Nitric Oxide as a Unique Nitric Oxide as a Unique Bioactive Signaling Messenger Bioactive Signaling Messenger

in Physiology and in Physiology and

PathophysiologyPathophysiology

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.com

Background InformationBackground Information

Prior to 1990: An air pollutantPrior to 1990: An air pollutant Named “Molecule of the Year” by Science magazine in 1992Named “Molecule of the Year” by Science magazine in 1992

Robert Furchgott, Louis J Ignore, Ferid Murad:Robert Furchgott, Louis J Ignore, Ferid Murad: Nobel Prize 1998Nobel Prize 1998 Properties of NO:Properties of NO:• Small water and lipid soluble gasSmall water and lipid soluble gas• Gaseous free radicalGaseous free radical

Three interchangeable forms:Three interchangeable forms: NO: Nitric OxideNO: Nitric Oxide NONO++: Nitrosonium cation: Nitrosonium cation NONO- - : Nitroxyl Radical: Nitroxyl Radical

NO: Unique messenger and play important role in following NO: Unique messenger and play important role in following functions:functions:

Neuronal signalingNeuronal signaling

Penile erectionPenile erection

Cardiovascular homeostasisCardiovascular homeostasis

Decompensation in atherogenesis Decompensation in atherogenesis

CVS also affected by NO production and regulate:CVS also affected by NO production and regulate:

Cardiovascular motor toneCardiovascular motor tone

Modulation of myocardial contractilityModulation of myocardial contractility

Inhibition of platelet activation aggregation and adhesion Inhibition of platelet activation aggregation and adhesion

Source of NO in CVS: eNOSSource of NO in CVS: eNOS Inhibition of chronic NO synthesis neurogenic and Inhibition of chronic NO synthesis neurogenic and

arterial hypertension Myocardial fibrosisarterial hypertension Myocardial fibrosis

Interaction of NO with Free RadicalsInteraction of NO with Free Radicals

Free radicals-unpaired electron –paramagneticFree radicals-unpaired electron –paramagnetic

Free radical formed by gaining an additional electron. NO radical Free radical formed by gaining an additional electron. NO radical can react with peroxyl radical (ROcan react with peroxyl radical (RO22),hydroxyl radical (OH),hydroxyl radical (OH..) and NO) and NO- -

to produce peroxinitrite, nitrous acid and nitrous oxide respectively.to produce peroxinitrite, nitrous acid and nitrous oxide respectively.

Synthesis of Nitric Acid

Contd…

Contd…

Contd..

Contd…

Nitric Oxide SignalingNitric Oxide Signaling

Enzyme Enzyme

GeneGene No. ofNo. ofexonsexons

No. ofNo. ofresiduesresidues

Subcellular Subcellular locationlocation

RegulationRegulation

nNOSnNOS NOS1NOS1 2929 1429-1429-14331433

Mainly Mainly soluble soluble (brain);(brain);

CaCa2+2+/Ca/CaMM

iNOSiNOS NOS2NOS2 2727 1144-1144-11531153

Mainly Mainly solublesoluble

CaCa2+2+ independentindependent

eNOSeNOS NOS3NOS3 2626 1203-1203-12051205

MainlyMainlyparticulateparticulate

CaCa2+2+/CaM/CaM

Neuronal Nitric OxideSynthase (nNOS)

Epithelial Nitric Oxide Synthase (eNOS)

Inducible Nitric Oxide Synthase (iNOS)

Mechanism of Action of NOMechanism of Action of NO

Various stimuli 5’- HTAcetylcholineThrombinCalcium ionophore A23187Arachidonic AcidChanges in AP &ES

NO Release

Platelet antiaggregation &Vasorelaxation effect

Prostacyclin

Contd…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.

Physiology of Nitric oxidePhysiology of Nitric oxide

• NO play important role :NO play important role :

• Penile erectionPenile erection • Lung vasodilatationLung vasodilatation

• Physiological stimuli for generation of NO are not fully understood, Physiological stimuli for generation of NO are not fully understood, but pulsatile flow and shear forces may be the main determinant.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 In biological system NO is not stored and diffuses freely to its site of action where it bind covalently to its effectors (taction where it bind covalently to its effectors (t1/21/2=3-5 second)=3-5 second)

• In coronary artery disease, basal level of NO as well as stimulated In coronary artery disease, basal level of NO as well as stimulated release of NO reducedrelease of NO reduced

Cond….Cond….

NO inhibitor of platelet activation

Alteration in formation of NO

Vasoconstriction, Platelet adhesion and Aggregation

Contd…Contd…

Isosorbide dinitrate

NITRIC OXIDE

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

The generation of NO in CVS dependent on constant vasodilation.The generation of NO in CVS dependent on constant vasodilation.

GTN (Glycerine trinitrate) = VasodilatorGTN (Glycerine trinitrate) = Vasodilator

Pharmacological Role of NO

Nitrovasodilators & No

Activation of sGC

cGMP in smooth muscle

Protein Phosphorylation with smooth muscle relaxation

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

NO in Ischemic myocardium: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

Contd…Contd…

Kitakaze Kitakaze et alet al.(2000) ACE I attenuate both reversible and .(2000) ACE I attenuate both reversible and irreversible myocardial cellular injury via bradykinin/ NO- dependentirreversible myocardial cellular injury via bradykinin/ NO- dependent

mannermanner

ACE I, enalaprilat, improves transmural myocardial perfusion at rest ACE I, enalaprilat, improves transmural myocardial perfusion at rest and after stress and restore impaired sub endothelial coronary flow and after stress and restore impaired sub endothelial coronary flow and vasodilator reserve .and vasodilator reserve .

The effect of Enalaprilat is bradykinin and NO dependent.The effect of Enalaprilat is bradykinin and NO dependent.

ACE I increase Bradykinin and NO:ACE I increase Bradykinin and NO:

Potent cardio protectionPotent cardio protection

Role of NO in Role of NO in HypertensionHypertension

In hypertension, morphological vascular alteration affecting In hypertension, morphological vascular alteration affecting • EndotheliumEndothelium• IntimaIntima• Vascular smooth muscleVascular smooth muscle Abnormalities of endothelial cells-- vascular resistance – Abnormalities of endothelial cells-- vascular resistance –

increase in Arterial Pressure.increase in Arterial Pressure. Endothelium produce contracting substances:Endothelium produce contracting substances:• O O 2- 2-

• Thromboxane AThromboxane A22

• Endothelin-1(Peptide)Endothelin-1(Peptide) Endothelin-1: Potent vasoconstrictor Endothelin-1: Potent vasoconstrictor

Contd…Contd…

Increase in endothelin plasma conc. observed in patients with Increase in endothelin plasma conc. observed in patients with primary hypertension compared to normal.primary hypertension compared to normal.

Mitogenic activation described in hypertension is induced by :Mitogenic activation described in hypertension is induced by : Increase in sympathetic activityIncrease in sympathetic activity Release of vasoactive agents such as endothelin, Release of vasoactive agents such as endothelin, Angiotensin II, PGAngiotensin II, PG Basal formation of NO decreased in Hypertension.Basal formation of NO decreased in Hypertension. Recently Das,U. N. (2004), the overall role of NO and ORecently Das,U. N. (2004), the overall role of NO and O2 2 (super (super

oxide anion ) in hypertension oxide anion ) in hypertension Patient with hypertension have elevated conc. Of super oxide Patient with hypertension have elevated conc. Of super oxide

anion , Hanion , H22OO2 2 ,Lipid peroxides, endothelin, with simultaneous ,Lipid peroxides, endothelin, with simultaneous decrease in eNO, SOD, Vit E and LCPUFAs.decrease in eNO, SOD, Vit E and LCPUFAs.

Nitroglycerine was used for many years to treat"angina" (chest pain) due to reduced blood flowin heart arteries without any knowledge of mechanism

Lumen diameter increases and resistance to blood flow decreases

Heart ("coronary") artery

NO

N

O O

O

N

O O

O

N

O O

O

C C C

H H H H H H

N-O

Nitroglycerine

We now know nitroglycerine does not act directly but is degraded to NO

Role of NO in ReproductionRole of NO in Reproduction

Contd…

ConclusionConclusion

NO is a universal messenger moleculeNO is a universal messenger molecule It is involved in a wide variety of pathophysiogical and biochemical It is involved in a wide variety of pathophysiogical and biochemical

reactions.reactions. In summary NO is involved in regulation of B.P., prevention of In summary NO is involved in regulation of B.P., prevention of

aggregation and adhesion of platelets, promotion of penile erection.aggregation and adhesion of platelets, promotion of penile erection. Other way to increase active concentration of endogenous NO such Other way to increase active concentration of endogenous NO such

as by prolonging its half life of duration of its actions.as by prolonging its half life of duration of its actions. NO donating compounds can be used as replacement therapy to NO donating compounds can be used as replacement therapy to

treat its impaired productiontreat its impaired production NO also as therapeutic potential for Ischemic CVS diseases, NO also as therapeutic potential for Ischemic CVS diseases,

pulmonary hypertension associated with cardiac and respiratory pulmonary hypertension associated with cardiac and respiratory diseases.diseases.

They are far from ideal because of the associated side effect mainly They are far from ideal because of the associated side effect mainly due to the catabolism of NO into NO2due to the catabolism of NO into NO2

Therefore a technology to regulate in vivo synthesis of NO by Therefore a technology to regulate in vivo synthesis of NO by genetic manipulation would be a welcome move.genetic manipulation would be a welcome move.