach in response to filling. When the stomach gets full, stretch receptors trigger smooth muscle relaxation through NO releasing neurons

New research ideas involving Nitric Oxide

The role NO might play in neuronal development The mechanism of NO inhibiting the different forms of NOS Diazeniumdiolates as NO releasing drugs Excessive NO release as the cause of most brain damage after stroke

Cond.
NO inhibitor of platelet activation

Alteration in formation of NO

Vasoconstriction, Platelet adhesion and Aggregation

Contd
Isosorbide dinitrate

NITRIC OXIDE

Reduced Platelet deposition & Increased survival time in patients with peripheral vascular disease

Nitric Oxide and Diseases

Hypertension
Neuronal function

Atherosc lerosis

Microcircu latory dysfunctio n

Diabetes

Vasospas m

+ +

NO
+ + +

+ +

NANC dysfunction

Ischemia reperfusion injury

Inflammati on

Vascular injury

Organ dysfunction

Coagulopa thies

Vascular actions of NO

Direct vasodilation (flow dependent and receptor mediated) Indirect vasodilation by inhibiting vasoconstrictor influences (e.g., inhibits angiotensin II and sympathetic vasoconstriction) Anti-thrombotic effect - inhibits platelet adhesion to the vascular endothelium Anti-inflammatory effect - inhibits leukocyte adhesion to vascular endothelium; scavenges superoxide anion Anti-proliferative effect - inhibits smooth muscle hyperplasia

NO, when its production is impaired or its bioavailability is reduced, the following can result:

Vasoconstriction (e.g., coronary vasospasm, elevated systemic vascular resistance, hypertension) Thrombosis due to platelet aggregation and adhesion to vascular endothelium Inflammation due to upregulation of leukocyte and endothelial adhesion molecules Vascular hypertrophy and stenosis

Conditions Associated with Abnormal NO Production and Bioavailability Hypertension Obesity Dyslipidemias (particularly hypercholesterolemia and hypertriglyceridemia) Diabetes (both type I and II) Heart failure Atherosclerosis Aging Cigarette smoking

Nitric Oxide and Cancer

NO, plays a variety of roles, which are at times

contradictory.

NO, has a dual complex action, at least dual, on

tumor growth that may depend on the local concentration of NO.

Additional factors such as the presence of ROS,

and the type of tumor and its susceptibility to NO.

Source: (Jenkins et al., 1995) (Wink et al., 1998)

Nitric Oxide
Low NO concentration Pro-angiogenic and protumor growth
Higher NO concentration Inhibit mitochondrial respiration,
the citric acid cycle glycolysis, and DNA replication. Locally high levels of reactive oxygen species (ROS), insufficient oxygen supply to the tumor tissue, may exacerbate these toxic effects by generating even more reactive compounds, such as peroxynitrite (ONOO-). The latter compound arises from the diffusion limited interaction of NO and O2 and is even more reactive than NO.

Source: (Vamvakas and Schmidt, 1997 and Masuda et al., 2002)

Nitric Oxide

Toxicology of NO

Inhalation of 25 ppm of mixed nitrogen

oxide, the recommended threshold limit, may cause pulmonary irritation.

Higher doses of NO may cause

minimal irritation initially but result in hemorrhagic pulmonary edema several days later.

Nitric Oxide

Individuals

exposed to nitrogen oxide should be carefully monitored and receive supportive care, including supplemental O2, morphine and steroid therapy.

NO must be handled with extreme caution.

On contact with air NO interacts with O2, producing a dimeric form of nitrogen dioxide, a reddish- brown gas (Archer., 1993).
Source: (Archer., 1993)

More about NO

Nitric oxide and hypertension (Leclercq et al., 2002) Nitric oxide and pulmonary system ( Persson et al., 1994) Nitric oxide and the nervous system (Kuwana, 1998) Nitric oxide and gastrointestinal tract (Miller and Sandoval, 1999) Nitric oxide in liver failure (Tomas et al., 1992) Nitric oxide levels in patients after trauma and during sepsis (Tylor et al., 1997). Nitric oxide as a cytostatic and cytotoxic agent (Moncada and Higgs, 1993). Nitric oxide in immunity and inflammation (Hadas et al., 2002) Nitric oxide and cancer. ..

Pharmacological Role of NO

The generation of NO in CVS dependent on constant vasodilation. GTN (Glycerine trinitrate) = Vasodilator
Nitrovasodilators & No

Activation of sGC

cGMP in smooth muscle Protein Phosphorylation with smooth muscle relaxation

Nitric Oxide Signaling

Relaxation of smooth muscle
1) Stimulated nerve releases Acetylcholine(ACh) at Nerve terminal

2) ACh binds to receptors on endothelial cells

Smooth muscle cell blood vessel wall

4) NO diffuses across membranes

GTP
NO

cGMP

Arg

NO

3) Activate NO synthase

5) NO binds to Guanylyl cyclase

NO in Ischemic myocardium:
ACE inhibitor
1.Stimulation of Bradykinin Receptor 2. Inhibit Kininase II

Inhibition in degradation of Bradykinin

Accumulation of Bradykinin and NO

Reduced Degdn. Of Bradykinin

Prevent coronary Vasoconstriction

Increase in coronary blood flow

Contd

Kitakaze et al.(2000) ACE I attenuate both reversible and irreversible myocardial cellular injury via bradykinin/ NO- dependent manner ACE I, enalaprilat, improves transmural myocardial perfusion at rest and after stress and restore impaired sub endothelial coronary flow and vasodilator reserve . The effect of Enalaprilat is bradykinin and NO dependent.

ACE I increase Bradykinin and NO: Potent cardio protection

Role of NO in Hypertension
In

hypertension, morphological vascular alteration affecting Endothelium Intima Vascular smooth muscle Abnormalities of endothelial cells-- vascular resistance increase in Arterial Pressure. Endothelium produce contracting substances: O 2 Thromboxane A2 Endothelin-1(Peptide) Endothelin-1: Potent vasoconstrictor

Contd

Increase in endothelin plasma conc. observed in patients with primary hypertension compared to normal. Mitogenic activation described in hypertension is induced by : Increase in sympathetic activity Release of vasoactive agents such as endothelin, Angiotensin II, PG Basal formation of NO decreased in Hypertension. Recently Das,U. N. (2004), the overall role of NO and O2 (super oxide anion ) in hypertension Patient with hypertension have elevated conc. Of super oxide anion , H2O2 ,Lipid peroxides, endothelin, with simultaneous decrease in eNO, SOD, Vit E and LCPUFAs.

Nitroglycerine was used for many years to treat "angina" (chest pain) due to reduced blood flow in heart arteries without any knowledge of mechanism NO

Heart ("coronary") artery

Lumen diameter increases and resistance to blood flow decreases We now know nitroglycerine does not act directly but is degraded to NO N-O

O O N N O

O O N O C H

Nitro O glycerine C
H
H H

C H

NO

Nitric oxide (NO) is a gaseous signaling molecule that readily diffuses across cell membranes and regulates a wide range of physiologic and pathophysiologic processes

including cardiovascular, inflammation, immune, and neuronal functions. Nitric oxide should not be confused with nitrous oxide (N2O), an anesthetic gas.

DISCOVERY OF ENDOGENOUSLY GENERATED NITRIC OXIDE

The first observations of the biologic role of endogenously generated NO -in rodent macrophages and neutrophils: In vitro exposure of these cells to endotoxin lipopolysaccharide resulted in the accumulation of significant amounts of nitrite and nitrate in the cell culture medium.

Furthermore, injection of endotoxin in animals elevated urinary nitrite and nitrate, the two oxidation products of NO.

The second observation was made by investigators in 1980 who found that the ability of acetylcholine to elicit relaxation of isolated strips of rabbit aorta was entirely dependent on the presence of the endothelium. If the endothelium was removed, the vessel still exhibited normal relaxation responses to nitroglycerin, but not to acetylcholine or carbachol. They discovered that following stimulation with acetylcholine or carbachol, the endothelium released a short-lived molecule that resulted in relaxation and dilation of surrounding vascular smooth muscle. The synthesis of this factor was not affected by cyclooxygenase inhibitors, indicating that it was distinct from endothelium-derived prostacyclin..

They named this vasodilator endothelium-derived relaxing factor (EDRF), since it promoted relaxation of precontracted smooth muscle preparations. In 1987, by comparing the pharmacologic and biochemical properties of the suspect molecule, three independent groups reported that EDRF and NO are the same molecule. It was later reported that other vasodilator molecules may be a part of EDRF, but it is clear that NO provides the major part of its activity. Subsequent studies revealed that NO was generated by many cells and was, like the eicosanoids , found in almost all tissues. The finding that NO is endogenously generated and elicits specific biologic effects explains why pharmacologic reagents that release NO (nitrates, nitrites, nitroprusside; are potent vasodilators

Synthesis

NO, written as NO to indicate an unpaired electron in its chemical structure, or simply NO, is a highly reactive signaling molecule that is made in a wide variety of cells, most prominently neurons, skeletal muscle, endothelial cells, and certain immune system cells. In these cells, NO is synthesized by one or more of three closely related NO synthase (NOS) isoenzymes, each of which is encoded by a separate gene and named for the initial cell type in which it was isolated These enzymes, neuronal NOS (nNOS or NOS-1), macrophage or inducible NOS (iNOS or NOS-2), and endothelial NOS (eNOS or NOS-3), despite their names, are each expressed in a wide variety of cell types, often with an overlapping distribution.

These isoforms generate NO from the amino acid Larginine in an O2- and NADPH-dependent reaction This enzymatic reaction involves enzyme-bound cofactors, including heme, tetrahydrobiopterin, and flavin adenine dinucleotide. In the case of nNOS and eNOS, NO synthesis is evoked by agents and processes that increase cytosolic calcium concentrations. Binding of calcium-calmodulin complexes to eNOS and nNOS leads to enzyme activation. On the other hand, iNOS is not regulated by calcium, but is inducible. In macrophages and several other cell types, inflammatory mediators induce the transcriptional activation of the iNOS gene, resulting in accumulation of iNOS and generation of increased quantities of NO.

Nitric oxide generation from L-arginine and nitric oxide donors and the formation of cGMP.

Signaling Mechanisms of NO

NO mediates its effects by covalent modification of proteins. There are three major effector targets of NO :
Metalloproteins Thiols Tyrosine Nitration

Metalloproteins NO interacts with metals, especially iron in heme. Soluble guanylyl cyclase (sGC), an enzyme that generates cyclic GMP from guanosine triphosphate (GTP), contains heme, which binds readily to NO. NO binding to heme results in activation of sGC and elevation in intracellular cGMP levels. cGMP activates protein kinase G (PKG), which phosphorylates specific proteins. Effects ,mediated : Vasodilatory effects, which are largely mediated by NOdependent elevations in cGMP and PKG activity. Inhibitory effect on enzymes that contain iron-sulfur clusters such as the tricarboxylic acid cycle enzyme aconitase. Inhibits mitochondrial respiration by inhibition of cytochrome oxidase. Inhibition of the heme-containing cytochrome P450 enzymes - major pathogenic mechanism in inflammatory liver disease.

Thiols NO reacts with thiols (compounds containing the SH group) to form nitrosothiols. In proteins, the thiol moiety is found in amino acid cysteine. Upon exposure to NO, certain proteins are found to accumulate nitrosothiols, which can activate or inhibit the activity of these proteins. This posttranslational modification, termed S-nitrosylation, is reversed by chemical reduction by intracellular reducing agents. The formation of nitrosothiols is not mediated by direct reaction of NO with thiols, but rather requires either metals or oxygen to catalyze the formation of this adduct. NO undergoes both oxidative and reductive reactions, resulting in the formation of a variety of oxides of nitrogen that can nitrosylate thiols, nitrate tyrosines,are stable oxidation products

Glutathione, a major intracellular sulfhydrylcontaining compound, also interacts with NO under physiologic conditions to generate Snitrosoglutathione, a more stable form of NO.
Nitrosoglutathione may serve as an endogenous long-lived adduct or carrier of NO.

Vascular glutathione is decreased in diabetes mellitus and atherosclerosis, and this may account for the increased incidence of cardiovascular complications in these conditions.

Tyrosine Nitration NO reacts very efficiently with superoxide to form peroxynitrite (ONOO), a powerful oxidant that leads to DNA damage, irreversible nitration of tyrosine, and oxidation of cysteine to disulfides or to various oxides (SOX). In several diseases, cellular degeneration, due to apoptotic mechanisms or due to ischemia, leads to excess superoxide production, and a consequent increase in peroxynitrite levels. Numerous proteins have been found to contain nitrotyrosines, and this modification can be associated with either activation or inhibition of protein function. However, it is not yet clear whether tyrosine nitration has essential roles in either physiologic signaling or in the pathology of any disease. Protein tyrosine nitration is also used as a marker for the presence of oxidative and nitrosative stress. Peroxynitrite-mediated protein modification is regulated by the cellular content of glutathione, which can protect against tissue damage by scavenging peroxynitrite. Factors that regulate the biosynthesis and decomposition of glutathione may have important consequences on the toxicity of NO.

Inactivation The lability of NO is related to its rapid reactions with metals and reactive oxygen species. Thus, NO reacts with heme and hemoproteins, including oxyhemoglobin, which catalyzes NO oxidation to nitrate. NO reactions with hemoglobin may also result in partial S-nitrosylation of hemoglobin, resulting in transport of NO throughout the vasculature. NO is also inactivated by superoxide, and scavengers of superoxide anion such as superoxide dismutase may protect NO, enhancing its potency and prolonging its duration of action.

NITRIC OXIDE IN DISEASE

VASCULAR EFFECTS

Apart from being a vasodilator, NO protects against thrombosis and atherogenesis through several mechanisms The antithrombotic effects of NO are also mediated by NO-dependent inhibition of platelet aggregation. Both endothelial cells and platelets themselves contain eNOS, which acts to regulate thrombus formation Thus, endothelial dysfunction and the associated decrease in NO generation may result in abnormal platelet function. As in vascular smooth muscle, cGMP mediates the effect of NO in platelets. an additional inhibitory effect on blood coagulation by enhancing fibrinolysis via an effect on plasminogen. reduces endothelial adhesion of monocytes and leukocytes, key features of the early development of atheromatous plaques. may act as an antioxidant, blocking the oxidation of low-density lipoproteins and thus preventing or reducing the formation of foam cells in the vascular wall. . Atherosclerosis risk factors, such as smoking, hyperlipidemia, diabetes, and hypertension, are associated with decreased endothelial NO production, and thus enhance atherogenesis.

NITRIC OXIDE IN DISEASE

SEPTIC SHOCK Increased urinary excretion of nitrate, the oxidative product

of NO, is a feature of gram-negative bacterial infection. Lipopolysaccharide components from the bacterial wall induce synthesis of iNOS, resulting in exaggerated hypotension, shock, and, in some cases, death. This hypotension is reversed by NOS inhibitors such as LNMMA (in humans as well as in animal models. A similar reversal of hypotension is produced by compounds that prevent the action of NO (such as methylene blue), as well as by scavengers of NO (such as hemoglobin). However, thus far there has been no correlation between the hemodynamic effects of relatively nonselective NOS inhibitors and survival rate in gram-negative sepsis. The absence of benefit may reflect the inability of the NOS inhibitors to differentiate between NOS isoforms or may reflect concurrent inhibition of beneficial aspects of iNOS signaling.

NITRIC OXIDE IN DISEASE INFLAMMATION

NO is an important microbicide and may have important roles in tissue adapting to inflammatory states.
However, overproduction of NO may exacerbate tissue injury in both acute and chronic inflammatory conditions. NO generated during inflammation is involved in the vasodilation associated with acute inflammation and can interact with superoxide to generate peroxynitrite and subsequently modify proteins, lipids, and nucleotides. In experimental models of acute inflammation, inhibitors of iNOS have a dosedependent protective effect, suggesting that NO promotes edema and vascular permeability. NO has a detrimental effect in chronic models of arthritis; dietary L-arginine supplementation exacerbates arthritis, whereas protection is seen with iNOS inhibitors. Recent studies have shown that NO stimulates the synthesis of inflammatory prostaglandins by activating cyclooxygenase isoenzyme II (COX-2). Thus, inhibition of the NO pathway may have a beneficial effect on inflammatory diseases, including joint diseases.

NITRIC OXIDE IN DISEASE THE CENTRAL NERVOUS SYSTEM NO has been proposed to have a major role in the central nervous systemas a neurotransmitter, as a modulator of ligand-gated receptors, or both. NO synthesis is induced at postsynaptic sites in neurons upon activation of the NMDA subtype of glutamate receptor, which results in calcium influx and activation of nNOS. In several neuronal subtypes, eNOS is also present and activated by neurotransmitter pathways that lead to calcium influx. NO synthesized postsynaptically may function as a retrograde messenger and diffuse to the presynaptic terminal to enhance the efficiency of neurotransmitter release through a cGMP or S-nitrosylation-dependent mechanism. It has been suggested that a major role for NO is in the regulation of synaptic plasticity, the molecular process that underlies learning and behavior

NITRIC OXIDE IN DISEASE THE PERIPHERAL NERVOUS SYSTEM Nonadrenergic, noncholinergic (NANC) neurons are widely distributed in peripheral tissues, especially the gastrointestinal and reproductive tracts NO as a mediator of certain NANC actions, and some NANC neurons appear to release NO. Penile erection is thought to be caused by the release of NO from NANC neurons; it is well documented that NO promotes relaxation of the smooth muscle in the corpora cavernosathe initiating factor in penile erectionand inhibitors of NOS have been shown to prevent erection caused by pelvic nerve stimulation in the rat. Thus, impotence is a possible clinical indication for the use of a NO donor, and trials have been carried out with nitroglycerin ointment and the nitroglycerin patch. An established approach is to inhibit the breakdown of cGMP by the phosphodiesterase (PDE isoform 5) present in the smooth muscle of the corpora cavernosa with drugs such as sildenafil , tadanafil

NITRIC OXIDE IN DISEASE RESPIRATORY DISORDERS NO has been shown to improve cardiopulmonary function in adult patients with pulmonary artery hypertension It s administered by inhalation. It has also been administered by inhalation to newborns with pulmonary hypertension and acute respiratory distress syndrome. NO may have an additional role in relaxing airway smooth muscle and thus acting as a bronchodilator. For these reasons, NO inhalation therapy is being widely tested in both infants and adults with acute respiratory distress syndrome. The adverse effects of this use of NO are being assessed.

PHARMACOLOGIC MANIPULATION OF NITRIC OXIDE

NITRIC OXIDE MODULATORS Inhibitors of Nitric Oxide Synthesis Nitric Oxide Donors Nitric oxide supplements

Different Class of Nitric Oxide Donors

SR Deshpande et al, 2012

NITRIC OXIDE DONORS NO donors, which release NO or related NO species, are used to elicit smooth muscle relaxation.. Organic Nitrates Nitroglycerin, which dilates veins and coronary arteries, is metabolized to NO by mitochondrial aldehyde reductase, an enzyme enriched in venous smooth muscle, accounting for the potent venodilating activity of this molecule. Other organic nitrates, such as isosorbide dinitrate are metabolized to an NO-releasing species through a currently unidentified enzyme. Organic nitrates have less significant effects on aggregation of platelets, which appear to lack the enzymatic pathways necessary for rapid metabolic activation. Organic nitrates are limited by the loss of therapeutic effect during continuous administration. This nitrate tolerance may derive from NO-mediated inhibition of mitochondrial aldehyde reductase.

Organic Nitrites Organic nitrites, such as the volatile antianginal isoamylnitrite, also require metabolic activation to elicit vasorelaxation, although the responsible enzyme has not been identified. Nitrites are arterial vasodilators and do not exhibit the rapid tolerance seen with nitrates.

Sodium Nitroprusside Sodium nitroprusside, which is used for rapid pressure reduction in arterial hypertension, generates NO in response to light as well as chemical or enzymatic mechanisms in cell membranes. Hybrid NO Donors Incorporation of NO-donating moieties onto currently available cardiovascular drugs. This approach is being tested with drugs such as aspirin and the angiotensin-converting enzyme inhibitor captopril. SNOCap, which incorporates a nitrosothiol moiety on captopril, is currently being examined for its efficacy in cardiovascular disorders.

NO Gas Inhalation NO itself can be used therapeutically. Inhalation of NO results in reduced pulmonary artery pressure and improved perfusion of ventilated areas of the lung. Inhaled NO has been used for acute respiratory distress syndrome, acute hypoxemia, and cardiopulmonary resuscitation with evidence for short-term improvements in pulmonary function. Alternate Strategies Another mechanism to enhance the activity of NO is to enhance the downstream NO signaling pathway. Sildenafil, an inhibitor of type 5 phosphodiesterase, results in prolongation of the duration of NO-induced cGMP elevations in a variety of tissues pulmonary hypertension in dogs..?

Different strategies to achieve NO-donating antiinflammatory drugs (CINOD)

Ennio Ongini and Manlio Bolla, 2006

NO donor drugs

NO gas is notoriously difficult to handle on account of the problems associated with complete exclusion of oxygen to prevent oxidation to nitrogen dioxide. Nevertheless, the gas itself can be used therapeutically, particularly in pulmonary hypertension (Griffiths and Evans, 2005) and in neonates (Greenough, 2000), where it is delivered to the lungs via inhalation. .

ORGANIC NITRATES
Most commonly used NO donor drugs. Glyceryl trinitrate (GTN; also known as nitroglycerin;) is the best-studied nitrate, used mainly in acute relief of pain associated with angina, whereas other slower release preparations, such as isosorbide mononitrate (ISMN), are used for the treatment of chronic angina. GTN ointments are also routinely used for the treatment of anal fissure (Fenton et al., 2006), transdermal patches in heart failure and chronic angina, whereas nebulized GTN may have benefits in certain subgroups with pulmonary hypertension Sodium nitroprusside (SNP). Used to provide rapid lowering of blood pressure in hypertensive crises. Relatively stable and does not release NO spontaneously in the physiological environment; instead, NO generation requires either light or a tissue-specific mode of release

Diazeniumdiolates (NONOates)

The first of this class to be described was an adduct of diethylamine and NO (diethylamine NONOate; DEA/NO), first synthesized in 1960 (Drago and Paulik, 1960). However, diazeniumdiolates only became the focus of attention in the NO world in the 1990s, when their NO donor properties were considered in biological settings Consist of a diolate group [N(O)N=O] bound to a nucleophile adduct (a primary or secondary amine or polyamine) via a nitrogen atom NONOates decompose spontaneously in solution at physiological pH and temperature, to generate up to 2 molar equivalents of NO. Feature of this class of compounds is that their decomposition is not catalysed by thiols or biological tissue, unless specifically designed to.
(Maragos et al., 1991).

The toxicity of by-products needs to be more fully confirmed (Lam et al., 2003), especially as subsequent reactions between decomposition products could lead to the formation of carcinogenic nitrosamines (Maragos et al., 1991). Incorporation of NONOates into polymers may represent a means of preventing the leaching of by-products (Mowery et al., 2000). At present, conjugated NONOates hold a great deal of promise, especially for the treatment of certain cancers, although further characterization of these drugs is essential before they reach larger clinical trials. The potential for oral preparations of NONOates

S-nitrosothiols

contain a single chemical bond between a thiol (sulphydryl) group (R-SH) and the NO moiety. Biological activity of S-nitrosothiols is highly influenced by the molecular environment of the parent thiol. That said, the complex chemistry of NO release from even the most basic S-nitrosothiol gives these compounds several means by which they can confer NO bioactivity. For instance, S-nitrosothiols are considered to be NO+donors (see below) and transfer of NO+ across the plasma membrane via protein disulphide isomerases (Zai et al., 1999) may allow even large molecule weight S-nitrosothiols to transfer oxides of nitrogen across cell membranes to subcellular targets. A vast number of factors are capable of releasing NO from Snitrosothiols, including light, heat, transition metals, thiols, superoxide (Williams, 1999; Al-Sa'doni and Ferro, 2000; Megson and Webb, 2002). and enzymes such as xanthine oxidase (Trujillo et al., 1998), superoxide dismutase (Jourd'heuil et al., 1999), protein disulphide isomerase (Ramachandran et al., 2001) and various dehydrogenases (Liu et al., 2001).

S-Nitrosothiols
Potential advantages over other classes of NO donor. Firstly, some examples show tissue selectivity: S-nitrosoglutathione (GSNO; ) is selective for arteries over veins, giving them a different haemodynamic profile of action than those of classical organic nitrates. Additionally, S-nitrosothiols are potent antiplatelet agents, inhibiting aggregation at doses that do not influence vascular tone (de Belder et al., 1994; Ramsay et al., 1995). Furthermore, the ability of S-nitrosothiols to directly transfer NO+ species allows biological activity to be passed on through a chain of other thiols without the release of free NO. This mechanism of bioactivation may make S-nitrosothiols less susceptible to conditions of oxidative stress by effectively protecting the NO moiety from attack by oxygen-centred free radicals. GSNO has at least the potential to concurrently boost

NO hybrid drugs (Hybrid NO donor drugs)

Represent a novel approach to the design of NO-releasing compounds. Structurally modified to incorporate NO-containing molecules. The aim of this strategy is to synthesize drugs that retain the pharmacological activity of the parent compound, but also have the biological actions of NO. Importantly, the release of NO must be balanced to provide sufficient activity within the concentration range of the parent compound (Bandarage et al., 2000

NO-NSAIDs

Low-dose aspirin is also routinely used prophylactically to reduce the risk of thrombotic events associated with a wide range of cardiovascular conditions. However, prolonged use of aspirin leads to serious side effects in the gastrointestinal tract that have been reported to cause 16 000 deaths each year in the USA (Keeble and Moore, 2002). A further increase in the risk of upper gastrointestinal bleeding attributed to the co-administration of multiple antithrombotic therapies regularly prescribed for cardiovascular conditions (Hallas et al., 2006). NO has a number of effects in the gastrointestinal tract that could counteract the loss of protective prostanoids caused by aspirin. NO increases secretion of protective gastric mucus (Brown et al., 1993), increases blood flow to the gastric mucosa, promoting repair and removal of toxins (Hallas et al., 2006), decreases interaction of neutrophils with the gastric microcirculation (Wallace, 1997) and may also promote the healing of gastric ulcers (Ma and Wallace, 2000). Therefore,

The first NO-NSAID compounds designed and released commercially were the NicOx compounds, NCX4016 and NCX4215. Both are derivatives of aspirin (often referred to as nitroaspirins') adapted to contain a nitrate group. These compounds have been shown to retain the ability of aspirin to inhibit inflammation and nociception without causing gastric ulcers seen with equivalent concentrations of aspirin (del Soldato et al., 1999; Fiorucci et al., 2003; Turnbull et al., 2006b). Also, several studies have shown that these compounds have comparable or greater antiplatelet effects than the parent NSAID, without causing excessive vasodilatation or hypotension (Lechi et al., 1996; del Soldato et al., 1999;Wallace et al., 1999a; Momi et al., 2000). Emerging novel NO-NSAID compounds.

In the past 5 years, there has been considerable investigation into the mechanism that underpins the anti-inflammatory properties of NO generated from nitroaspirins (Keeble and Moore, 2002). Caspases are a family of proteases involved in cytokine release and apoptosis. NO from NCX4016 inhibits the action of capsase-1, and, subsequently, the propagation of other cytokines such as IL1 and IL-8 (Fiorucci et al., 2000). NCX4016 induced inhibition of capsase-1 is mediated through S-nitrosylation of a sulphydryl group (Dimmeler et al., 1997), therefore, other NO-NSAIDs, such as S-nitrosodiclofenac (see Figure 6a) may be more effective inhibitors of capsase-1 through transnitrosation reactions. Nitroaspirins also inhibit the release of TNF- from lipopolysaccharide-stimulated macrophages (Minuz et al., 2001), although it is difficult to determine whether this is a direct effect of NO or through inhibition of other cytokines.

NO-NSAIDs have attractive properties in a number of cardiovascular conditions. On top of the antiplatelet actions of NO-NSAIDs, the NOmediated anti-inflammatory properties would be useful in vascular injury and atherosclerosis, given the central role of inflammatory cells in the process (Ross, 1999). NCX4016, but not aspirin, has been shown to reduce experimental restenosis in hypercholesterolemic (Napoli et al., 2001) and aged (Napoli et al., 2002) mice. NCX4016 also reduces infarct size in several different models of myocardial ischaemia-reperfusion injury (Rossoni et al., 2000,2001; Wainwright et al., 2002; Burke et al., 2006) and has been shown to reduce plateletmonocyte interaction in humans to a greater extent than aspirin alone (Fiorucci et al., 2004).

Nitroaspirins

also show potential in cancer therapy. Upregulation of cyclooxygenase-2 leading to enhanced prostaglandin output is a feature of a number of cancers (Baron, 1995). Both the gastro-sparing properties of nitroaspirins and the direct effect of NO on cell proliferation could be beneficial, although obtaining suitable balance between the different facets of a nitroaspirin might prove difficult. That said, NCX4016 have been shown to be 2506000-fold more effective at inhibiting the growth of a number of different cancer cell lines (Kashfi and Rigas, 2005). Additionally, the compound also reduced tumour growth in an in vivo rat model of colonic adenocarcinoma to a greater extent than aspirin itself (Bak et al., 1998). Aside from cancer, NO-NSAIDs may have applications in other areas of the body (Keeble and Moore, 2002). A NO-releasing derivative of flurbiprofen has been shown to have beneficial actions in models of renal ablation (Fujihara et al., 1998), bone degeneration (Armour et al.,

Given the tolerance issues associated with organic nitrates, it is surprising that the majority of nitroaspirins investigated so far exploit the same NO donor moiety, especially as it has been shown for at least one example that the mechanism of NO release is the same in hybrid drugs (Turnbull et al., 2006a). Many new NSAIDs/anti-inflammatory/analgesic agents with nitrates groups Paracetamol (Marshall et al., 2006), flurbiprofen (Fujihara et al., 1998), naproxen (Young et al., 2005), mesalamine (Wallace et al., 1999b), gabapentin (Wu et al., 2004), predisolone and other steroids (Tallet et al., 2002). However, the nitrate ester of nitroaspirin has been replaced with a furoxan moiety (Cena et al., 2003; Turnbull et al., 2006a) and S-nitroso- (Bandarage et al., 2000) and diazeniumdiolate (Velazquez et al., 2005) forms of other NSAID drugs (e.g. aspirin, diclofenac, indomethacin and ibuprofen) have also

Naproxcinod: A first-in-class cyclooxygenase-inhibiting nitric oxide donor for osteoarthritis pain

Naproxcinod, a cyclooxygenase-inhibiting nitric oxide donor, is pending FDA approval for the indications of knee and hip osteoarthritis
This agent consists of the nonselective NSAID naproxen, linked through a butyl group to a nitric oxide donating moiety.12 Nitric oxide may contribute to maintaining gastric mucosa integrity and circulation by inhibiting gastric acid secretion and enhancing gastric mucus alkaline secretion. Furthermore, nitric oxide may counteract NSAID-induced gastric damage secondary to increased leukocyte adherence, impairment of mucosal repair, and decreased gastric blood flow.13,14 Nitric oxide has been shown to activate guanylyl cyclase, which allows for the conversion of GTP into cyclic GMP, the second messenger primarily responsible for nitric oxide signaling.14 In addition to its gastroprotective effects, nitric oxide signaling has been shown to exert potentially beneficial cardiovascular effects, because vascular nitric oxide generation controls blood pressure. The inhibitory effects of nitric oxide on

Other nitrate-hybrid drugs There are several other drugs of note that have been successfully adapted to contain a nitrate to provide NO bioactivity. The first, nicorandil (Figure 5b), a nicotinamide derivative with a nitrate group that has both NO donor and mitochondrial K+ATP channel-opening properties. Although existing organic nitrates are extremely effective at relieving the acute symptoms of angina, clinical trials have not found that nitrates improve outcome (Yusuf et al., 1988; GISSI-3 study group, 1994; ISIS-), indeed, they may even worsen long-term prognosis (Ishikawa et al., 1996; Nakamura et al., 1999). Nicorandil, however, has been shown to decrease coronary events in over 5000 patients with stable angina during a mean follow-up period of 1.6 years (IONA study group, 2002), and subsequently, this drug is beginning to overshadow classical nitrates in the minds of many physicians.

The additional beneficial properties of nicorandil have been largely attributed its actions on K+ channels, causing dilatation of peripheral arteries and coronary resistance vessels, reducing the afterload of the heart (Simpson and Wellington, 2004; Herman and Moncada, 2005). This proposal is somewhat puzzling, as it is regularly argued that the reason behind organic nitrate effectiveness in the relief of angina, is their ability to selectively dilate peripheral veins over arteries (Kojda, 2000), and large coronary arteries over small (Winbury et al., 1969; Kurz et al., 1991). Should the dual action of nicorandil on preload and afterload be the reason for its success, it would suggest that other NO donors with more balanced arteriovenous selectivity may also be useful in the treatment of angina and ischaemic heart disease. Alternatively, actions other than those on blood vessels may be important; for example, encouragement of fibrinolysis by decreasing the activity of type-1 plasminogen

Nipradilol (K-351; Figure 5c) is an orally active compound that was first described in the early 1980s as a nonspecific receptor antagonist containing a nitrate group (Uchida et al., 1983). It was hypothesized that combining a NO donor with an adrenoceptor antagonist would provide additional beneficial cardiovascular actions by counteracting the undesirable side effects of the individual drugs. Addition of the nitrate group instils the drug with vasodilator actions similar to those of GTN, as well as enhancing the antagonistic potency and, surprisingly, providing a degree of antagonism for -receptors (Uchida et al., 1983). There is conflicting evidence as to whether nipradilol shows selectivity for conduit (Uchida et al., 1983) or resistance (Lamping and Bloom, 1995) arteries. More recently, Thakur et al., (2002) demonstrated that nipradilol inhibits the development of atherosclerotic lesions in a rabbit model of hypercholesterolemia and eNOS inhibition. Interestingly, the

Lastly, it is worth briefly mentioning another type of nitrate hybrid drug that, while only preliminary experimental data have been published so far, will undoubtedly come under close scrutiny. Statins are highly effective inhibitors of 3hydroxy-methylglutaryl CoA reductase that are used to lower cholesterol. They have had a dramatic impact on clinical outcome in a number of cardiovascular conditions. It is now clear that statins have a number of additional molecular mechanisms beyond lipid-lowering, that ultimately modulate inflammation and smooth muscle cell proliferation. Ongini et al., (2004) have synthesized nitrate-linked forms of pravastatin (Figure 5d) and fluvastatin, which they believe could complement the existing statin effects in conditions where there is endothelial dysfunction, such as diabetes and atherosclerosis. In vivo effects have yet to be tested, but experiments in cell lines showed that the NO-statins had a far greater ability to inhibit cell proliferation compared to their

Other S-nitroso-hybrid drugs A hybrid approach has also been applied to inhibitors of angiotensin-converting enzyme (ACE). Captopril is an example of an ACE inhibitor that contains a SH group, which can be nitrosated, forming S-nitrosocaptopril (SNOCap; Figure 6b) (Loscalzo et al., 1989). SNO-Cap has sGCmediated vasodilator and antiplatelet actions, yet retains the ability to inhibit ACE (Cooke et al., 1989; Loscalzo et al., 1989). Additionally, SNO-Cap appears to preferentially dilate coronary arteries over peripheral vessels (Cooke et al., 1989). Intravenous SNO-Cap produces a long-lasting hypotensive effect in vivo (Shaffer et al., 1991) and, similarly to other S-nitrosothiols, is less susceptible to tolerance (Shaffer et al., 1991; Zhang et al., 1994; Matsumoto et al., 1995). ACE inhibition may also contribute to the NO-mediated actions of SNO-Cap, as the enzyme also inactivates bradykinin, an endogenous endothelium-dependent

Tissue-type plasminogen activator (t-PA) is an endogenous enzyme synthesized by the endothelium. Fibrin, a constituent of thrombus, binds to t-PA stimulating the conversion of plasminogen into plasmin: a powerful fibrinolytic agent. t-PA contains a single SH group which can be S-nitrosated allowing t-PA to directly inhibit platelets as well as exerting a slightly greater fibrinolytic activity than native t-PA (Stamleret al., 1992). The combined antithrombotic and antiinflammatory action of SNO-t-PA has been shown to reduce cardiac necrosis following ischaemia-reperfusion injury in vivo (Delyani et al., 1996). Von Willebrand factor (vWF) is synthesized and released by damaged blood vessels. The binding of vWF to platelet glycoprotein receptors induces platelet activation, causing adhesion to damaged regions of blood vessels. Recombinant fragments of vWF, such as AR545C, bind to platelet receptors, competing with the binding of endogenous vWF to platelet receptors and

Other NO-hybrid drugs Furoxans are a group of compounds that has been of considerable interest to chemists for decades, yet have received relatively little attention from biologists, despite their NO-releasing properties. The complicated chemistry of NO release from the many different compounds in this class are reviewed elsewhere (Gasco and Schoenafinger, 2005), here we focus on existing pharmacological agents that can be modified to contain the pentavalent furoxan group. The dihydropyridine class of calcium antagonists can be linked a furoxan group (Figure 6c) and these compounds cause vasodilatation through sGC stimulation as well as inhibition of voltage-dependent Ca2+ ion channels on VSMCs (Di Stilo et al., 1998). The same group have also linked furoxans to 1- (Fruttero et al., 1995) and 1-antagonists (Boschi et al., 1997). Both produce vasodilatation of isolated aortic strips through a combination of adrenoceptor

Outside the cardiovascular system, furoxans have also been linked to a histamine H2 receptor antagonist, and provide a greater protection against gastric ulcers than antihistamine drugs alone (Coruzzi et al., 2000). Furoxan-linked H3antagonists have also been described (Bertinaria et al., 2003b). Protein pump inhibitors (PPIs) have been a major success in a range of gastric pathologies. A furoxan form of the PPI, rabeprazole, has been shown to reduce histamine secretion and indomethacin-induced gastric lesion development in rats in vivo (Sorba et al., 2003). Remaining in the gastrointestinal system, furoxan derivatives linked to the antimicrobial drug metronidazole have been shown to have potent activity against Helicobacter pylori in metronidazoleresistant strains, although it is unclear if this property is owing to NO release per se (Sorba et al., 2003; Bertinaria et al., 2003a

Zeolites A novel approach to storage and delivery of NO has recently been adopted using ion-exchanged zeolites (Wheatley et al., 2006). These microporous insoluble materials form a framework containing metal ions that can bind NO. Exposure of the solid to NO gas results in NO binding to the metal ions within the pores, facilitating highly efficient packing of NO within the solid. NO-zeolites of this type are very stable in the anhydrous state, but NO is displaced by water on immersion in an aqueous environment. The beauty of these materials is that they constitute very high capacity stores for NO and the rate of release can be modulated by altering the porosity of zeolite, the metal ion in the framework and the composition and nature of the binder (Frost et al., 2005; Wheatley et al., 2006).

This infinite flexibility with respect to NO release allows for development of a range of different NO donor materials for different purposes ranging from fast-acting antimicrobial coatings for urinary catheters and wound dressings to durable, slow acting antithrombotic coatings for stents, bypass tubing, cannulae and catheters. Although at an early stage, this approach represents a novel means of siteselective delivery of NO that optimizes the benefits of NO as a local mediator.

Summary and conclusions Depression of the NO:sGC pathway is a feature of many cardiovascular conditions and delivery of low concentrations of exogenous NO is an attractive therapeutic option. In contrast, delivery of high concentrations might have completely different therapeutic targets and act via the cytotoxic actions of NO. The chemical versatility of NO has led to the synthesis of a wide range of NO donors, each with different modes and rates of NO release. Subsequently, NO donor drugs can be chosen or tailor-made to suit the disease target. Long-term use of current NO donors is limited by development of tolerance and toxicity issues, ensuring that there is a clinical need for novel alternatives

NONOates have proved extremely popular in the experimental setting due to the predictable nature of NO release. However, use of these compounds should not be restricted to the bench; NONOates have been successfully applied in a number of cardiovascular conditions, especially where a slow prolonged release rate is desirable. In pulmonary hypertension, for example, NONOates are less likely to cause rebound hypertension on cessation than inhaled NO (Butler and Russell, 2005). There has been an explosion of interest in the possibility of delivering cytotoxic levels of NO to cancer cells to promote tumour regression. Subsequently, a series of NONOates have been designed with protected diolate groups that release NO upon metabolism by factors specific to tumour cells. However, the potential toxicity of by-products of NONOate metabolism still remains to be fully investigated before the clinical potential of these compounds can be fully assessed

Role in Treatment

Polymers containing tiny silica particles that release low

levels of NO gas. The U-M polymers are designed to mimic human endothelial cells, which produce NO to relax blood vessels and inhibit blood coagulation to reproduce the body's natural NO production system, which prevents clots from forming inside blood vessels (Meyerhoff, 2000).

Currently, exogenous NO sources constitute a powerful

way to supplement NO when the body can not generate enough for normal biological functions (novel NO donors, NO releasing devices) (Hou et al., 1999).

NOS inhibitors

Role in Treatment

Nitric Oxide

Important NO NO

messenger molecule involved in many pathological and physiological processes within the mammalian body both beneficial and detrimental. has a potent antimicrobial and antiviral activity against wide array of organisms. is also mediating endothelial cell growth. contributing to DNA damage and carcinogenesis.

NO is promoting angiogenesis.

..

Conclusion

NO is a universal messenger molecule It is involved in a wide variety of pathophysiogical and biochemical reactions. In summary NO is involved in regulation of B.P., prevention of aggregation and adhesion of platelets, promotion of penile erection. Other way to increase active concentration of endogenous NO such as by prolonging its half life of duration of its actions. NO donating compounds can be used as replacement therapy to treat its impaired production NO also as therapeutic potential for Ischemic CVS diseases, pulmonary hypertension associated with cardiac and respiratory diseases. They are far from ideal because of the associated side effect mainly due to the catabolism of NO into NO2 Therefore a technology to regulate in vivo synthesis of NO by genetic manipulation would be a welcome move.

THANK YOU

References and Further Reading

Amin A.R., Attr M.L., Vyas P.S., Leszczynska P.J., Vora K.N. and Abramason S.D. (1997): Expression of nitric oxide synthase in human peripheral blood mononuclear cells and neutrophil. J. Inflamm.; 47(4); 190-205. Archer S. (1993): Measurement of nitric oxide in biological models. FASEB; February (7): 349-360. Azza Kamel., Adel El Missiry., Salwa Teama. (2002). Peripheral blood mononuclear cell inducible nitric oxide synthase as a potential tumor marker in hepatocellular carcinoma. Journal of National Cancer Institute.14(2): 145-151. Banerjee D., Rodriguez M., Nag M. and Adamson J.W. (2000): Exposure of endothelial cells to recombinant human erythropoietin induces nitric oxide synthase activity. Kidney International; 57: 1895-1904. Chinje E.C. and Stratford I.J. (1997): Role of nitric oxide in growth of solid tumors: a balancing act. Essays Biochem.; 32: 61-72. Davies, S.A., Stewart, E.J., Huesmaan, G.R and Skaer, N. J. (1997): Neuropeptide stimulation of the nitric oxide signaling pathway in Drosophila melanogaster Malpighian tubules. Am. J. Physiol.; 273, R823-827. Denninger J.W. and Marletta M.A. (1999): Guanylate cyclase and the No/cGMP singling pathway. Biochem. Biophys. Acta, May; 1411 (2-3): 334-356. Freid Murad.(1998). Discovery of some of the biological effect of Nitric oxide and role in cell signaling. Nobel Lecture. December 8. 1998. Furchgott R.F.(1990) : Studies on endothelium- dependent vasodilation and the endothelium- derived relaxing factor. Acta Physiol Scand; 139: 257-270 Hadas E., Derda M., Wandurska-Nowak E. (2002): Effect of exogenous nitric oxide in experimental trichinellosis. Parasitol Res.; 88(1): 86 - 88.

Hou Y.C., Janczuk A. and Wang P.G. (1999): Current trends in the development of nitric oxide donors. Curr. Pharm. Des. June, 5 (6): 417- 471.
Ignarro L.J. (1989): Endothelium-derived nitric oxide. actions and properties. FASEB. J. , 3:31- 36. Jenkins D.C., Charles I.G., Thomsen L.L., Moss D.W., Holmes L.S., and Baylis S.A. (1995): Roles of nitric oxide in tumor growth. Proc. Natl. acad. Sci. USA; 92: 4392-4396. Kaibori M., Sakitani K., Oda M., Kamiyama Y., Masu Y. and Okumura T.(1999). Immunosuppressant FK56 inhibits iNOS gene expression at a step of NK-Kappa B activation in rat hepatocytes. J. Hepatol. Jan; 30 (6): 11381145.

Kane J.M., Shears L.L., Hierholzer C., Ambs S., Billair T.R. and Posener M.C. (1997): Chronic hepatitis C virus infection in humans: induction of hepatic nitric oxide synthase and proposed mechanisms for carcinogenesis. Surg. Res.; 69: 321-324.

References and Further Reading

Koshland E.D. (1992): The molecule of the year 1992. Science, 258. 18 December; 1861-1861. Kuwana E. (1998): NO is for Nobel. December. http://www. kron.. Com. Leclercq B., Jaimes E.A. and Raij L.(2002): Nitric oxide synthase and hypertension Curr. Opin. Nephrol. Hypertens. Mar; 11(2):185-189. Majano P.L., Garcia M., Kope C.M., Lara P.E., Garcia I., Borque M.J. and Moreno A.R. (1998): Inducible nitric oxide synthase expression in chronic viral hepatitis. Evidence for a virus induced gene upregualtion. J. Clin Invest.; Apr, 101 (7): 1343 -1352.

Masuda M., Nishino H., Ohshima A. (2002): Formation of 8-nitroguanosine in cellular RNA as a biomarker of exposure to reactive nitrogen species. Chem Biol Interact. Feb 20; 139 (2): 187-197
Meyerhoff E.M. (2000): Scientists Develop Polymers With Nitric-Oxide Releasing Particles For Use In Biomedical Devices. Science Daily Magazine August 21, http://www. sciencedaily. com/. htm Miller M.J. and Sandoval M. (1999): Nitric oxide. III. A molecular prelude to intestinal inflammation. Am. J. Physiolo. Apr; 276(4pt1): G795-G799. Moncada S., Palmer M.J., and Higgs E.A. (1991): Nitric oxide: physiology., pathophysiology and pharmacology. Pharmacol Rev.; 43:109 -142. Moncada S. and Higgs A. (1993): Mechanisms of the L-arginine nitric oxide pathway disease. New Eng. J. Med.; 329 (27): 2002-2011. Persson M.G., Zettertrom D., Agrenius V., Zhre E. and Gustagsson L.E. (1994): Single breath measurement of nitric oxide. Lancet.; 343:146 - 150. Rocha M., Kruger A., Rooijen N.V., Schirrmacher V. and Umansky V. (1995): Liver endothelial cells participate in T-cell dependent host resistance to lymphoma metastasis by production of nitric oxide in vivo. Int. J. Can.; 63: 405-411.

References and Further Reading

Shami P.J., Moore J.O., Cockerman J.P., Halhorn W.J., Misukonis M.A., and Weinberg J.B. (1995): Nitric oxide modulation of the growth and differentiation of freshly isolated acute non-lymphocytic leukemia cells. Leukemia Research; 19(8): 527 - 534. Skvaril J. (2000): Nitrates in cardiology. Cas Lek. Cesk. Jun 7; 139(11): 343 -349. Tomas A., Sorians G. and Guarnen E. (1992): Increased serum nitrite and nitrate in cirrhosis. J. Hepatol.; 16 (Suppl): 4. Tomas A., Sorians G. and Guarnen E. (1992): Increased serum nitrite and nitrate in cirrhosis. J. Hepatol.; 16 (Suppl): 4

Tylor B.S., Kion Y.M., Wang Q.I., Sharpio R.A., Billiar T.R. and Geller D.A. (1997): Nitric oxide down regulates hepatocyte-inducible nitric oxide synthase gene expression. Arch. Surg. 1, (32). Nov.; 1177-1182.
Vamvakas S., and Schmidt H.W. (1997): Just say NO to cancer. J. Natl. cancer. Institute. 1997, 89(6): 406-407. Wink D.A. and Mitchell J.B. (1998): Chemical biology of Nitric oxide: Insights into regulatory, cytotoxic, and cytoprotective mechanisms of nitric oxide. Free Radic Biol. Med. Sep; 25 (4-5): 434- 456. Xu W., Charles I.G., Moncada S., Gorman P., Sheer D., Liu L. and Emson P. (1994): Mapping of the genes encoding human inducible and endothelial nitric oxide synthase (NOS2 and NOS3) to the pericentric region of chromosome 17 and to chromosome 7, respectively. Genomics; 21: 419 - 421. Yoon Y., Song U., Hong S.H. and Kim J.Q. (2000): Plasma nitric oxide concentration and nitric oxide synthase .gene polymorphism in coronary artery disease. Clinc. Chem.; 46(10): 1626 -1630.

Nitric Oxide is the most important signaling molecule in the body, and it affects a wide range of physiological functions including circulation, inflammation, learning and memory, digestion and oxygen release in tissues from red blood cells. In all dogs, particularly dogs performing in any performance event, it prevents injury and aides in injury recovery. Learning and memory are key in the dog and nitric oxide is able to assist in this process by increasing the physiologic function of the brain and neurological system

PharNitroCoQ10 improves: The hearts pumping ability and hearts muscle tone Blood circulation Increase tolerance to exercise Lung function and oxygen transfer to the vital organs Production of ATP- lifes master energy molecule for endurance, agility, and peak performance Recovery from disease

What is it exactly? NitroCoQ10 is a combination of L-arginine, to generate nitric oxide, and Co Q10, which is lifes master energy molecule. The benefit of generating nitric oxide in the system is that it enhances the bioavailability of Co Q10 due to its vasodilatory effect. CoQ10 can be viewed as the bodys cellular spark plug because the key role it plays is the production of energy, also

Reversing Nitroxidative Stress

Cox-2 inhibitors Vitamin C Allopurinol (xanthine oxidase)

GTN, nicorandil