1. NITRIC OXIDE

2. NITRIC OXIDE BY
SUKHENDU CHOWDHURY
MODERATOR
Dr. ULLAS KAMATH

3. NITRIC OXIDE INTRODUCTION HISTORICAL CONSIDERATION
CHEMISTRY OF NITRIC OXIDE
BIOSYNTHESIS OF NITRIC OXIDE
NITRIC OXIDE SYNTHASE (NOS)
NITRIC OXIDE RELEASE
METABOLIC FATE
MECHANISM OF ACTION OF NITRIC OXIDE
PHYSIOLOGICAL ACTION OF NITRIC OXIDE
NITRIC OXIDE IN DISEASE AND TREATMENT
NITRIC OXIDE, THE FUTURE
CONCLUSION

4. INTRODUCTION molecular oxygen and L-Arginine. signaling molecule.
immune defense.
glyceryl trinitrate in angina via NO.
inhalation to relieve pulmonary hypertension.
“MOLECULE OF THE YEAR” by Science (1992)

5. HISTORICAL CONSIDERATION
1916 ,Tonnenbaum and his colleagues - nitric oxide was being biosynthesized mainly outside the intestine.
In 1977, Ferid Murad - vasodilatory effect of nitroglycerine due to the release of NO.
In 1980, Robert Furchgett showed that ‘endothelium derived releasing factor (EDRF) is required for arterial dilatation.
In 1987, Louis Ignarro showed that EDRF is chemically NO.

6. Murad, Furchgett and Ignarro were awarded Nobel Prize in 1988.
Robert Furchgott
Louis Ignarro
Ferid Murad

7. CHEMISTRY OF NITRIC OXIDE
diatomic, colorless gas.
uncharged molecule having an unpaired electron.
highly reactive free radical
correctly written as NO.
freely diffusible across the membranes.
short half-life about 0.1 secs. N O

8. CONT..... high affinity for haem and other iron-sulfur groups.
activation of guanylate cyclase which contains a heme group.
inactivated by binding to hemoglobin.
circulates in the mammalian plasma bound to SH-groups of albumin.

10. BIOSYNTHESIS OF NO
L- Arginine and molecular oxygen catalyzed by Nitric Oxide Synthase (EC )
initial step is a hydroxylation of nitrogen in the guanidine group of L-Arginine.
incorporates molecular oxygen into NO and citrulline.
Reduced pyridine nucleotides, reduced biopteridines and calmodulin.

11. By product of reaction, citrulline is recycled back to L-Arginine incorporating one nitrogen. This modified urea cycle thus has 2 functions- secretary role to regenerate L-Arginine for NO synthase and an excretory role to eliminate excess nitrogen created by the cell’s metabolism.
COO-
COO-
COO-
+ O2
+H3N C H
+H3N C H
NADPH
NAD+
+H3N C H + NO
(CH2)3
(CH2)3
(CH2)3
NOS
NOS NH NH NH + C
NH2+ C N OH C H
H2N
H2N O
NH2
Arginine
N-w-Hydroxyarginine
Citrulline

12. NITRIC OXIDE SYNTHASE (NOS)
Are central to the control of NO biosynthesis.
first identified and described in 1989.
have 3 isoforms.
product of 3 different genes.
dimers.
similar to cytochrome P-450 enzymes.

13. Each isoform contains
Iron Protoporphyrin IX (Heam)
FAD
FMN
tetrahydrobiopterin(BHP) as bound to prosthetic groups.
Also contain binding sites for
L-Arginine
NADPH
Calcium Calmodulin
These prosthetic groups and ligands control the assembly of the enzyme into active dimer.

14. ISOENZYMES OF NOS NOS have 3 isoforms which are:-
NOS I - Neuronal NOS (n NOS)
NOS II - Inducible NOS (i NOS)
NOS III – Endothelial NOS (e NOS)

15. NOS-I or Neuronal NOS (n NOS)
central and peripheral neurons.
cytoplasmic enzyme.
Activated by calcium
Chromosome 12.

16. NOS-II or inducible NOS ( I NOS)
macrophages, neutrophils, kupffer cell, fibroblasts, and vascular smooth muscles.
Induced by cytokines (IL 1, TNF), during inflammation / micro organisms.
cytoplasmic enzyme.
Calcium does not activate.
Chromosome 17.

17. NOS III or Endothelial NOS (e NOS)
endothelium cells, platelets, endocardium, myocardium, renal mesanglial cells, osteoblasts and osteoclasts.
constantly produced and released.
Localized in plasma membrane.
activated by calcium.
Chromosome 7.

18. NO RELEASE
CONSTITUTIVE NO RELEASE
INDUCED NO RELEASE

19. Constitutive NO Release
NOS I ( n NOS) & NOS III (e NOS)
Synthesize small amount of NO on demand.
stimulus – receptor activation – Ca NOS NO
act on target cells e.g.- vascular smooth muscle, platelets etc.

21. Induced NO release Stimulus – cytokine receptor – iNOS – NO
iNOS isoenzyme.
large amount of NO continuously.
acts as a killer molecule.
Stimulus – cytokine receptor – iNOS – NO
NO + superoxide anion -- cGMP & DNA

23. METABOLIC FATE With oxygen form nitrites - excreted through urine.
With Hb, NO is converted to nitrates - excreted in urine.
Very low quantity expelled through lungs.
With superoxide anion (O2-), - peroxy nitrite (OONO.) which causes lipid peroxidation, cell injury & cell death.

24. MECHANISM OF ACTION OF NO
Constitutive NOS acts via Calcium-calmodulin mechanism to release NO which acts on smooth muscle endothelium & regulate the level of Guanylate Cyclase, producing cGMP& thus muscle relaxation.

25. Endothelial
cells
Smooth Muscle cells
sukhendu

26. PHYSIOLOGICAL ACTIONS OF NITRIC OXIDE
Circulatory and cardiovascular system
Pulmonary system
Nervous system
Muscular system
Immune system
Digestive system
Other actions

27. NO in the circulatory and cardiovascular system
As a vasodilator
NO -- GC -- cGMP -- relaxation
Stimulus -- high blood flow rate and signaling molecules (Ach and bradykinin).
Highly localized and effects are brief.
If NO synthesis is inhibited, blood pressure skyrockets.

28. Inhibits platelet aggregation and adhesion.
mechanism dependent on cGMP & synergies with prostacyclins.
Platelets themselves generate NO -- negative feed back mechanism.
beneficial effect on blood coagulation by enhancing fibrinolysis.

29. NO in the pulmonary system
Nerve dependent bronchodilatation.
treating pulmonary hypertension.
inhaled NO (18-16 ppm) - adult respiratory distress syndrome.
Specific vasodilatation - well ventilated segments of the lung.
inactivation by binding to Hb in blood.

30. NO in the nervous system
As a neuro transmitter
signaling molecule, but not necessary a neurotransmitter.
signals inhibition smooth muscle contraction, adaptive relaxation & localized vasodilatation.
Role in vision, feeding behavior, nociception & olfaction.

31. Role in long term memory -- retrograde messenger.

32. Is NO a neurotransmitter?
Differences :
synthesized on demand vs. constant synthesis.
diffuses out of the cells vs. storage in vesicles and release by exocytosis.
does not bind to surface receptors, but with intracellular guanylate cyclase.
Similarities :
Present in presynaptic terminal
Natural removal from synaptic junction

33. NO in the muscular system
Originally called EDRF
Signals inhibition of smooth muscle contraction
Ca2+ -- eNOS – NO – cGMP – Ca Pump -- relaxation

35. NO in the immune system
NOS II catalyzes synthesis of NO used in host defense reactions.
independent of Ca 2+
most nucleated cells, macrophages
potent inhibitor of viral replication.
may damage neighbouring cells
Bactericidal agent
kills bacteria in the mouth

36. NO in the digestive system
Adaptive relaxation
Promotes stretching of the stomach in response to filling.
stretch receptors trigger muscle relaxation through NO releasing neurons.

37. Other Actions In diabetes
Induced NO formation may play a role in the dysfunction of the pancreatic beta cells during the development of type I diabetes.

38. Impotence
Physiological mediator of penile erection. NO causes relaxation of corpora cavernosa & thus development of penile erection in humans.

39. Apoptosis able to induce and inhibit apoptosis.
inhibits - leukocytes, hepatocytes, trophoblasts & endothelial cells.
antiapoptotic effects via nitrosylation & inactivation of caspases, activating p53, cGMP signalling etc.

40. NO

41. CLINICAL SIGNIFICANCE OF NITRIC OXIDE
NO in Pathophysiology
underproduction of neuronal NO - babies with hypertrophic pyloric stenosis.
reduced Endothelial NO production - hyper cholestrolemia and arteriosclerosis, contribute to atherogenesis.
overproduction of NO - neurodegenerative diseases, septic shock, & dysfunction of photoreceptor cells.

42. NO in diseases and treatment
Nitroprusside, nitroglycerine – Angina pectoris
Inhalation of NO (18-36 ppm) – Pulmonary hypertension, Acute Respiratory Distress syndrome.
Arginine – Coronary vascular dysfunctions
Sildenafil – Erectile dysfunction

43. Cont….
NOS inhibitors( L-NMMA- NG –monomethyl -L-arginine) – Septic shock
Increase NO activity in brain – Alzheimer’s disease, Multiple Sclerosis, Huntington's disease.
S-nitro glutathione – inhibits platelet aggregation.
N-acetyl Cysteine – protects and enhances NO activity

44. Antagonists of NO Glucocorticoids - biosynthesis of iNOS.
Synthetic arginine analogue - NOS
Eg; L-NMMA – (NG-Monomethyl-L-Arginine)
L-NAME – (NG-Nitro-L-Arginine Methyl ester).
ADMA (Asymmetric dimethyl arginine) - eNOS.
7-Nitroimidazol – nNOS.
Heme – NO Scavenger.
Protein inhibitor of NOS(PIN) – Endogenous inhibitor found in brain.

45. MEASUREMENT OF NO Electrochemical detection.
analysis of nitrite and nitrate by ion exchange chromatography & spectrophotometry.
reversed phase HPLC with fluorescence detection.

46. Nitric Oxide, The future
preserve endothelial integrity and boost failing L-Arg /NO system.
new NO donors having both vasodialating & antiadhesive effects.
treatment of incontinence.
inhalation of NO – treating pulmonary hypertension & bronchial asthma.
specific inhibitor of iNOS – septic shock, inflammation, stroke & diabetes.

47. CONCLUSION
mediator of homeostatic processes and host defence mechanisms.
gaseous signaling molecule
Generated from L-Arginine by the action of an enzyme NOS having 3 isoforms.
Regulates physiological and pathophysiological processes involving CVS, Inflammation, Immune & Neuronal function.

48. Cont…. NO donors - as replacement therapy.
Selective inhibitors of i NOS - treatment of hypotension of shock.
Selective inhibitors of brain NOS -degenerative disease of the nervous system.
Nitrite, nitrate, L- Citrulline, may become clinical markers for monitoring certain diseases and their progresses.

49. THANK YOU

50. References Lancet – 1994;343:1199-1206
New England Journal Of Medicine – 1993; 329:
American Journal Of Surgery – 1995; 170:
D M Vasudevan, Sreekumari S: Text Book Of Biochemistry,4th edition,Jaypee,2005
Rang: Pharmacology,5th edition,Elsevier,2003
Katzung: Basic & Clinical Pharmacology,9th edition,McGrawHill,2004
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