M.Prasad Naidu
MSc Medical Biochemistry,
Ph.D.Research Scholar

Introduction:
Nitric oxide a free radical which is both
cytoprotective as well as tumor promoting agent
is formed from L-arginine by converting it to L-
citrulline via nitric oxide synthase enzymes.
The reaction product of NO with superoxide
generates potent oxidizingagent ,peroxynitrite
which is the main mediatorof tissue and cellular
injury.
Peroxynitrite is reactive towards many biomolecules
which includes aminoacids,nucleic acid
bases,metal containing compounds,etc.

 Nitric oxide is also capable of directly interacting
with mitochondria through inhibition of respiration
or by permeability transition.
 Reaction of NO with metal ions include its direct
interaction with the metals or with oxocomplexes
there by reducing them to lower valent state.
 Excessive production of NO can be inhibited by
inhibiting synthetic pathway of NO using both
selective or specific NO synthase inhibitor or non
selective NO synthase inhibitor with respect to
isoforms of NO

INTRODUCTION
 NO was first discovered as colourless toxic gas.
 By 1987 NO was shown to be actually produced
in the body and its role in regulating blood
pressure became well established
 Two years later research revealed that NO is used
by macrophages to kill tumor cells and bacteria
 The discovery has opened up newer ways of
treatment for millions of people

 NO plays an important role in the protection
against the onset and progression of
cardiovascular diseases.
 The cardioprotective effects of NO include
regulation of blood pressure and vascular
tone,inhibition of platelet aggregation and
leukocyte adhesion,and prevention of smooth
muscle cell proliferation.
 Any disturbance in the bioavailability of NO leads
to a loss of cardioprotective actions and in some
cases may even increase disease progression

Endothelial NOS
 It is constitutively expressed in endothelial lining of
blood vessels and depends on calcium
 The NO produced by eNOS diffuses into smooth
muscle cells of blood vessels and elicits cGMP
dependent smooth muscle relaxation and thus
increasing blood flow
Inducible NOS
 This is type 2 NOS and is induced by inflammatory
stimuli eg. Cytokines or LPS
 It is mainly expressed in macrophages and
possess tighly bound calmodulin
 Its synthesis can also be induced in Gial cells, liver
and cardiac muscle

I
Neuronal NOS
 nNOS is constitutively expressed in post synaptic
terminus of neurons and is calcium dependent
 It is activated by calcium influx caused by binding
of neurotransmittor Glutamate to receptor in cell
membrane

 DIRECT EFFECT OF NO
 NO protects tissue from peroxide mediated
damage by scavenging metal oxo species
 It has been shown to inhibit lipid oxygenase
activity by reacting with non heme iron at the
active site.
 A heme protein ,cyclooxygenase ,involved in the
conversion of arachidonic acid to
prostaglandin,and other related enzymes is also
influenced by NO radical reactions and metal-NO
interaction
 A possible mechanism accounted for
cyclooxygenase inhibition by superoxide involves
the reduction of ferric form to ferrous state

 The presence of NO results in scavenging
superoxide which besides preventing enzyme
inactivation also converts any ferrous oxy adducts
to active ferric state.
 It is also reported that at low concentration of
NO direct effects will predominate,while at higher
concentrations indirect effects mediated by
ONOO-
 The production of NO in brain is very well
established and it is quite different from other
neurotransmitters like acetyl choline.
 The later ,after a release from synapses,lasts for a
few milliseconds whereas NO persists for
seconds,coupled with its rapid diffusion,enables it
to encompass several million synapses.

 NO mediates DNA damage by three mechanisms.
 1)formation of nitrosamines.
 2)inhibition DNA lesions repair systems which is also
mediated by other genotoxic systems.
 Modification of DNA not directly by NO but by its
oxidation products.
 NO production is increased in patients with
SLE,where up regulation of I NOS in normal
appearing vascular endothelium and over epression
of soluble vascular molecules like ICAM-1,VCAM and
E- selectin were noted
 There fore it appears that endothelium plays an active
role in leukocyte and antibody mediated inflammation.

INDIRECT EFFECT OF NO
The indirect effect NO involves the reactions between
superoxide and NO which leads to the production
ofperoxynitrite,a powerful oxidant.
Formation of peroxynitrite is governed by the relative
amount of NO and superoxide produced and also on
reaction of these radicals with other biological
components.
In presence of excess NO or superoxide, peroynitrite
gets converted to nitrogen dioxide.
An intracellular source of peroynitrite is mitochondria
where aerobic respiration results in production of
superoxide and as NO concentration is higher in lipid
layers than in cytosol,most peroxynitrite formed is in
the hydrophobic region.

 It has also been reported that quinone derivative
of catechol oestrogen,which is produced by NO
mediated oidation may form covalent adducts with
nucleophilic groups of DNA.
 Since human uterus and breast are site for
hydroxylation,a possible mechanism of hormonal
carcinogenesis associated with these organs can
be related with increased production of 4-
hydroyestradiol.
 The peroxynitrite can also influence protein and
enzyme function,this occurs by nitration of tyrosine
residuesin tissues contributing to pathological
dysfunction.

 The indirect effect of NO can be further divided as
oxidationand nitrosation
 those reactions in which RNOS donate NO to
nucleophilic groups e.g.thiols and amines ,lead to
formation of nitrosonium adducts known as
nitrosation reactions and condition termed as
nitrosative stress
Where as when removal of electrons or hydroylation
reactions occur,similar to those for ROS ,leading
to oxidative stress,they are termed as oxidation
reactions.
Both these reactions have different effects on
biological systems.

 Pro-tumour effects of NO
 Excess production of NO has been linked to
endogenous human carcinogenesis.
 Induction of apoptosis by NO has also been observed
in culture of macrophages and pancreatic beta cells.
 Macrophages exposed to nitric oxide exhibited typical
morphology and showed typical DNA fragmentation
indicating apoptotic cell death.
 NO also induced cell death and showed toxic effects
in two different cell lines viz.,CHO-AA8 and TK6 cells
highlighting the role of NO in the onset of
mutagenesis and celldeath and the involvement of
these processes in cancer and inflammatory
diseases.

 The endothelial cells synthesize NO by e NOS
which helps in vascular permeability and relaxation .
 Various tumours over express NOS.
 A study between the relationship of malignancy and e
NOS expression in endothelial cells of tumor vessels
showed that astrocytic tumor vessels possess higher
level of NO than do normal vessels and found that
there was significant correlation with the proliferative
potential and e NOS expression in tumor vessels.
 NO perse is not capable of reacting with
biomolecules; only its reaction products lead to the
production of RNOS e.g.ONOO which can result in
DNA lesions.

 Nitrosamines are formed under conditions of
inflammation which can lead to cancer
 NO also enhances tumor production by
increasing the production of prostaglandin
PGE2 which increases the permeability tumor
vasculature and thus facilitate angiogenesis.
 Further ,tumor growth is supported by increased
uptake of nutrients.
 Cells lacking Cu, Zn-SOD are reported to be
more susceptible to NO and ONOO-.
 RNOS have high affinity towards aminoacid with
thiol residues required for their function. e.g.
DNA alkyl transferases, DNA
ligase, formamidopyrimidine glycosylase.

Anti tumour effect of NO
 NO is capable of protecting cell from apoptosis or
mediating apoptosis depending on the cell type.
 NO is reported to protect tissue from peroxide
mediated damage by scavenging metallooxo
species.
 It is also been reported that animal subjects having
tumorous growth acquire the ability through which
their tumour tissues suppress the expression of i
NOS and thus reduce the concentration of NO.
 NO is capable of suppressing metastasis by
reducing intracellular stores of GSH or by blocking
the adhesion of tumour cells to venular side of
microcirculation

 It has also been reported that NO produced in
vasculature of brain limits the spread of colon cancer
to the brain.
 Liver endothelial cells produce NO which curbs the
metastases of melanoma cells to the lungs.
 NO is also reported to inhibit platelet aggregation and
it reduces platelet adhesion to endothelial
monolayers.
 It is also reported that when cells were exposed to NO
it resulted in DNA single strand breaks .However
when purified DNA was exposed NO at
concentrations as high as 1.0M,single strand breaks
were not observed.
 NO is also reported to protect DNA against oxidative
stress by inhibiting fenton reaction of hydrogen
peroxide which leads to single strandgeneration.

 EFFECT OF NO ON MITOCHONDRIA
 NO synthesis may occur in organelle itself or NO
produced outside may diffuse inside.NO may effect
mitochondria by three main pathways.
 1).Inactivation of mitochondrial enzymes which is
irreversible.
 2)induction of mitochondrial permeability transition.
 3)inhibition of respiration which is reversible.
I In addition ONOO formed outside
mitochondria diffuses into matrix .The
increasedconcentration of onoo- inactivates Mn-SOD
resulting in increase of superoxide level and hence
activating a destructive cascade of NO which
includes;

 1)Irreversible damage of enzymes of citric acid cycle e.g.
aconitase,iron-sulfur centres,etc
 2)Inhibition of glycolysis by inactivating glyceraldehyde _3
phosphate dehydrogenase thus impairing ATP synthesis.
 3)Under inflammatory conditions,NADH:ubiquinone oxido
reductase(complex 1) and succinate:ubiquinone
oxidoreductase(complex 2) are irreversibly inhibited by
NO.
It has been found that NOS is present in mitochondria
and that under normal conditions ,production of NO is well
regulated.Induction of mitochondrial permeability
transition is mainly caused by ONOO-which oxidizes thiols
and NADPH of mitochondria and induces calcium efflux
along with oxidative efflux.As a consequence,ca
homeostasis is disrupted.Mitochondrial decrease in
membrane potential in induction of permeability transition
leads to increase in cytoplasmic ca.This is accompanied
by formation of a protein pore in mitochondrial membrane
resulting in the leakage of its contents.

 Interaction with metal ions
 NO forms iron nitrosyl complexes by binding to iron-
sulfur clusters.e.g.NO is capable of inactivating
aconitase due to its direct interaction with the
enzymes heme iron nitrosyl complex.
 These reactions of NO with metals involve covalent
interactions.
 In addition various ,various metal oxygen complexes
e.g.reaction of NO and oxyhemoglobin to form met-hb
and nitrate also occur.This is one of the primary
detoxification mechanisms of NO.
 NO also rapidly reacts with hypervalent metal
complexes and results in their conversion to lower
valent state, thereby scavenging the metallo oxo
species and protecting cells from peroxide mediated
damage.

Inhibitors of NO
 In order to study the over production of
NO, inhibitors of NOS have been synthesized and
investigated for their probable role in controlling the
overproduction of NO under disease condition
 NMMA (N- mono methyl arginine) competitively
inhibits NOS
 ADMA (Assymetric dimethyl arginine) an
endogenous Arginine analogue acts as an NOS
inhibitor
 ADMA is seem to be increased in
Hyperhomocystenemia, Preeclampsia
 In eclampsia the hypertension is due to lowered
production of NO
 Among the synthetic analogue of L-Arginine only
Homo-L-Arginine and Agmatine are active

 NO may play a significant role in articular damage
in OA as NO stimulates synthesis of MMP s by
chondrocytes.
 Chondrocytes are a major source of NO ,the
synthesis of which is stimulated by IL-1 and TNF
and shear stress.
 In an experimental model of OA,treatment with a
selective inhibitor of i NOS reduced severity of
cartilage damage

 Enhancers of NO activity
 S-nitroglutathione (GS-NO) is a NO donor
which inhibits platelet agglutination.
 N-acetyl cysteine(NAC) is a glutathione
precursor.It protects NO from being
metabolised by free radical scavengers and
hence enhances NO activity
 Measurement of nitric oxide
 The levels of metabolic products
(nitrites,nitrates and 3-nitrotyrosine) in blood
and urine are markers of nitric oxide
production

Biosensors
 These are chemical sensors having an optical
device or transducers and a biological recognition
element
 The concentration of the analyte is recognized by
an enzyme based biosensor (catalytic reaction) or
affinity based sensor (binding specificity)
 When the recognition element interacts with the
analyte a product formed or reactant consumed on
the surface of the sensor
 This change in property is converted by a tranducer
to an electrical signal and quantified
 Eg. Implantable subcutaneous glucose sensor-
adjust dose of insulin
 Intravascular sensors- release NO- have been
developed to decrease the possibility of thrombosis

 Within the kidney eNOS and iNOS
isoenzymes are present
 Activation of NOS has been shown to occur
as a result of shear stress
 Eg. Increased arteriolar tone
 NOS has been shown to play an important
role in regulation of human vascular tone and
crucial role in control of blood pressure and
kidney function
 It has also been found in macula densa and
has been implicated in the regulation of
Renin release

Endothelial NOS
 It is constitutively expressed in endothelial lining of
blood vessels and depends on calcium
 The NO produced by eNOS diffuses into smooth
muscle cells of blood vessels and elicits cGMP
dependent smooth muscle relaxation and thus
increasing blood flow
Inducible NOS
 This is type 2 NOS and is induced by inflammatory
stimuli eg. Cytokines or LPS
 It is mainly expressed in macrophages and
possess tighly bound calmodulin
 Its synthesis can also be induced in Gial cells, liver
and cardiac muscle

 conclusion
 In higher vertebrates NO has key roles in maintaining
homeostasisin vascular smooth muscle,neurons and
in GI tract.
 It has a definite role in regulating all aspects of our
lives from walking,digestion,sexual function,pain
perception and pleasure,memory recall and sleeping.
 Finally ,the way it continues to function in our bodies
will influence how we degenerate with age.
 It has a likely role in deaths through cardio vascular
disease,stroke,diabetes and cancer.
 Our ability to control NO signalling and to use NO
effectively in therapy must there fore have a major
bearing on the future quality and duration of human
life.

Inhibitors of NO
 In order to study the over production of
NO, inhibitors of NOS have been synthesized and
investigated for their probable role in controlling the
overproduction of NO under disease condition
 NMMA (N- mono methyl arginine) competitively
inhibits NOS
 ADMA (Assymetric dimethyl arginine) an
endogenous Arginine analogue acts as an NOS
inhibitor
 ADMA is seem to be increased in
Hyperhomocystenemia, Preeclampsia
 In eclampsia the hypertension is due to lowered
production of NO
 Among the synthetic analogue of L-Arginine only
Homo-L-Arginine and Agmatine are active