Nitric oxide (NO) is a diatomic free radical and a critical signaling molecule in various body systems, acting as a neurotransmitter, vasodilator, bactericide, and more. It is produced by nitric oxide synthase (NOS) isoforms, each with distinct properties and functions, with roles in the nervous, circulatory, muscular, immune, and digestive systems. NO plays a major role in processes such as vasodilation, memory formation, and host defense, highlighting its essential presence in biological functions.

2. Robert Furchgott Louis J. Ignarro
Ferid Murad

3. • Nitric oxide is a diatomic free radical consisting of one atom of
nitrogen and one atom of oxygen
• Lipid soluble and very small for easy passage between cell
membranes
• Short lived, usually degraded or reacted within a few seconds
• The natural form is a gas

4. •First described in 1979 as a potent relaxant of peripheral
vascular smooth muscle.
•Used by the body as a signaling molecule.
•Serves different functions depending on body system. i.e.
neurotransmitter, vasodilator, bactericide.
•Environmental Pollutant
•First gas known to act as a biological messenger
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5. Functional NOS is a dimer formed of 2 identical subunits. Each subunit consists of 3 distinct
Domains.
1. The Reductase Domain: C-terminal, 600-
residue that supplies the electrons to
oxygenase domain for the NOS reaction.
2 . Calmodulin Binding Domain: Calmodulin
binding is required for the activity of all the
NOS isoforms.
3. The Oxygenase Domain: N-terminal, 500
residue, heme domain that catalyzes 2 steps
for conversion of arginine into citrulline and
nitric oxide.

6. NOS-catalyzed two-stage oxidation of L-arginine (Arg) to L-citrulline (Cit) and NO via
the stable intermediate Nω-hydroxy-L-arginine (NOHA).

7. Types of NOS Isoforms
1. nNOS- (Neuronal NOS / Type I / NOS-1) isoform first found in neuronal tissue.
2. iNOS- (Inducible NOS / Type II / NOS-2) isoform which is inducible in a wide range
of cells and tissues.
3. eNOS- (Endothelial NOS / Type III / NOS-3) isoform first found in vascular
endothelial cells.
4.Encoded by different genes with different localization, , catalytic properties and
inhibitor sensitivity.
5.They exhibit 51-57% homology.

8. CONT….
Human NOS Isoform
nNOS iNOS eNOS
Expression Constitutive Inducible Constitutive
Protein size 1434 aa 1153 aa 1203 aa
Protein weight 161kDa 131kDa 133kDa
Enzyme activity Ca2+ dependent Ca2+ independent Ca2+ dependent
Chromosomal location 12 17 7

9. • 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

10. Nitric oxide as a neurotransmitter
NO is a signaling molecule, but not necessarily a neurotransmitter
NO signals inhibition of smooth muscle contraction, adaptive relaxation, and
localized vasodilation
Nitric oxide believed to play a role in long term memory
Memory mechanism proposed is a retrograde messenger that facilitates long
term potentiation of neurons (memory)
Synthesis mechanism involving Ca/Calmodulin activates NOS-I
NO travels from postsynaptic neuron back to presynaptic neuron which
activates guanylyl cyclase, the enzyme that catalyzes cGMP production
This starts a cycle of nerve action potentials driven by NO

11. • NO serves in the body as a neurotransmitter, but there are
definite differences between other neurotransmitters used
commonly in the body
• NO is synthesized on demand vs. constant synthesis
• NO diffuses out of the cells making it vs. 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
• Similarities to normal NTs
• Present in presynaptic terminal
• Natural removal from synaptic junction

12. • 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
• 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
• Excess NO is picked up by HGB with CO2

13. NO was orginally 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 guanylyl cyclase to produce cGMP
A rise in cGMP causes Ca+2 pumps to be activated, thus reducing Ca+2
concentration in the cell
This causes muscle relaxation

15. • NOS II catalyzes synthesis of NO used in host defense reactions
• Activation of NOS II is independent of Ca+2 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

16. •NO 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

17. •Which post-translational modifications are significant in
the regulation of the three isoforms ?
•What is the significance and basis of the sub-cellular
localization of the NOSs ?
•Will selective iNOS, nNOS or dual iNOS-nNOS
inhibitors prove to be of value in the treatment of human
diseases, and if so, which diseases, and what side-effects
might result ?

18. 1st enzyme to synthesize a gas Nitric Oxide, a signalling molecule.
A very complex enzyme with six co-factors and three distinct
isoforms.
It has bidomain structure and formation of a dimer is essential for its
activation.
There are a number of NOS signalled pathways associated with many
biochemical and physiological processes

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