Neuronal NOS

Three isoforms of NO synthesizing enzymes ( nitric oxide synthase (NOS)) were isolated, purified, and cloned neuronal NO synthase ( neuronal nitric oxide synthase (nNOS) or isoform (I), immunological or inducible NOS ( inducible (immunological) nitric oxide synthase (iNOS) or isoform (II), and endothelial NOS ( endothelial nitric oxide synthase (eNOS) or isoform... 

NO synthases (NOS, L-arginine, NADPH oxygen oxi-doreductases, nitric oxide forming EC 1.14.13.39) represent a family of enzymes that catalyze the formation of nitric oxide (NO) from the amino acid L-arginine. In mammals, three isoforms of NOS have been identified. They are termed neuronal NOS (nNOS, NOS I, NOS1), inducible NOS (iNOS, NOS H, NOS2), and endothelial... 

Neuronal NO synthase (nNOS) is constitutively expressed in neurons of the brain. Its activity is regulated by Ca2+ and calmodulin. Half-saturating L-arginine concentrations are around 2 pM. cDNAs encoding nNOS have been cloned from rat and human brain. The open reading frame of human nNOS consists of 4299 bp, corresponding to 1433 aa. This predicts a protein of 160 kDa, which is in accordance with the molecular mass of the purified protein. 

We have successfully used the lentivims to infect neurons cultured from 1 to 12 days. However, infection efficiency decreases with the age of the culture. Thus, it is recommended to use neurons no more than 1 week... 

In addition to nitric oxide, superoxide, and peroxynitrite, NO synthases are able to generate secondary free radicals because similar to cytochrome P-450 reductase, the reductase domain can transfer an electron from the heme to a xenobiotic. Thus it has been found [158,159] that neuronal NO synthase NOS I catalyzed the formation of CH3CH(OH) radical from ethanol. It was suggested that the perferryl complex of NOS I is responsible for the formation of such secondary radicals. Miller [160] also demonstrated that 1,3-dinitrobenzene mediated the formation of superoxide by nNOS. It was proposed that the enhancement of superoxide production in the presence of 1,3-dinitrobenzene converted nNOS into peroxynitrite-produced synthase and may be a mechanism of neurotoxicity of certain nitro compounds. 

A more complete analysis of human neuronal NOS gene was made by Hall s group71. It is a gene of 29 exons a flanking region of over 1500 bp was determined 5- and several... 

Using neuronal NOS, Matsuoka and collaborators107 observed that arginine apparently reacted with the heme to reduce the rate of CN or CO reacting. The reaction of L-arginine... 

The term NOS is used to denote a family of three related but distinct isoenzymes neuronal NOS (nNOS) endothelial NOS (eNOS, endothelium and platelets) and inducible NOS (iNOS, endothelium, vascular smooth muscle and macrophage). In addition to reduced nicotinamide adenine dinucleotide phosphate (NADPH) shown in Figure 5.5, NOS enzymes also require flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN) and tetrahydrobiopterin (BH4) as coenzymes. 

O Hearn E, Zhang P, Molliver ME. (1995). Excitotoxic insult due to ibogaine leads to delayed induction of neuronal NOS in Purkinje cells. Neuroreport. 6(12) 1611-16. 

Fig. 1. The overall reaction catalyzed by NOS and a schematic representation of the three main NOS isoforms. L-Arg is first converted to JV-hydroxy-L-Arg in a P450-like monooxygenation reaction requiring two electrons and one O2 molecule. In the second step, Ai-hydroxy-L-Arg to NO and L-citrulline, only one electron and one O2 molecule are required. nNOS, neuronal NOS (neural system) iNOS, inducible NOS (immune system) eNOS, endothelial NOS (cardiovascular system).

Nitric oxide (NO) and carbon monoxide are atypical neurotransmitters. They are not stored in synaptic vesicles, are not released in by exocytosis, and do not act at postsynaptic membrane receptor proteins. NO is generated in a single step from the amino acid arginine through the action of the NO synthase (NOS). The form of NOS initially purified was designated nNOS (neuronal NOS), the macrophage form is termed inducible NOS (iNOS), and the endothelial from is called eNOS. 

NOS-containing neurons have a very discrete localization in the CNS, representing only 1% of neuronal cells. However, their axons ramify so extensively that virtually every cell in the brain may encounter a NOS nerve terminal. As a diatomic gas, NO is freely diffusible and thus can readily enter adjacent neuronal cells. Once inside the target cell, NO binds the iron in heme contained within the active site of soluble guanylyl cyclase, activating the enzyme to form cyclic guanosine monophosphate (GMP). The activity of NO is therefore mediated by an enzyme receptor. In neurons, NO is formed in response to calcium influx reminiscent of calcium-dependent exocytotic release of neurotransmitters. 

Overall sequences, similarity between the constitutive endothelium and neuronal NOS isoforms is approximately 50%, and both are roughly 50% identical to the inducible macrophage NOS (Xie et al., 1992 Lamas et al., 1992). Studies have located the genes for human neuronal, endothelial, and inducible NOS on... 

How the NOS isoforms compare to these related dual flavin enzymes is a matter of ongoing investigation. Characterization of the neuronal NOS revealed that it normally exists in its one-electron reduced form and maintains an air-stable, flavin semiquinone radical (Stuehr and Ikeda-Saito, 1992), as seen for NADPH-cytochrome P450 reductase. It is unknown which flavin in NOS contains the odd electron, although precedent argues that it probably resides on... 

Calmodulin binds to and activates neuronal NOS (Bredt and Snyder, 1990 Schmidt et al., 1991), and also functions as a tightly bound prosthetic group to keep macrophage NOS in its active state (Cho et al., 1992). Work with the neuronal NOS has uncovered the basis for its calmodulin activation Calmodulin binding triggers electrons to transfer onto the NOS heme iron (Abu-Soud and Stuehr, 1993) (Fig. 10). Because this transfer is associated with initiation of... 

Other names nNOS (neuronal NOS) iNOS (inducible NOS) eNOS (endothelial NOS)... 

The CNS contains a substantial amount of nitric oxide synthase (NOS), which is found within certain classes of neurons. This neuronal NOS is an enzyme activated by calcium-calmodulin, and activation of NMDA receptors, which increases intracellular calcium, results in the generation of nitric oxide. Although a physiologic role for nitric oxide has been clearly established for vascular smooth muscle, its role in synaptic transmission and synaptic plasticity remains controversial. Perhaps the strongest case for a role of nitric oxide in neuronal signaling in the CNS is for long-term depression of synaptic transmission in the cerebellum. 

NOS catalyses the generation of nitric oxide (nitrogen monoxide, NO) from L-arginine. Three major isoforms occur in mammals neuronal NOS (nNOS or type I) and endothelial NOS (eNOS or type III) require Ca2+ for activity,... 

In spite of remarkable efforts to develop sodium channel blockers which are selective for subtypes preferentially expressed in primary sensory neurons, no such pain-selective compound has as yet been found. 

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