BIOCHEMISTRY OF NITRIC OXIDE

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Biochemistry of Nitric Oxide
M.Prasad Naidu MSc Medical Biochemistry, Ph.D.Rese
arch Scholar
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• 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.

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• 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

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• 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

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• 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

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• 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

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• 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

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• 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

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• 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.

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• 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.

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• 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.

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• 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.

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• 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.

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• 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.

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• 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.

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• 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.

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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

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• 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.

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• 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

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• 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,NADHubiquinone
  oxido reductase(complex 1) and succinateubiquinon
  e 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.

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• 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.

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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

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• 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

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• 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

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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

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• 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

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• 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

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• 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.

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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

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