NITRIC OXIDE

1. M.Prasad Naidu MSc Medical Biochemistry, Ph.D.Research Scholar Biochemistry of Nitric Oxide

2. 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.

3. 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

4. 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

5. 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

9. 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

10. 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

12. 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

13. 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.

14. 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.

16. 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,mostperoxynitrite formed is in the hydrophobic region.

17. It has also been reported that quinone derivative of catecholoestrogen,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.

18. The indirect effect of NO can be further divided as oxidationandnitrosation • 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.

22. 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.

23. 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.

24. 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 thiolresidues required for their function. e.g. DNAalkyl transferases, DNAligase, formamidopyrimidineglycosylase.

25. 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

26. 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.

27. 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;

28. 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:ubiquinoneoxidoreductase(complex 1) and succinate:ubiquinoneoxidoreductase(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 cytoplasmicca.This is accompanied by formation of a protein pore in mitochondrial membrane resulting in the leakage of its contents.

29. 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 metallooxo species and protecting cells from peroxide mediated damage.

30. 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 (Assymetricdimethylarginine) 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

32. 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

33. 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

34. 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

35. Within the kidney eNOS and iNOSisoenzymes 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

36. 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

37. 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,sexualfunction,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.

38. 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 (Assymetricdimethylarginine) 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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