Nitric Oxide and Its

UtilityBy

Dr Ketan Asawalle

JR1

Dept of Pharmacology

SVNGMC Yavatmal

Formula: NO

IUPAC ID: Nitric oxide

Molar mass: 30.01 g/mol

Density: 1.34 kg/m³

Boiling point: -152 °C

Soluble in: Water

Nitric Oxide (NO) is a novel mediator with diverse function

It is generated from L-argenine by nitric oxide synthase(NOS)

NOS occurs in endothelial, neuronal and inducible isoforms

Nitric oxide is a FREE RADICAL gas that forms during lightening storms

NO

NO

NO

NO

NO

NONO

NO

NO NO

NO

It was suspected that NO is produced in human body when cultures of MACROPHAGES were subjected to INFLAMMATORY mediators like

VASCULAR STUDIES also indicated towards the same

Mediators like acetylcholine were known to relax blood vessels

But this was seen only when LUMINAL ENDOTHELIAL CELLS near SMOOTH MUSCLES were preserved

They secreted EDRF

The physiological function

of NO was discovered

in the vasculature when

it was shown that the

endothelium derived

relaxing factor was

NOFURCHGOTT

ZAWADZKI

In the human body NO is formed by an enzymatic catalysed reaction between oxygen and L

Key role as a signalling molecule in

• CARDIOVASCULA SYSTEM

• NERVOUS SYSTEM

Also has a role in HOST DEFENCE

NO is endogenous activator of Guanylyl cyclase

Activates cGMP

Secondary Messenger

Nerves, smooth muscles, monocytes and platelets

Nitrogen and oxygen are neighbours in periodic table

so they have a few similar properties

For instance

HAEM

This is important for activation of guanylyl cyclase as it has a haem group

Similar mechanism acts in inactivation of NO by Haemoglobin

BIOSYNTHESIS OF NITRIC OXIDE

NITRIC OXIDE SYNTHASE

Enzyme in central control of Nitric Oxide biosynthesis

iNOS

NOS-II

nNOS

NOS-I

eNOS

NOS-III

i = Inducible n = Neuronale = Endothelial

iNOS

NOS-II

nNOS

NOS-I

eNOS

NOS-III

•Macrophages

•Kupffer cells

•Neutrophills

•Fibroblasts

•Vascular

smooth muscles

•Endothelium

•Cardiac myosites

•Osteoblasts

•Osteoclasts

•Neurons

PHYSIOLOGICAL

CONSTITUTIVE FORMS

PATHOLOGICAL

The activity of constitutive isoforms of NOS is controlled by

CALCIUM CALMODULIN

by TWO ways

Endothelium dependent Agonists like

Acetycholine,bradykinin,Substance P

Cytoplasmic Calcium ions

Calcium Calmodulin

Activates eNOS and nNOS

In the absence of any change in calcium ion concentration

Phosphorylation of specific resides on eNOS

Controls Calcium-Calmodulin concentration

The most important stimulus controlling endothelial NO synthesis is

SHEAR STRESS

Shear stress is sensed by mechanoreceptors and transduced via SERINE-THREONINE protein kinase channel Akt

cAMP

eNOS

Protein

Kinase A-

mediated

phosphorylation

e.g. ß2 agonists

Protein C kinase

eNOS

Phosphorylation

of residues in

calmodulin

binding domain

INSULIN

eNOS

Tyrosine Kinase

Activation

Unlike constitutive NOS, iNOS does not depend on Calcium for activation

iNOS is activated by

• Bacterial Lipopolysaccharides

• Cytokines synthesised in response to Lipopolysaccharides

Tumour Necrosis factor alpha

Interlukin-1

Involved in induction of iNOS

But Not Alone

INTERFERON GAMMA

DEGRADATION OF NITRIC OXIDE

2NO + O2 ——> N2O4

N2O4 + H2O ——> NO3- + NO2- + 2H+

NO2- + HbO ——> NO3- + Hb

Nitric Oxide is inactivated by

• Combination with haem of haemoglobin

• Oxidation to Nitrite or Nitrate

Excreted through

MAJOR ROUTE

NO is unstable and combine reversibly with CYSTINE residues

NO + Cystine residues of Albumin and Globulin

Stable Nitrosothiols

They act as circulation O2 sensitive NO carriers

REDUCTIVE reactions with a variety of oxides of nitrogen that can nitrosylate thiols and nitrate tyrosine

NO + PEROXISIDES ––> PEROXYNITRATE(ONOO-)

• DNA damage

• Nitration of tyrosine

• Oxidation of Cystine

EFFECTS OF

NITRIC OXIDE

Nitric Oxide is a volatile gas and it reacts with various metals, thiols and oxygen

One of the most important function of NO is activation of soluble Gyanylyl Cyclase which activates cGMP

NO activates enzymes by combining with haem group which is important for many physiological functions

BIOCHEMICAL AND CELLULAR ASPECTS

Pharmacological properties of NO can be studied with NO gas dissolved in deoxygenated salt solution

Various donors od NO can also be used to study the same e.g.

• Nitroprusside

• S-nitrosoacetylpenicillamine(SNAP)

• S-nitroglutathine(SNOG)

BIOCHEMICAL AND CELLULAR ASPECTS

NO can activate Gyanylyl Cyclase in the same cell that produces it

AUTOCRINE EFFECTS

e.g. Barrier function of endothelium

BIOCHEMICAL AND CELLULAR ASPECTS

NO can diffuse away from site of synthesis and activate Guanylyl Cyclase

This affects protein G, cyclic nucleotide phosphodiesterase, ion channels, etc.

INHIBITS calcium induced smooth muscle contraction and platelet aggregation

ANTIATHEROSCLEROTIC

BIOCHEMICAL AND CELLULAR ASPECTS

Inhibits

Smooth muscle

and

Fibroblast

proliferation

Migration

adhesion and

aggregation of

platelets

Monocyte

adhesion

BIOCHEMICAL AND CELLULAR ASPECTS

NMDA receptors when stimulated excessively produce NOS

Nitric Oxide

HOST

DEFENCE

NEURONAL

DESTRUCTION

Through

DIRECT EFFECT

PEROXYNITRATE ANIONS

VASCULAR EFFECTS

Argenine/NO pathway is tonically active in resistance vessels

The continuous release of NO keeps vasodilation in resistance vessels thus maintaining Blood Pressure

It is Anti-Atherosclerotic

Anti-Thrombotic

NEURONAL EFFECTS

Cholinergic mediators in many tissues like upper airway, GIT, Penis(errection)

Important for neuronal development

Helps in synaptic Plasticity

HOST DEFENCE

NO has Cytotoxic/Cytostatic effects

This has been proved experimentally in mice

Mice without iNOS were susceptible to Leishmania major

SEPTIC SHOCK

Systemic inflammatory response

Endotoxins

TNF-alpha

Cytokines

Macrophages

Neutrophils

T cells

Hepatocytes

Smooth muscle cells

Endothelial cells

Fibroblasts NO

HYPOTENSION

Actions of NO

Actions of NO

THERAPEUTIC

APPLICATIONS

NITRIC OXIDE

Inhalation of large quantities of nitric oxide causes

• ACUTE PULMONARY OEDEMA

• METHAEMOGLOBINEMIA

NO between 5 - 300 ppm has bronchodilatory action in guinea pigs

MAIN ACTION

PULMONARY VASODILATATION

Inspired NO preferentially acts on VENTILATED ALVEOLI

ARDS

ARDS is caused by different reasons causing shunting of arterial blood to venous blood

NO acts on arteries of PERFUSED areas and causing VASODILATION

ETHYL NITRIC gas is being used to reduce toxicity

NITRIC OXIDE DONORS/PRECURSORS

• GLYCERYL TRINITRATE(GTN)

• S-NITROSOGLUTATHIONE(SNOG)

• S-NITROSOACETYLPENICILLAMINE(SNAP)

• NITROPRUSIDE

The common mode of action of these drugs is

DONATION of NO

GTN is more potent on VASCULAR SMOOTH MUSCLES

SNOG selectively INHIBITS PLATELET FUNCTION

Dietary Nitrates (Beetroot) lowers arterial BP parallel to rise in plasma Nitrate concentration

SALIVARY conversion od Nitrate to Nitrite causes lowering of nitrites in body leading to rise in BP and abolishe

Rapidly acting Consistantly acting

Sodium Nitropruside

Brief duration of action - 2 to 5 minutes

Dose can be TITRATED

Relaxes both RESISTANCE and CAPACITANCE vessels

REDUCES both TOTAL PERIPHERAL RESISTANCE and CARDIAC OUTPUT

Myocardial work Reduced

Sodium Nitropruside

Sodium Nitropruside

Endothelial cells RBCs

Na Nitropruside

NITRIC OXIDE

EnzymaticallyNon

Enzymatically

50mg in 500 ml of 5% Dextrose

Slow IV

The bottle is covered with dark cloth to avoid oxidation of NO

USES

• Hypertensive emergencies

• Maintenance of patients with hypertension

• CONTROLLED HYPOTENSION in REFRACTORY CCF, Pump failure in MI and Acute MR

Sodium Nitropruside

SIDE EFFECTS

Palpitations

Nervousness

Vomiting

Perspiration

Abdominal pain

Disorientation

Weakness

Lacti acidosis

Sodium Nitropruside

GTN

It a VOLATILE LIQUID

SUBLINGUAL route of administration is preferred

Acts in 1 - 2 minutes and peaks in 3 - 6 minutes

T1/2 is 2 minutes

Duration of action depends on the amount of time the drug is in contact with the sublingual surface

GTN

GTN

GTN is readily absorbed from skin

OINTMENT produces hemodynamic effects in 4

TRANSDERMAL PATCH produces effect in 60 minutes

IV administration produces stable titrable effects

GTN

ISOSORBIDE DINITRATE

SUBLINGUAL route gives peak in5 - 6 minutes

ORALY T1/2 is 40 minutes but sustained release can give effect up to 8 hours

ISOSORBIDE MOMONITRATE

Active metabolite of Isosorbide dinitrate

USES

• Angina Pectoris

• Acute Coronary Syndrome

• MI

• CHF with acute LVH

• Biliary colic

• Esophagial spasm

• Cyanide poisoning

CYANIDE POISONING

Haemoglobin

Methaemoglobin

CYANIDE

Cyanomethhemoglobin

Methaemoglobin + Na Thiocynate

SODIUM

NITRITE

AMYL

NITRITE

SODIUM

THIOSULPHATE

INHIBITORS OF

NITRIC OXIDE

SYNTHESIS

N-MONOMETHYL-L-ARGENINE(L-NMMA)

NITRO-L-ARGENINE METHYL ESTER(L-NAME)

ASSYMETRIC DIMETHYL GLYCERENE

NON SELECTIVE

NMMA when infused slowly in brachial artery causes LOCAL VASOCONSTRICTION

L-NMMA acts through nNOS

When Given IV

Vasoconstriction of Renal, Mesenteric and Cerebral vessels

Sustained muscle resistance and increased BP

SELECTIVE

• iNOS - N-iminomethyl-L-lysine

•nNOS - 7-nitroindazole

S-methyl-L-thiocitruline

POTENTIATORS OF NITRIC OXIDE

SELECTIVE NO DONORS

GTN

SNOG

SNAP

DIETARY SUPPLIMENTS

BEETROOT

ANTIOXIDNTS

Reduce free oxygen radicals and stabilise NO

ACE inhibitors, Statins, Insulins, Oestrogens

RESTORE ENDOTHELIUM FUNCTION

ß2 AGONISTS

Activate L-Argenine/NO Pathway

PHOSPHODIESTERASE V INHIBITORS

Sildenafil, Tadanafil, Vardenafil

PHOSPHODIESTERASE V INHIBITORS

Used in treatment of ERECTILE DYSFUNCTION

SILDENAFIL

TADANAFIL

VARDENAFIL

NOGenerates cGMP

Dephosphorylates

MLCK

PD-5SILDENAFIL

TADANAFIL

VARDENAFIL

5-GMP

SILDENAFIL

Selective PD-5 Inhibitor

Enhances NO activity in CORPORA CAVERNOSA

Oral bioavailability is about 40%

Peak blood levels attained in about 1 - 2 hours

Metabolised by CYP3A4

SILDENAFIL

SIDE EFFECTS

Headache

Nasal congestion

Dizziness

Facial flushing

Fall in BP

Pulmonary HTN

TADANAFIL

Faster onset of action

Longer lasting

T1/2 18 hours

Duration of action 24-36 hours

Peak Plasma levels in 80 - 120 minutes

UTILITY OF NITRIC OXIDE

HYPERTENSION

Nitroprusside

Maintenance

Emegencies

Na Nitroprusside

GTN

Isosorbide

UTILITY OF NITRIC OXIDE

ERECTILE DYSFUNCTION

Sildenafil

Tadanafil

Vardenafil

UTILITY OF NITRIC OXIDE

PULMONARY HTN and GASTRIC STASIS

NO is under investigaton

ADULT and NEONATAL RESPIRATORY DISTRESS SYNDROME

INHALED NO and ETHYL NITRIC GAS

UTILITY OF NITRIC OXIDE

NO inhibitors are under investigation for conditions where there is excess NO production e.g. INFLAMMATION, SEPSIS, NEURODEGE

THANK

YOU

References1. Rang and Dale’s Pharmacology 6th edition

2. Basic and Clinical Pharmacology; Katzung 12th

edition

3. Essential of Medical Pharmacology; K D Tripathi

7th edition