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Nitric oxide

Metabolism

Biosynthesis

NO biosynthesis is carried out from L-arginine and is catalyzed by NO-synthases, NOS, an heminic enzyme whose structure resembles that of P-450 cytochrome. In the presence of cofactors ( NADPH, oxygen, iron, tetrahydrobiopterine, FAD and FMN), enzyme NO-synthases, convert arginine into hydroxyarginine and finally into citrulline and NO according to the following reactions:

Citrulline, in presence of arginosuccinate synthethase and aspartate, is then converted into arginosuccinate, fumarate and arginine. Thus, arginine comes from an endogenous renewal and an exogenous source, food.

There are three types NO-synthases isoenzymes: type I, present in neurons and epithelial cells, type II, present in various types of cells, among which macrophages, after induction by cytokines, and type III, present primarily in endothelial cells.

NO-synthases of type I and III are constitutive. They are present naturally in endothelial cells and called eNOS and neurons and called nNOS. They are activated by the Ca2+ /calmodulin complex and induce an immediate release of NO of short duration.

Inducible NO-synthase of type II, called iNOS, is present in macrophages, neutrophils and hepatocytes after activation by cytokines, in particular interleukin-1, Tumor necrosis factor, interferon gamma and lipopolyssaccharides. The induction of this NO-synthase implies a genomic effect and requires several hours but the induced NO-synthase is immediately active after its synthesis, in the absence of calcium, and induces a delayed- and very important release of NO. Inducible NOS synthesis is inhibited by glucocorticoids.

Several derivatives of arginine, such as monomethyl-L-arginine which is present in the plasma of renal insufficient patients, are inhibitors of NO-synthases.

Notice

Arginine by decarboxylation catalyzed by arginine decarboxylase is converted into agmatine which is found in various tissues, in particular brain. Agmatine appears to have features of a transmitter, it reduces addiction and morphine tolerance and could have an antalgic effect. It inhibits NO-synthase. Agmatine is metabolized by agmatinase which transforms it into putrescine and by diamine oxidase which transforms it into guanido-butanoïc acid.

Release

As soon as it is formed, NO diffuses in gas form; synthesis and release are simultaneous and there is no storage of NO in tissues. There is a continuous basal release of NO which, by its vasodilator effect, takes part in arterial pressure regulation. The mechanisms controlling NO synthesis are complex:

• Transmitters like acetylcholine, histamine, serotonin, adenosine, bradykinin, glutamate, activate constitutive NO-synthase, already present in the cell. For example, acetylcholine activates muscarinic receptors coupled to G proteins which, via phospholipase C and formation of IP3, elicits an increase of intracellular calcium, which interacts with calmodulin to activate NO-synthase.
• Cytokines, TNFalpha (tumor necrosis factor alpha) interleukin-I, interferon-gamma, activate the synthesis of inducible NO-synthases. The hypotension observed during certain septic shocks and certain cirrhoses might result from an excessive release of NO.
• The direct formation of NO, without enzymatic intervention, is also possible from nitrite when the pH of the medium is acid as during ischemia.

NO diffuses through membranes and penetrates into all cells close to those which release it. Released by the vascular endothelium, it penetrates into smooth vascular fibers. Released by the neuronal presynaptic terminations, it diffuses into postsynaptic elements and, in a retrograde way, into the presynaptic terminations which released it and increases glutamate release.

NO can be in neutral form NO (which can be writen NO · because it is a radical, i.e. of a molecule having an unpaired electron), in the form of cation NO+ by loss of one electron, and in the form of anion NO- by gain of one electron. It can be thus involved in reduction and oxidation reactions.

Catabolism

NO is an unstable gas molecule which in the body is transformed spontaneously, in the presence of oxygen, into nitrite NO2- then into nitrate NO3 .

NO ¾ ¾® NO2- ¾ ¾® NO3

Not long ago, it was thought that nitrate present in the body was only of exogenous origin, food. The diet, especially plants, supply from 30 to 200 mg of nitrate per day. Now it is known that part of the nitrate is of endogenous origin, its production being increased by infections and by activation of the immune system. It is also increased physiologically in the second part of the menstrual cycle by estradiol increase.

Cysteine could protect NO by slowing down its oxidation.

Effects

Nitric oxide, NO, like carbon monoxide, CO, has a great affinity for iron; it modulates the activity of numerous enzymes containing iron.

1. NO activates guanylate cyclase, an heterodimeric heminic enzyme (iron bound to nitrogen atoms).
   This activation elicits transformation of guanosine triphosphate, GTP, into cyclic guanosine mono-phosphate, cyclic GMP, which increases activity of many protein kinases leading to cellular hyperpolarisation by decrease of intracellular potassium and of calcium. These effects have many consequences:
   • relaxation of vascular smooth muscles, i.e. vasodilation, including that of cavernous bodies leading to erection and that of cerebral vessels during migraine
   • bronchodilatation which is not, however sufficiently important for using NO in the treatment of asthma
   • relaxation of stomach after meals to adapt it to food contents
   • inhibition of platelet aggregation and of adhesion of the platelets to the endothelium.

    

   NO: biosynthesis by the endothelial cell and effects on smooth fibers

   Cyclic GMP formed is converted by phosphodiesterases into inactive 5 ' GMP. Inhibition of these phosphodiesterases causes an increase of active cyclic GMP concentration

2. NO can inhibit nonheminic enzymes having an iron atom bound to sulfur atoms:
• ribonucleotide reductase, necessary to DNA synthesis, because it transforms a ribonucleotide into a deoxyribonucleotide
• NADPH/ubiquinone-oxidoreductase, called mitochondrial complex I, and succinate-ubiquinone-oxidoreductase, called mitochondrial complex II.
3. NO is involved in intracellular metabolism of iron by complex mechanisms. The biosynthesis of transferrin, ferritin and ALA-synthase receptors is controlled by the intracytoplasmic protein IRP-1 (iron regulatory protein) which, by interaction with mRNA sequences called IRE (iron response elements), controls proteins translation. NO excess has the same effect as an intracytoplasmic iron deficiency and stimulates the biosynthesis of transferrin receptors and inhibits that of ferritin and of ALA-synthase, which induces an anemia. NO produced in excess during a chronic inflammatory state could induce anemia.
4. NO interacts with a certain number of molecules other than enzymes:
   • with superoxide, O2 radical ion, to give peroxynitrite anion, ONOO-, which, after protonation, decomposes into radical ·OH and nitrogen dioxide NO· 2
   • with hemoglobin: the affinity of NO for hemoglobin is 100 000 times more important than that of oxygen. NO transforms oxyhemoglobin into methemoglobine and is simultaneously inactivated according to the following reaction:
     Hb (Fe2+) O2 + NO ¾ ¾® Hb (Fe3+) + NO3
   • with thiol groups, RSH, which are transformed into R-S-NO or nitrosothiol.
5. NO modulates activity of many signalling pathways leading to genomic effects.
6. By the preceding mechanisms, and probably others not yet identified, NO participates in many effects:, sleep regulation, cellular differentiation, maturation, apoptosis, angiogenesis, inflammation and cytotoxic effect of lymphocytes and macrophages.

Whereas NO was regarded initially as having only beneficial effects, more recent studies showed that an excessive endogenous production of NO can have harmful effects: for example, in high concentration, it elicits cerebral lesions, perhaps by inducing glutamate release responsible for opening of cation channels. It could be involved in pathophysiology of Parkinson's disease and septic shock. NO excess could be involved in damaging pancreas beta cells leading to diabetes development. It could also stimulate angiogenesis and development of tumors. The anion peroxynitrite, ONOO-, can damage cellular membranes, DNA and RNA. The presence of nitrotyrosine residues in proteins can be a marker of production, perhaps excessive, of NO. Consequently, drugs inhibiting its synthesis or neutralizing its effects, like cobalt or iron chelates, could have, in particular circumstances, therapeutic applications.

Notice

Carbon monoxide, CO, whose chemical reactivity is quite similar to that of NO, in particular in its affinity for iron, is produced in the body from heme, in the presence of heme oxygenase. Endogenous CO could have a biological role which remains to be specified.

Hydrogen sulfide, SH2, is a gas formed from cysteine in cells such as neurons in the presence of 2 enzymes, cystathionine-gamma-lyase and cystathionine-beta-synthase. Like NO, SH2 has a vasodilator effect but its pathophysiological role is not well known.

Use

Nitric oxide, diluted in nitrogen, is presented in cylinders of 5 and 20 liters filled under a pressure of 200 bars. It is used by inhalation in very low concentration, diluted in air/oxygen mixtures. It is used in treatment of pulmonary arterial hypertension, in particular neonatal pulmonary hypertension and in that of refractory hypoxemia during acute respiratory distress syndrome.

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