Nitric oxide (NO) is produced in the body by nitric oxide synthase (NOS) enzymes from the amino acid L-arginine. NO acts as both an intracellular and extracellular signaling molecule and is involved in many physiological processes like neurotransmission and smooth muscle relaxation. There are three isoforms of NOS - neuronal NOS, inducible NOS, and endothelial NOS. NO stimulates soluble guanylate cyclase which increases cyclic GMP levels and triggers smooth muscle relaxation. In addition to cGMP signaling, NO can also signal through S-nitrosylation of proteins or by forming nitrite and nitrate storage pools.

1. PRESENTED BY :-KINJAL S. GAMIT
DEPARTMENT OF PHARMACOLOGY
M.PHARM,SEM-1
EN.NO :- 172280825005
L.M. COLLAGE OF PHARMACY
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2.  During the 1980s, a new type of messenger was discovered that was
neither an organic compound, such as cAMP, nor an ion, such as Ca
2+ ; it was an inorganic gas—nitric oxide ( NO).
 NO is unusual because it acts both as an extracellular messenger,
mediating intercellular communication, and as a second
messenger, acting within the cell in which it is generated.
 NO is formed from the amino acid l ‐arginine in a reaction that
requires oxygen and NADPH and that is catalyzed by the enzyme
nitric oxide synthase ( NOS ).
 Since its discovery, it has become evident that NO is involved in a
myriad of biological processes including anticoagulation,
neurotransmission, smooth muscle relaxation, and visual
perception.
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3.  Nitric oxide synthase (NOS) enzymes produce NO by
catalyzing a five electron oxidation of a guanidino
nitrogen of L-arginine (L- Arg).
 Oxidation of L- Arg to L- citrulline occurs via two
successive mono-oxygenation reactions producing
NGhydroxy L-arginine as an intermediate.
 Two moles of O2 and 1.5. moles of NADPH are consumed
per mole of NO formed.
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5.  NOS enzymes are the only enzymes known to simultaneously
require five bound cofactors/prosthetic groups: FAD, FMN,
heme, tetrahydrobiopterin and Ca2+-calmodulin.
 There are three isoforms of NOS, the genetic sequence of each
residing on three distinct chromosomes.
 One type is constitutive,cytosolic, Ca2+/calmodulin dependent
and releases NO for short time periods in response to receptor
or physical stimulation.The NO released by this enzyme acts as
a transduction mechanism underlying several physiological
responses.
 The other enzyme type is induced after activation of
macrophages, endothelial cells and a number of other cells by
cytokines and once expressed, synthesizes NO for long
periods of time.
 Furthermore, this enzyme is cytosolic, Ca2+ independent since
calmodulin is already bound to the enzyme, and its induction is
inhibited by glucocorticoids. 5

6.  Endothelial NOS/ eNOS
 Neuronal NOS/ nNOS
 which are both constitutively expressed in mammalian cells have
now been well characterized in the cardiovascular system and
nervous system respectively.
 Inducible NOS/ iNOS
 which was first believed to be expressed only when activated by an
immune response.
 To clarify the nomenclature they referred as
 NOSI (neuronal)
 NOSII (inducible)
 NOSIII (endothelial)
 Which is based on the order in which they were first purified and
cloned.
 Bacterial NOS/ bNOS in several bacterial species(notorious
pathogens Bacillus anthracis and Staphylococcus aureus.) 6

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9.  All three isoforms of the enzyme function as a homodimer
consisting of two identical monomers, which can be functionally
and structurally divided into two major domains:
 a C-terminal reductase-carboxy domain, and
 an N-terminal oxygenase-amino domain.
 iNOS has been described as calcium-insensitive, likely due to its
tight non-covalent interaction with calmodulin (CaM) and Ca2+.
 Bacterial NOS (bNOS) plays a key role in the transcription
of superoxide dismutase (SodA). Bacteria late in the log phase who
do not possess bNOS fail to upregulate SodA, which disables the
defenses against harmful oxidative stress.
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10.  1. cGMP dependent signaling
 2. PDE inhibitors
 3. Splice forms
 4. Allosteric regulators of sGC
 cGMP independent signaling
 1. Nitrite and nitrate
 2. Nitrosothiols
 3. Nitrotyrosine
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11.  Many of the physiological functions of NO in the cardiovascular, neuronal,
gastrointestinal and other systems are mediated through its primary
receptor, soluble guanylyl cyclase.
 sGC is a heme-containing, heterodimeric NO receptor.
 TWO SUBUNITS :
 α and β(which make up the active enzyme)
 Four sGC isoforms, products of four genes :
 α1, α2,β1 and β2
 Only α1/β1 and α2/β1 heterodimers are activated by NO.
 The heme-containing heterodimer sGC converts guanosine triphosphate
into the secondary messenger guanosine 3’:5’-cyclic monophosphate.
 Through the production of cGMP, sGC can exert many physiological
effects such as mediating vascular smooth muscle tone and motility,
phototransduction, and maintaining fluid and electrolyte homeostasis.
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12.  Phosphodiesterase (PDEs) are intracellular enzymes that
specifically catalyze the hydrolysis of the second messengers
cAMP and cGMP to the inactive metabolites AMP and GMP.
 The discovery of NO as a second messenger has also led to the
development ofViagra (sildenafil).
 During sexual arousal,nerve endings in the penis release NO,
which causes relaxation of smooth muscle cells in the lining of
penile blood vessels and engorgement of the organ with blood.
 NO mediates this response in smooth muscle cells by activation of
the enzyme guanylyl cyclase and subsequent production of
cGMP.
 Viagra (and related drugs) has no effect on the release of NO or
the activation of guanylyl cyclase, but instead acts as an inhibitor
of cGMP phosphodiesterase, the enzyme that destroys cGMP. 12

13.  Inhibition of this enzyme leads to maintained, elevated levels of
cGMP, which promotes the development and maintenance of an
erection.
 Viagra is quite specific for one particular isoform of cGMP
phosphodiesterase, PDE5, which is the version that acts in the
penis.
 Another isoform of the enzyme, PDE3, plays a key role in the
regulation of heart muscle contraction, but fortunately is not
inhibited by the drug.
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14.  The absence of sGC protein resulted in a significant increase in
blood pressure, complete loss of NO-dependant aortic relaxation
and platelet aggregation in animals.
 sGC function is affected not only by NO, but also by regulation of
the expression of sGC subunits at transcriptional and post-
transcriptional levels.
 The steady state mRNA levels of α1 and β1 subunits decrease with
hypertension, ageing and vary during embryonic development.
 The expression of sGC subunits is regulated by estrogen, cAMP-
elevating compounds, cytokines and NO donors.
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15.  There are also many allosteric regulators of sGC which
provide NO independent activation.
 Impaired bioavailability and/or responsiveness to
endogenous NO has been implicated in the pathogenesis
of cardiovascular and other diseases.
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16. 1. Nitrite and nitrate :
 Inorganic nitrite (NO2-) and nitrate (NO3-) are known
predominantly as undesired residues in the food chain or as inert
oxidative end products of endogenous NO metabolism.
 it is now apparent that nitrate and nitrite are physiologically
recycled in blood and tissues to form NO and other bioactive
nitrogen oxides.
 As a result, they should now be viewed as storage pools for NO-like
bioactivity to be acted upon when enzymatic NO production
from NOS is insufficient.
 The recognition of this mammalian nitrogen cycle has led
researchers to explore the role of nitrate and nitrite in physiological
processes that are known to be regulated by NO.
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17. 2. Nitrosothiols :
 S-nitrosothiols are thio-esters of nitrite with the general structure
R-S-N=O; naturally occurring examples include S-nitrosocysteine, S-
nitrosoglutathione and Snitrosoalbumin, in which R is an amino acid,
polypeptide and protein respectively.
 S-nitrosothiols can be synthesized from the reaction between
thiols and nitrous acid in extremely acidic condition.
 S-nitrosation is a ubiquitous redox-related modification of
cysteine thiol which transduces NO bioactivity.
 S-nitrosothiols as an intermediate in nitric oxide–dependent and
guanylyl cyclase–independent signaling processes.
 Reactive protein thiols are becoming regarded as major
intracellular target of nitric oxide.
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18.  S-nitrosation has since been implicated in the control of a wide
array of protein functions and cell activities, Among the growing
list of proteins whose activities are regulated by s-nitrosation are
included, ion channel proteins, kinases, proteolytic enzymes,
transcription factors and proteins involved in energy transduction.
 Through s-nitrosation of these proteins, nitric oxide has been shown
to regulate apoptosis, G-protein-coupled receptor based signaling,
vascular tone and inflammatory responses.
 However cellular signaling events are dictated by specificity and a
transient modification that can quickly and specifically be
inactivated to turn off the signal.
 Whereas s-nitrosation produces the effects of nitric oxide inside
the body, denitrosation pathways inside the cells terminate the
cellular effects of nitric oxide. 18

19. 3. Nitrotyrosine :
 The discovery of nitric oxide focused on reactive nitrogen species,
which includes NO that can under go interconversion to form
NO+ or nitrosonium and NO- or nitroxyl anion.
 NO reacts with O2•- to form peroxynitrite that can further form
peroxynitrous acid, a very unstable and reactive oxidizing
species.
 Involvement of ONOO is the most widely studied mechanism of
protein nitration, and the formation of NO2-Tyr has been
detected in various pathological conditions including
atherosclerosis, myocardial infarction, myocarditis, heart failure,
shock, diabetic complication and neurodegenerative and
inflammatory disorders.
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20.  The binding of acetylcholine to the outer surface of an
endothelial cell (step 1, Figure 15.36 ) signals a rise in cytosolic
Ca 2+ concentration (step 2) that activates nitric oxide synthase
(step 3).
 The NO formed in the endothelial cell diffuses across the plasma
membrane and into the adjacent smooth muscle cells (step 4),
where it binds and stimulates guanylyl cyclase (step 5), the
enzyme that synthesizes cyclic GMP (cGMP), which is an important
second messenger similar in structure to cAMP.
 Cyclic GMP binds to a cGMP‐dependent protein kinase (a PKG),
which phosphorylates specific substrates causing relaxation of
the muscle cell (step 6) and dilation of the blood vessel.
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22.  All three forms of NOS :NOSI (neuronal),NOSII (inducible),NOSIII
(endothelial) found in cardiomyocytes produce NO involved with
cGMP- dependent and cGMP independent signaling.
 NO stimulates sGC, which produces cGMP.
 cGMP activates protein kinase G (PKG), which activates multiple
targets includingTroponin І (TnІ) and L-type calcium channels.
 In addition NO produced by each isofrom of NOS can react with
superoxide (o2-) to from peroxynitrite (ONOO-).
 NOS1 and NOS3 are constitutively expressed in cardiomyocytes,
while NOS2 is expressed under immunopathological conditions
(e.g. cardiac ischemia, aging, cardiac failure) that often result in an
inflammatory response.
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23.  Murad and colleagues ultimately demonstrated that azide was
being converted enzymatically into nitric oxide, which served as
the actual guanylyl cyclase activator.
 This also explained the action of nitroglycerine.
Nitroglycerine is metabolized to nitric oxide,
which stimulates the relaxation of the smooth muscles lining the
blood vessels of the heart,
increasing blood flow to the organ.
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24.  Recent investigations have revealed that NO has a variety
of actions within the body that do not involve production
of cGMP.
 For example, NO is added to the — SH group of certain
cysteine residues in well over a hundred proteins,
including hemoglobin, Ras, ryanodine channels, and
caspases.
 This posttranslational modification, which is called
S‐nitrosylation , alters the activity, turnover, or
interactions of the protein.
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25.  https://en.wikipedia.org/wiki/Nitric_oxide_synthase
 www.abcam.com
 karp’s cell and molecular biology concepts and experiments 8th
edition
 1Institute of Molecular Medicine,The University ofTexas-Houston
Health Sciences Center, Houston,TX 77030, USA, 2The Murad
Research Institute for Modernized Chinese Medicine and E-Research
Institute of Nitric oxide and Inflammatory Medicine of Shanghai
Universities, 1200 Cailun Rd. Shanghai,China
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