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

3. 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 Nobel Prize 1998  Properties of NO: Small water and lipid soluble gas Small water and lipid soluble gas Gaseous free radical Gaseous free radical  Three interchangeable forms: NO: Nitric Oxide NO: Nitric Oxide NO + : Nitrosonium cation NO + : Nitrosonium cation NO - : Nitroxyl Radical NO - : Nitroxyl Radical

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

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

6. Interaction of NO with Free Radicals  Free radicals-unpaired electron –paramagnetic  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.

7. Synthesis of Nitric Acid

8. Contd…

10. Contd..

11. Contd…

12. Nitric Oxide Signaling EnzymeGene No. of exons residues Subcellular location Regulation nNOSNOS129 1429- 1433 Mainly soluble (brain); Mainly soluble (brain); Ca 2+ /Ca M Ca 2+ /Ca M iNOSNOS227 1144- 1153 Mainly soluble Mainly soluble Ca 2+ ­ independent eNOSNOS326 1203- 1205 Mainly Mainlyparticulate Ca 2+ /CaM

16. Mechanism of Action of NO Various stimuli5’- HT Acetylcholine Thrombin Calcium ionophore A23187 Arachidonic Acid Changes in AP &ES NO Release Platelet antiaggregation & Vasorelaxation effect Prostacyclin

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

18. Physiology of Nitric oxide NO play important role :NO play important role : Penile erection Penile erection Lung vasodilatation Lung vasodilatation Physiological stimuli for generation of NO are not fully understood, but pulsatile flow and shear forces may be the main determinant.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 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 reducedIn coronary artery disease, basal level of NO as well as stimulated release of NO reduced

19. Cond…. NO inhibitor of platelet activation Alteration in formation of NO Vasoconstriction, Platelet adhesion and Aggregation

20. Contd… Isosorbide dinitrate NITRIC OXIDE Reduced Platelet deposition & Increased survival time in patients with peripheral vascular disease

21.  The generation of NO in CVS dependent on constant vasodilation.  GTN (Glycerine trinitrate) = Vasodilator Pharmacological Role of NO Nitrovasodilators & No Activation of sGC cGMP in smooth muscle Protein Phosphorylation with smooth muscle relaxation

22. Arg NO GTP cGMP 5) NO binds to Guanylyl cyclase Relaxation of smooth muscle NO Smooth muscle cell blood vessel wall 4) NO diffuses across membranes 2) ACh binds to receptors on endothelial cells 3) Activate NO synthase 1) Stimulated nerve releases Acetylcholine(ACh) at Nerve terminal Nitric Oxide Signaling

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

24. Contd…  Kitakaze et al.(2000) ACE I attenuate both reversible and irreversible myocardial cellular injury via bradykinin/ NO- dependent manner 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 Potent cardio protection

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

26. 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 O 2 (super oxide anion ) in hypertension  Patient with hypertension have elevated conc. Of super oxide anion, H 2 O 2,Lipid peroxides, endothelin, with simultaneous decrease in eNO, SOD, Vit E and LCPUFAs.

27. Nitroglycerine was used for many years to treat "angina" (chest pain) due to reduced blood flow in heart arteries without any knowledge of mechanism Lumen diameter increases and resistance to blood flow decreases Heart ("coronary") artery NO N-O Nitro glycerine We now know nitroglycerine does not act directly but is degraded to NO

28. Role of NO in Reproduction Role of NO in Reproduction

29. Contd…

30. Conclusion 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.