Nitric Oxide as a Unique Bioactive Signaling Messenger

Nitric Oxide as a Unique Bioactive Signaling Messenger in Physiology and Pathophysiology Speaker: Dr. R. A. Siddique B. V. Sc. , M. V. Sc. , Ph. D Scholar Division of Animal Biochemistry National Dairy Research Institute, Karnal INDIA , 132001 E-mail- riazndri@hotmail. com

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

v NO: Unique messenger and play important role in following functions: Neuronal signaling Penile erection Cardiovascular homeostasis Decompensation in atherogenesis

v CVS also affected by NO production and regulate: Cardiovascular motor tone Modulation of myocardial contractility Inhibition of platelet activation aggregation and adhesion v Source of NO in CVS: e. NOS v Inhibition of chronic NO synthesis neurogenic and arterial hypertension Myocardial fibrosis

Interaction of NO with Free Radicals v Free radicals unpaired electron –paramagnetic v Free radical formed by gaining an additional electron. NO radical can react with peroxyl radical (RO 2), hydroxyl radical (OH. ) and NO to produce peroxinitrite, nitrous acid and nitrous oxide respectively.

Synthesis of Nitric Acid

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Nitric Oxide Signaling Enzyme Gene No. of exons No. of residues Subcellular location Regulation n. NOS 1 29 1433 Mainly soluble (brain); Ca 2+/Ca M i. NOS 2 27 1144 1153 Mainly soluble Ca 2+ independent e. NOS 3 26 1203 1205 Mainly particulate Ca 2+/Ca. M

Mechanism of Action of NO Various stimuli Changes in AP &ES NO Release Prostacyclin Platelet antiaggregation & Vasorelaxation effect 5’ HT Acetylcholine Thrombin Calcium ionophore A 23187 Arachidonic Acid

Contd… NO NO bind to Fe 2+ haem group of Guanylyl Cyclase Active Guanylate Cyclase Increased c. GMP Increased intracellular Ca 2+ Relaxes muscle Dilating the vessel & lowering B. P.

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 (t 1/2=3 5 second) • In coronary artery disease, basal level of NO as well as stimulated release of NO reduced

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

Pharmacological Role of NO v The generation of NO in CVS dependent on constant vasodilation. v GTN (Glycerine trinitrate) = Vasodilator Nitrovasodilators & No Activation of s. GC c. GMP 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 Arg NO 3) Activate NO synthase GTP c. GMP NO 5) NO binds to Guanylyl cyclase

v NO in Ischemic myocardium: ACE inhibitor 1. Stimulation of Bradykinin Receptor Inhibition in degradation of Bradykinin Accumulation of Bradykinin and NO Prevent coronary Vasoconstriction Increase in coronary blood flow 2. Inhibit Kininase II Reduced Degdn. Of Bradykinin

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

Role of NO in Hypertension v In hypertension, morphological vascular alteration affecting • Endothelium • Intima • Vascular smooth muscle v Abnormalities of endothelial cells vascular resistance – increase in Arterial Pressure. v Endothelium produce contracting substances: • O 2 • Thromboxane A 2 • Endothelin 1(Peptide) v Endothelin 1: Potent vasoconstrictor

Contd… v Increase in endothelin plasma conc. observed in patients with primary hypertension compared to normal. v Mitogenic activation described in hypertension is induced by : v Increase in sympathetic activity v Release of vasoactive agents such as endothelin, v Angiotensin II, PG v Basal formation of NO decreased in Hypertension. v Recently Das, U. N. (2004), the overall role of NO and O 2 (super oxide anion ) in hypertension v Patient with hypertension have elevated conc. Of super oxide anion , H 2 O 2 , Lipid peroxides, endothelin, with simultaneous decrease in e. NO, 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 Nitro glycerine N-O

Role of NO in Reproduction

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Conclusion v NO is a universal messenger molecule v It is involved in a wide variety of pathophysiogical and biochemical reactions. v In summary NO is involved in regulation of B. P. , prevention of aggregation and adhesion of platelets, promotion of penile erection. v Other way to increase active concentration of endogenous NO such as by prolonging its half life of duration of its actions. v NO donating compounds can be used as replacement therapy to treat its impaired production v NO also as therapeutic potential for Ischemic CVS diseases, pulmonary hypertension associated with cardiac and respiratory diseases. v They are far from ideal because of the associated side effect mainly due to the catabolism of NO into NO 2 v Therefore a technology to regulate in vivo synthesis of NO by genetic manipulation would be a welcome move.