This document provides an overview of nitric oxide (NO) including its chemistry, synthesis via nitric oxide synthase (NOS) enzymes, mechanisms of action, roles in various organ systems, and therapeutic applications. Key points discussed include the 3 isoforms of NOS (eNOS, nNOS, iNOS), NO's vasodilatory effects via soluble guanylate cyclase activation, roles in inflammation and the cardiovascular, nervous, and reproductive systems, and use of NO donors and NOS inhibitors for conditions like pulmonary hypertension and erectile dysfunction.

1. Dr PRASHEETAV PRAVIRAJ
FINALYEAR
DEPARTMENTOF PHARMACOLOGY
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2.  Introduction
 Chemistry of NO
 Synthesis of NO
 NOS enzymes
 Pharmacokinetics of NO
 Mechanism of action
 Role of NO on various organ systems
 NO therapeutics
 Conclusion
 References
2

3.  Fig 1 : Polygraph reading ( tension under isometric
contraction)- rabbit aorta strip precontracted with
noradrenaline ( Courtesy : Rang & Dale)
3

4.  Until 1980 : NO as an air pollutant.
 1980: EDRF
 1992 :Molecule of the year
 1998 : Nobel prize in Physiology/Medicine
Robert F Furchgott
Louis J Ignarro
Ferid Murad
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5.  Small diatomic molecule
 It has unpaired electron in its valence shell
 Highly reactive – looses the extra e- or gaining the
missing e-.
 Paracrine mediator : able to rapidly (lipid soluble)
and easily penetrate across cell membrane.
 Biological t1/2 = 1-100nmin
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6. Other sources of nitrates :
1. Dietary nitrates : beetroot juice , dark chocolate,
pepper .
2. Nitric oxide donors
6
Fig 1 : Biosynthesis of NO

7.  Dimeric flavoproteins ~ CYP 450
 3 distinct domains :
- Reductase domain
- Calmodulin binding domain
- Oxygenase domain
 3 isoforms of NOS
- eNOS (endothelial)
- nNOS (neuronal)
- iNOS (inducible)
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constitutional

8. Characteristics eNOS nNOS iNOS
Distribution Cardiac myocytes ,
platelets , osteoblasts ,
osteoclasts , renal
mesangial cells
,endothelial cells
Peripheral and
central neurons
Macrophages ,
fibroblasts , smooth
muscles , endothelial
cells and other
inflammatory cells
Activation Ca2+ -calmodulin
binding dependent
Same as eNOS Ca2+ -calmodulin
binding independent
Duration of
release
Short lasting Short lasting Long lasting
Concentration Picomoles Picomoles Nanomoles
Location Membrane bound Cytosolic Cytosolic
Stimuli Shear mechanical strain
, Histamine,
Acetylcholine ,5HT
Bradykinin ,ATP
Substance P
Same as eNOS Pathological stimuli
-Microorganisms,
endotoxins
- Cytokines
Inhibitors L-NAME
7-nitroindazole
Same as eNOS Glucocorticoids
TGF-β
8

9.  Absorbed : systemically after inhalation
 Distribution : low
 Metabolism : inactivated by haem , oxidation ,
superoxide dismutase
 Elimination : as nitrates in urine
: small amount is exhaled via lungs
 No known storage mechanism - nitrites
9

10.  Haemoglobin
2NO + O2 = N2O4
N2O4 + H2O = NO3- + NO2- + 2H+
NO2- + HbO = NO3- + Hb
 Superoxide dismutase
NO + O2 x ONOO
GLUTATHIONE
Inactivates –SH containing enzymes
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11. 11
Fig 2 : activation of guanylyl cyclase enzyme ( major mechanism)

12. Target Mechanism Effect
Heme protein Nitrosoheme Activates soluble GC
Metheme Inhibits hemoglobin and myoglobin
- SH proteins Nitrosothioproteins Activates t-PA : plasma nitroso albumin
Inhibit dehydrogenases
Fe-S protein Nitroso-iron sulfur
proteins
•Inhibit NADH : Ubiquinone
oxidoreductase
•NADH : succinate oxidoreductase
•Cis – aconitase
•Ferritin
•Ribonucleotide reductase
Fe 2+ & free thiols Dinitrosyl Fe(II)
complexes
Depletion of iron in cells
Acts as a transport form of NO
Free amine Nitrosamine Tumorigen
DNA Deamination of DNA
DNA binding protein
Mutagen
Induction/Suppression of expression
(Carcinogen)
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13. NAD dehydrogenase Auto ADP ribosylation Inhibits glyceraldehyde – 3-
phosphate dehydrogenase
O2 Nitrites , nitrates Inactivation of NO
O2
- Peroxynitrite ,hydroxyl Toxifications of NO
13
Fig 3 : Alternative pathway for vasodilatation (courtesy :
Golan )

14.  Cardiovascular system
 Inflammation &
Immunity
 Nervous system
 Respiratory system
 Gastrointestinal
system
 Musculoskeletal
system
 Reproductive system
 Mutagenesis
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15.  Vasodilatation
 Antithrombotic
 Antiproliferative
 Anti-inflammatory
 Embryonic fetal heart development
 Gaseous exchange between Hb & tissue
 Pregnancy : generalised vasodilatation
Vascular integrity
Anti –
atherosclerotic
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16. 16

17.  Decreased levels of glutathione = increased levels of
peroxynitrite ions(ONOO)
 Pre-eclampsia
 Hypertension : mutant mice lacking NOS3
 Diabetes mellitus (endocrine disorder)
 Atherosclerosis
 Cardiovascular complications secondary to other
 CAD : Coronary artery diseases
 Sickle cell disorders : heme scavanges NO .
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18. Pro-inflammatory cytokines Endotoxins
Increased inducible NOS expression
1000 fold increase in the NO levels
(monocytes , macrophages, neutrophils
& granulocytes)
NO diffuses into ECF Destroys the microbes without
prior phagocytosis
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19. “Double edged sword”
 Physiological levels : anti-inflammatory
- kills invading pathogens ( antibacterial , antiviral , tumoricidal
,antifungal , antiprotozoal , apoptosis , antiparasitic )
- non specific immunity
 Pathological levels: pro-inflammatory
-bacterial sepsis
-transplant rejection
-inflammation
-autoimmune disorders
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20.  Neurotransmitter/neuromodulator
-- it enhances the release of other neurotransmitters by
activating soluble guanylyl cyclase .
-- learning and memory (inc glutamate release - NMDA)
* NO as NT vs other NTs
-- not regulated by storage , release , degradation of NO
-- doesnot require receptors
-- Ca2+ requires for NO synthesis
-- synthesized on demand
* PNS : NANC neurotransmitter
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21.  Increases cerebral blood flow
 Nociception : decreases the pain threshold by sensitizing the
neurons
-- chronic pain : increased NO levels
 Over production of NO during inflammation
Activation of glial cells in brain Cytokines , fatty acid
metabolites , free
radicals
Neurodegeneration Excitotoxicity
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22.  Retrograde messenger
- acting via mGlu receptors
it blocks caspase 3 – PCD inhibited
 Recently hypothesis ,
- receptors for NO is present
intracellularly in presynaptic
neuron .
 Stroke
 AIDS dementia
 Huntington disease
 Parkinsons disease
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Fig 4 : Retrograde messenger
Courtesy:textbook on glutamate
receptors (Pelkey & Mcbain) Pg 182

23.  NOS2 is present in alveolar epithelial cells and smooth
muscles
 Vasodilatation of bronchial vessels
 Bronchodilatation
 Lung inflammatory disorders : increased NO dependent
oxidative stress indices
 Asthmatic patients : NO levels high in expired breath
 IND : NO containing steroid moeity
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24.  Pulmonary vasodilatation
-- useful in pulmonary hypertension
-- children & adult respiratory distress syndrome
-- current modality Noenatal ARDS : ECMO
-- when inhaled it has minimal systemic effects.
 Clinical trial
-- Cystic fibrosis and NO donor drugs
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25.  ENS : NO is NANC (Non adrenergic Non cholinergic )
neurotransmitter
 NOS 1 enzyme concentrated in pyloric region of stomach
--Hastens gastric emptying
--Infants with hypertrophic pyloric stenosis lack NOS1 in the
stomach.
 Effect on GI secretion
-- NO increases mucus and epithelial cell fluid
--thus improves host defences against microbes, toxins and
irritants
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26.  Effect on Mucosal blood flow
-- NO potent vasodilator
-- buffering of acid
-- dilution of toxins
-- stimulates angiogenesis
 Inflammatory bowel diseases
--Crohn’s disease
--Ulcerative colitis
-- Rx : cortocosteroids has shown decreased urinary nitrates
Protective influence over
gastric mucosa
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27.  Physiological levels :
-- inhibit bone resorption by osteoclasts
-- acute protective effect in cartilage breakdown
 Pathological amounts
--increase bone resorption
-- bone proliferation decreased
-- induces chondrocyte apoptosis
 Location of NOS : synoviocytes , osteoblasts, chondrocytes,
synovial membrane , fibroblasts
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28.  Rheumatoid arthritis , osteoarthritis , ankylosing spondylitis
 Increased serum concentration of nitrates
 Increased urinary concentration of nitrates , cGMP.
 Index of NO-dependent oxidative damage : Nitrotyrosine
 ---- peroxynitrite of tyrosine
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29.  Vasodilatation of blood vessels in corpus cavernosum
 Helps in development and maintainence of penile erection.
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Fig 5 : Action of PDE 5 inhibitors ( Courtesy : Furchgott.at. al
1984)

30.  Mutagen , tumorigen , carcinogen
 The nitric oxide binds to the free amines ,DNA , DNA binding
proteins
 Thus they induce or suppress the expression of a particular
gene
 Procarcinogen = carcinogen
 Inactivation of tumor suppressor gene (p53)
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31.  Nitric oxide gas
 Nitric oxide donors
 Nitric oxide inhibitors
 Nitric oxide potentiators
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32.  Persistent pulmonary hypertension (newborns)
 Adult & Neonatal respiratory distress
-- dilates the blood vessels in ventilated alveoli
-- should not be associated with sepsis
-- injected into the inspiratory circuit of ventilator
-- NO is stored in aluminium / stainless cylinder in N2
(100/1000/2000ppm)
-- 5-300ppm : safe in humans
-- monitoring : chemiluminescence
electrochemical analyser
-- high conc. : acute pulmonary edema and methaemoglobinaemia
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33. Anti-anginal drugs : amyl nitrite , glyceryl nitrate ,isosorbide
dinitrate , isosorbide mononitrate , erythrityl tetranitrate ,
pentaerythritol tetranitrate.
 Lauder Brunton – 1867
 Nitrates are converted into NO by an enzymatic reaction
involving tissue –SH groups.
 Subsequently NO activates soluble guanylyl cyclase.
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34.  Nitrovasodilators
- Sodium nitroprusside , Nicorandil ,hydralazine
 β adrenergic blockers
- Nebivolol , Celiprolol
 Experimental tools
- SNAP : S-nitro-acetylpenillcillamine
-SNOG : S - nitrosoglutathione
- NOC-18 : long acting
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35.  COX inhibitory NO donors
 Addition of an NO releasing moiety
 +/= analgesic & anti-inflammatory
 Protective effect on gastric mucosa (NO)
 NO-Aspirin (NCX4016)
 Its more potent in inhibiting platelet activation induced by
thrombin
 + antiproliferative , antiapoptic action
 Cancer treatment trials – colon cancer
 NO-Statins
NO Pravastatin ,NO fluvastatin
 NO- ACE inhibitors
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36.  PhosphodiesteraseV inhibitors
-Sildenafil ,Tadalfil , vardenafil
- pulmonary hypertension , erectile dysfunction.
 Antioxidants
 Statins – restore endothelial function (pleotropic effect)
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37.  NMMA : N-Mono Methyl Arginine
 ADMA : Asymetric DiMethyl l- Arginine
 L-NAME : N-Nitro-L-Arginine Methyl Ester
 Arginine analogues compete with arginine for NOS
 Experimental tools
 ADMA : endogenously present in plasma
--hypercholestrolaemia , haemodialysis patients ; increased
levels
-- plasma conc α vascular mortality
 Other inhibitors : trials have failed to produce favourable
results
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38.  Nonspecific NO inhibitors
 Glucocorticoids : inhibit the expression of inducible NOS
 Cyclosporin : same as above – decreases IL1 production
 Salicylates : scavengers of NO
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39.  Psychiatric diseases and NOS1
Psychiatric disorder related to implusive behaviour and
impaired prefrontal brain functioning .
Jail inmates and nitric oxide study
No drugs so far .
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40.  Nitric oxide was promoted from the status of an air
pollutant to “molecule of the year” .
 NO was followed by discovery CO ,H2S as a signaling
molecules in CVS and CNS.
 NO as a central neurotransmitter is not yet targeted
even though its role is quite well understood.
 Emergence of new branch “gasotransmitters”(2002)
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41.  https://www.nobelprize.org/nobel_prizes/medicine/laurea
tes/1998(1)
 Schmidt, Harald H. H.W. Et al,The nitric oxide and cGMP
signal transduction system: regulation and mechanism of
action , Biochimica et Biophysica Acta (BBA) - Molecular
Cell Research , vol 1178, 153-175.
 Rang H.P et al , Nitric oxide and related mediators, Rang &
Dales Pharmacology;8th edition; Chapter20; Pg 237-244
 David.E.Golan; Nitric oxide; Principles of Pharmacology ;
edition 4;chapter 21;pg356-357
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42.  Samie R Jaffrey ; Nitric oxide ;; Basic & Clinical Pharmacology
(13th edition) ; chapter 19 ;Pg 329-334
 Sharma J.N.et.al. ; Role of nitric oxide in infl ---ammatory
diseases; Inflammopharmacology 15 (2007) 252–259
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