Pharmacology of Nitric oxide

RVS Chaitanya koppala

ENDOTHELIUM

ENDOTHELIUM The endothelium is the thin layer of cells that lines the interior surface of

Blood vessels and lymphatic vessels forming an interface between circulating

blood and lymph in the lumen and the rest of the vessel wall.

The cells that form the endothelium are called endothelial cells.

Endothelial cells in direct contact with blood are called vascular endothelial

cells where as those in direct contact with lymph are known as lymphatic

endothelial cells.

• Endothelial cells release substances acting directly on vascular

smooth muscle cells, causing either contraction or relaxation.

In 1980 Furchgott & Zawadzki first described endothelium- dependent relaxation of

the blood vessels by acetylcholine.

Further studies in 1984 revealed that other factors such as bradykinin, histamine

and 5-hydroxytryptamine release endothelium derived relaxing factor (EDRF),

which can modulate vessel tone.

In 1987 Furchgott proposed that EDRF might be nitric oxide (NO) based on a study

of the transient relaxations of endothelium-denuded rings of rabbit aorta to ‘acidified’

inorganic nitrite (NO-) solutions and the observations that superoxide dismutase

(SOD, which removes O2-) protected EDRF.

ENDOTHELIUM DERIVED RELAXING FACTOR

•The following year in 1989 the enzyme responsible for NO production, NO synthase, was

discovered and NO pathway was proposed.

• Neuronal and humoral mediators, e.g. Ach, adrenaline , noradrenaline , histamine ,5-

HT, ATP, Adenosine, Substance P , Arginine Vasopressin (AT/VP), Bradykinin,

Thrombin and Ca++ , Insulin, Angiotensin , TNF-α, IL-6 , Arginase , Asymmetric

Dimethylarginine (ADMA), Dimethylarginine Dimethylaminohydrolase (DDAH), etc.

acting in their corresponding receptors or cellular structures can affect production and

release of NO.

NITRIC OXIDE

A chemical compound with formula NO is a free radical gas.

It is first identified as endothelial derived releasing factor(E D R F ).

At high concentration , fight against infectious organism and cancer cell.

At lower concentration helps in regulating the circulatory and central nervous system.

Nitric oxide differs from other neurotransmitter and hormones in a way that it is not

regulated by storage, release , or targeted degradation.

NO does not require receptor for its action when synthesized immediately utilized.

Ca++ clamudulin complex is necessary for nitric oxide synthesis.

Synthesis of Nitric Oxide

Nitric oxide is synthesized from L-arginine.

This reaction is catalyzed by nitric oxide synthase, a 1,2,9,4 amino acid enzyme.

Release & MOA of NO-

INTRACELLULAR MECHANISM:

Nitric oxide also binds to the heme moiety of hemoglobin and heme moiety of

enzyme gunayl cyclase , which is found in smooth muscle cell and most other

cells of body.

When NO formed by vascular endothelium it rapidly diffuses into the blood

where it binds to hemoglobin & subsequently broken down.

which serve as a second messenger for many important cellular function , particular

for signaling smooth muscle contraction.

cGMP induces smooth muscle relaxation by multiple mechanism including-

Increased intracellular cGMP which inhibit ca++ entry into the cell and decrease

intracellular ca++ concentration.

Activates k+ channel which leads to hyper polarization & relaxation .

• Stimulates a cGMP dependent protein kinase that activates myosin light chain

phosphate (MLCK) the enzyme that dephosphorylate myosin light chain leads to

smooth muscle relaxation

Types of NOS

NOS I or n NOS

Central and peripheral neuronal cells, brain, spinal cord, platelets.

Ca++ dependent, used for neuronal communication

Constitutive

NOS II or iNOS

Most nucleated cells, particularly macrophages

Independent of intracellular Ca++.

Inducible in presence of inflammatory cytokines, bacterial liposaccharides.

NOS III or e NOS

Present on Vascular endothelial cells and neuronal cells

Ca+2 dependent

Vascular regulation

NOSConstitutive

Inducible

Nitric Oxide in the human body has many uses which are best

summarized under five categories.

NO in the nervous system

NO in the circulatory system

NO in the muscular system

NO in the immune system

NO in the digestive system

No in the reproductive system

NO in the gene toxicity

No in the apoptosis

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Role of Nitric oxide

NO is a signaling molecule, but not necessarily a neurotransmitter.

NO signals inhibition of smooth muscle contraction, adaptive relaxation, and

localized vasodilation.

NO diffuses out of the cells making it vesicular storage in vesicles and release by

exocytosis

NO does not bind to surface receptors, but instead exits cytoplasm, enters the target

cell, and binds with intracellular guanylyl cyclase

Present in presynaptic terminal

Natural removal from synaptic junction 15

Nitric oxide in the Nervous system NO serves in the body as a neurotransmitter, but there are definite differences.

Role in Neurodegenerative disease Implicated in :- Alzheimer disease

Parkinson disease

All are related to the excessive release of NO & glutamate both.

But in Parkinson's disease Glial cells produce excessive levels of nitric oxide, which may be

neurotoxic for a sub population of dopaminergic neurons.

The presence of glial cells expressing nitric oxide synthase in the substantia nigra of patients

with Parkinson's disease represents a consequence of dopaminergic neuronal loss.

Play a role in long term memory

As a messenger that facilitates long term potentiation of neurons (memory)

Synthesis mechanism involve Ca2+/Calmodulin

activates NOS-I

activates Guanyl cyclase

cycle of nerve action potentials

catalyzes cGMP production

Nitric oxide in the circulatory system

NO serves as a vasodilator

Released in response to high blood flow rate and signaling molecules (Ach and

bradykinin)

Highly localized and effects are brief

If NO synthesis is inhibited, blood pressure increases

NO aids in gas exchange between hemoglobin and cells

Hemoglobin is a vasoconstrictor, Fe scavenges NO

NO is protected by cysteine group when O2 binds to hemoglobin.

During O2 delivery, NO locally dilates blood vessels to aid in gas exchange

Nitric oxide in the Muscular system

NO was originally called EDRF (endothelium derived relaxation factor)

NO signals inhibition of smooth muscle contraction

Ca 2+ is released from the vascular lumen activating NOS

NO is synthesized from NOS III in vascular endothelial cells

This causes guanyl cyclase to produce cGMP

A rise in cGMP causes Ca2+ pumps to be activated, thus reducing Ca2+

concentration in the cell

This causes muscle relaxation

Role in Blood vessels

Role in the Immune system NOS II catalyzes synthesis of NO used in host defense reactions

Activation of NOS II is independent of Ca2+ in the cell

Synthesis of NO happens in most nucleated cells, particularly macrophages.

NO is a potent inhibitor of viral replication.

NO is a bactericidal agent

NO is created from the nitrates extracted from food near the gums.

This kills bacteria in the mouth that may be harmful to the body.

Role In the Digestive systemNO is used in adaptive relaxation

NO promotes the stretching of the stomach in response to filling.

When the stomach gets full, stretch receptors trigger smooth muscle

relaxation through NO releasing neurons.

Role In the Reproductive system:

NOS localized in pelvic nerve neuron innervating the corpora cavrinosa

and the neuronal plexuses of the adventitial layer of the penile arteries –

proven most effective for erectile dysfunction.

NO and its derivatives produced in inflamed tissue contribute to the

carcinogenesis process due to direct or indirect DNA damage.

Direct DNA damage: DNA deamination , peroxynitrite induced adult

formation single strand break in the DNA

Indirect DNA damage: interaction of NO reactive species with other

molecule like amines , thioles , lipids.

-NO after reaction with O2/superoxide forms genotoxicity .

Role In Genotoxicity:

Role in wound healing & tissue repair

No is powerful stimulator of cell division maturation and differentiation .

Necessary mediator of neuro vascularization i.e. angiogenesis and lymph ducts to nourish

the healing of tissue.

Role in Apoptosis

Nitric oxide and its reaction products either promotes or prevent apoptosis .

Pro-apoptosis effect of NO- induction of apoptosis by NO resulting in the accumulation of

tumor suppressor protein p-53.

Anti-apoptotic effect of NO- some studies suggest that endogenous iNOS expression or

exposure to low dose of NO donors inhibits apoptosis.

Role in Inflammation

NO has shown to act as a mediator of inflammatory processes.

This process has enhanced the effect of cyclooxygenases and stimulates

the production of pro-inflammatory eicosanoids.

Mechanism of action of NO

Oxides of nitrogen

Name Known function

Nitric oxide (NO) Vasodilator, Platelet inhibitor, immune regulator, neurotransmitter.

Peroxynitrite (NO3-) Oxidant and nitrating agent

Nitroxyl anion (NO-) Exhibit NO like action by oxidising like NO

Nitrous oxide (N2O) Anesthetic

Dinitrogen trioxide (N2O3) Auto oxidation product of NO that can nitrosylate protein binds

Nitrite (NO2-) Decomposes to NO at acidic Ph

Nitrate (NO3-) Stable oxidation of NO-

Nitric oxide synthesis inhibitors Primary strategy to reduce NO generation is to use NO synthesis

inhibitors.

Majority of inhibitors are L-arginine analogs which bind to the NOS arginine binding site.

No selectivity for specific NOS and hence acts generally on all the NOS

NOS Approach for inhibition

n NOS Neurodegenrative diseases

e NOS Heamostatic signalling

i NOS Inflammatory disorders

Nitric oxide Donors NO donors which release NO or related NO species , are used to elicit

Smooth muscle relaxation.

Different classes of NO donors have different biologic response.

Organic nitrates

Organic nitrites

Sodium nitroprusside

NO gas inhalation

Alternate strategies (type 5 phosphodiesterase)

Organic nitrates

Nitroglycerin

dilates veins and coronary arteries is metabolized to NO by aldehyde reductase.

Venous dilation --- decrease cardiac preload

Arterial dilation ---antianginal effects.

Isosorbide dinitrate

metabolized to NO releasing species through a poorly understood enzymatic path.

Less significant effect on aggregation of platelets.

Organic nitrates exhibits rapid tolerance during continuous administration.

Organic nitritesSodium nitroprusside: Dilates arterioles and venules.

Used for rapid pressure reduction in arterial hypertension

In response to light/chemical/enzymatic mechanism in cell membranes SNP breaks down to generate five cyanide molecules and a single NO.

NO gas inhalation NO itself can be used therapeutically,

Results in reduced pulmonary artery pressure

Improved perfusion of ventilated areas of the lung.

Inhaled NO- pulmonary hypertension/ acute hypoxemia and cardiopulmonary resuscitation.

Alternate strategiesAnother mechanism to potentiate the action of NO is to inhibit the

Phosphodiesterase enzymes that degrade cGMP

Inhibitors of type 5 phosphodiesterase such as Sildenafil result in prolongation of the duration of NO- induced cGMP elevations in various tissues.

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