Nitric oxide

(KEGG pathway MAP00330 for arginine and proline metabolism)

The small, free radical molecule nitric oxide (NO; N=O) has been identified as a major signal transduction molecule in vertebrates (animals). NO is derived from arginine in two steps catalyzed by nitric oxide synthase (NOS; EC NOS catalyzes the net reaction:

            L-Arginine + n NADPH + m O2 = Citrulline + Nitric oxide + n NADP+

with the intermediate N-(omega)-Hydroxyarginine (C05933). The catalytic activity of nitric oxide synthase is related to the monooxygenase activity of cytochrome P450. This catalytic relationship becomes apparent when comparing the gene structure of both enzymes. Nitric oxide synthase is the larger of the two enzymes. Its C-terminal domain is identical to the smaller Cyt P450 protein.

There are three forms of nitric oxide synthase - a neuronal type called nNOS, an epithelial type called eNOS, and an inducible form called iNOS. The latter is only expressed under certain conditions like immune system regulation by cytokines or pathological induction in the presence of endotoxins (bacterial lipopolysaccharide) and cytotoxins (which affect cytokine secretion). NO production is a stress response and can lead to either tissue injury because of its radical chemistry, or be cytoprotective, protecting cells from damage by destroying pathogenic microorganisms first. For example, stomach ulcers have lately been associated with a bacterial infection. Helicobacter pylori (H.pylori) causes ulcers and gastric cancers. Nitric oxide and particularly its superoxide derivative peroxynitrite cause DNA damage in the bacteria.

Free radicals as antimicrobials: ONOO induces DNA damage through chemical modifications (mutations) while NO inhibits ribonucleotide reductase. Both DNA damage and reductase inhibition keep the cell in a state of energy costly nucleotide synthesis and repair mode. This leads eventually to cell death by energy depletion of bacterial cells.

Such defense mechanism, however, have their draw backs. Inducible NOS, which is expressed as an emergency mechanism to suppress tumor growth in gastric epithelia, breast tissue, and the brain,  is linked to septic shock. Bacterial endotoxins (e.g. from H.pylori or E.coli infections) induce the iNOS gene, which in turn produces high levels of NO damaging pathogenic DNA and inhibiting respiration (inhibits metabolic energy production needed for cell division). The free radicals, however, cannot discriminate pathogenic DNA from host DNA and overstimulation of iNOS therefore induces cell and tissue damage, sometimes leading to a fatal development (septic shock) in the course of bacterial infections. This is a well known situation in hospitals affecting patients with an already suppressed immune system.

The neuronal and epithelial NOS isoforms are constitutively expressed and regulated by calcium concentration via calmodulin interaction. The calcium-calmodulin complex stabilizes the homodimer. Each monomer contains a reductase and oxygenase subunit containing FAD, NADPH & FMN or heme & tetrahydrobiopterine (B4H) cofactors, respectively. B4H is essential for NOS dimer formation. (H4biopterine is an important cofactor of aromatic amino acid hydroxylases.) Nitric oxide synthase is a membrane bound protein, anchored to the cytoplasmic side of endoplasmatic reticulum, Golgi, or plasma membrane by myristoylation or palmitoylation. The lipid anchored NOS are preferentially found in cholesterol and glycolipid rich membrane domains. The compartmentalization of NOS appears to be crucial for its functionality by providing local NO levels.

NOS regulation

The activity of eNOS and nNOS is controlled by tetrahydrobiopterin and Ca/CaM availability because these two cofactors are needed for the proper dimer formation of an active synthetase. The dependence on calmodulin has been used as a model to explain the role of glutamate in neurotoxicity in the central nervous system. Neurotoxicity is a mechanism of glutamate induced neuronal cell death. The immediate effect of glutamate on neurons is its role in activating glutamate receptor, namely to pharmacological subtypes known as NMDA Receptors (NMDA is a methylated derivative of aspartate). Glutamate receptors are selective for calcium ions. Thus, prolonged activation of glutamate receptors stimulates eNOS via Ca/CaM complex binding to the synthetase. The formation of NO is implicated in cell death as described above: DNA damage, suppressed mitochondrial respiration, leading to energy depletion. Neurons are particularly sensitive to impaired mitochondrial ATP synthesis capacity, because neurons depend almost exclusively on the oxidative degradation of glucose and ketone bodies. The formed ATP is used by ion selective pumps to maintain the proper ion gradients for action potential generation and neurotransmitter release of presynaptic membranes.

NO can only be synthesized, however, if the amino acid arginine is available. Neuronal NOS critically depends on this substrate, which is mainly synthesized in adjacent glial cells and is transported into neurons. Arginine uptake into neurons is controlled by non-NMDA glutamate receptors. This became evident when these receptors were blocked by arginine-uptake inhibitors such as L-lysine which functions as antagonist of these glutamate receptors. The physiological role of nNOS in mechanisms such as long term potentiation has been shown to involve retrograde transport (diffusion) of NO synthesized in post synaptic neurons across the synaptic cleft into synapses, where they stimulated guanyl cyclase.

Nitric oxide and free radical biochemistry

Nitric oxide is a free radical molecule and its major effect is the activation of cytoplasmic, soluble guanyl cyclase (sGC; EC This enzyme catalyzes the cyclization of GTP to cGMP + PPi. Cyclic GMP is a signaling molecule (similar to cAMP) by virtue of activating protein kinases. .

Nitric oxide binds to the heme group of cyclase. Other protein targets are metallo enzymes, where NO binds to Fe-S clusters. Aconitase is inactivated by NO, as is complex IV, the cytochrome oxidase in the inner membrane of mitochondria. Thus NO as an inhibitory effect on oxidative phosphorylation by blocking the electron transport chain and controlling the levels of citrate in the Krebs cycle essentially blocking the oxidative degradation of acetyl-CoA.

NO is a short lived chemical transmitter, which is freely diffusible across membranes. The molecule possesses a small dipole moment because of the similar electronegativity of oxygen and nitrogen, making it essentially hydrophobic. Its reactivity is due to the unpaired electron in the outer valence orbital of its oxygen constituent.  NO is almost unreactive as free radical as compared to other oxygen radicals. Indeed, NO decays within seconds after its synthesis if left unbound in solution because it reacts with either molecular oxygen or superoxide.

NO strongly interacts with molecular oxygen to form dinitrotrioxide (N2O3), or with superoxide O2-. to form peroxynitrite (ONOO-). NO also binds to sulfhydryl groups (SH) and unsaturated fatty acids. The reaction with superoxide can be diminished by superoxide dismutase (SOD) which removes O2-. to form hydrogen peroxide (H2O2). NO can be 'stored' by covalent interaction to glutathione to form S-nitroso-glutathion. Both H2O2 and S-nitrosoglutathion can have a stimulatory effect on guanine cyclase. Superoxide dismutase thereby prevents the loss of nitric oxide to peroxynitrite forming hydrogen peroxide instead and increasing the cyclase stimulatory capacity of the cell. See glutathione metabolism for details MAP00480.

NO can potentially be regenerated from ONOO- in two steps; a first reduction of peroxynitrite by cytochrome C oxidase to nitrite (NO2), followed by a reduction of nitrite to NO by the enzyme nitrate reductase. The latter enzyme exists in two isoforms, a mitochondrial type and an endoplasmatic reticulum resident protein. Both receive their electrons needed for nitrite reduction to NO from either NADH or NADPH, and interact with flavoproteins (FAD prosthetic groups) and cytochromes (cytochrome c oxidase in mitochondrial membrane; cytochrome P450 in ER membrane).

Cytochrome C oxidase and nitrite reductase therefore reduce the concentration of highly reactive, secondary metabolites, and potentially contribute to NO signaling. The latter has only been shown in plant cells, where nitrate reductase reaction appears to be a considerable contributor to this signaling molecule.

Peroxynitrite, hydrogen peroxide, and dinitrotrioxide all have been linked to cell death (apoptosis = programmed cell death) through protein nitration and increased mutagenesis. The latter is a consequence of DNA stand breakage and guanine nitration. For example, acute neural toxicity is linked to the overproduction of peroxynitrite, which inhibits respiratory enzymes and also damages DNA by covalent bond formation to DNA and removal of bases. Inhibitors of nitric oxide synthase and antioxidants are known to have neuroprotective properties because the limit the formation of highly reactive nitrogen containing radicals.


The free radical chemistry in cells can be prevented or at least diminished by adding antioxidants or free radical scavengers, molecules which have a high affinity and strongly react with these free radicals. Antioxidants are either hydrophilic or hydrophobic. Hydrophilic antioxidants include glutathione peroxidase, Fe(II) chelators like the proteins ceruloplasmin and transferrin, and hydroxylated aromatic molecules like uric acid or ascorbate (vitamin C). Hydrophobic antioxidants include flavin-nucleotide or carotene containing proteins and vitamin E.

Melatonin too is a major physiological antioxidant (and hormone) by directly reacting with hydroxyl and peroxyl radicals, or by stimulating the expression of superoxide dismutase, glutathione peroxidase, or glutathione reductase. Melatonin has also been reported to inhibit nitric oxide synthetase.

Physiological role of NO as neurotransmitter

In epithelial cells, NO causes vascular dilatation by controlling smooth muscle contractility. In the central nervous system it affects synaptic transmission stimulating learning and memory capacity. Glutamate is produced and released by a synapse and activates the NMDA receptor subtype of glutamate receptors. This leads to an influx of calcium ions which in turn bind to calmodulin, activating the neuronal NOS. NOS synthesizes NO depending on the availability of L-arginine, which is mainly supplied from extra-neuronal sites (mainly glial cells). NO not only activates the postsynaptic guanyl cyclases, but can diffuse across the synaptic cleft back into the synapse that originally released the glutamate. This retrograde transport of NO is thought to reinforce the capability of glutaminergic signaling. Such a prolonged reinforcement of synaptic stimulatory activity is known as long term potentiation and is implicated as a possible molecular mechanism promoting long term memory and learning.

In blood plasma NO induces platelet aggregation, an important factor in wound healing and blood coagulation. It has been shown that hemoglobin is a major transport vehicle for NO in blood.

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