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Isolation of nitric oxide synthetase, a calmodulin-requiring enzyme (endothelium-derived relaxing factor/arginine/cGMP)

TL;DR: In this paper, it was shown that NO synthetase is a calmodulin-requiring enzyme, and showed that NO formation is accompanied by the stoichiometric conversion of arginine to citrulline.
Abstract: Nitric oxide mediates vascular relaxing effects of endothelial cells, cytotoxic actions of macrophages and neu- trophils, and influences of excitatory amino acids on cerebellar cyclic GMP. Its enzymatic formation from arginine by a soluble enzyme associated with stoichiometric production of citruline requires NADPH and Ca2 . We show that nitric oxide synthetase activity requires calmodulin. Utilizing a 2',5'-ADP affmity col- umn eluted with NADPH, we have purified nitric oxide synthetase 6000-fold to homogeneity from rat cerebellum. The purified enzyme migrates as a single 150-kDa band on SDS/PAGE, and the native enzyme appears to be a monomer. Endothelium-derived relaxing factor, a labile substance formed by endothelial cells, which mediates vasodilation, has been shown to be identical to nitric oxide (NO) (1-3). In addition to relaxing blood vessels, NO has multiple messen- ger functions as it has been demonstrated in macrophages (4) and in brain tissue (5-7). NO appears responsible for the cytotoxic effects of macrophages and neutrophils (8). We have obtained direct evidence for NO as a messenger for the influences of the excitatory amino acid glutamate on cGMP in the cerebellum (7). We showed a striking enhancement by glutamate and other excitatory amino acids of the conversion of arginine to NO and the associated formation of citrulline. Moreover we observed that Nw-monomethyl-L-arginine (MeArg), an inhibitor of the enzymatic conversion of arginine to NO, inhibits glutamate-elicited cGMP formation, an influ- ence selectively reversed by excess arginine. Evidence that NO mediates functions of tissues as diverse as the brain, endothelium, and blood cells suggests a wide- spread role for NO as a messenger molecule. Localizing NO formation at a cellular level throughout the body would be greatly facilitated by immunohistochemical identification of NO synthetase, the NO-forming enzyme. The mechanism of conversion of arginine to NO, presently unclear (9), would be greatly clarified by purification of NO synthetase. Charac- terization of this enzyme has been hampered by the complex assays required to assay the enzyme by monitoring NO formation directly. We have shown that NO formation is accompanied by the stoichiometric conversion of arginine to citrulline, permitting a simple, sensitive, and specific enzyme assay measuring the transformation of (3H)arginine to (3H)citrulline (7). Utilizing this assay in the present study, we have purified NO synthetase to homogeneity. We demon- strate that NO synthetase is a calmodulin-requiring enzyme, explaining numerous reports of a crucial role for calcium in endothelium-dependent smooth muscle relaxation.
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Journal ArticleDOI
TL;DR: Current evidence indicates that most of the cytotoxicity attributed to NO is rather due to peroxynitrite, produced from the diffusion-controlled reaction between NO and another free radical, the superoxide anion, which is presented in detail in this review.
Abstract: The discovery that mammalian cells have the ability to synthesize the free radical nitric oxide (NO) has stimulated an extraordinary impetus for scientific research in all the fields of biology and medicine. Since its early description as an endothelial-derived relaxing factor, NO has emerged as a fundamental signaling device regulating virtually every critical cellular function, as well as a potent mediator of cellular damage in a wide range of conditions. Recent evidence indicates that most of the cytotoxicity attributed to NO is rather due to peroxynitrite, produced from the diffusion-controlled reaction between NO and another free radical, the superoxide anion. Peroxynitrite interacts with lipids, DNA, and proteins via direct oxidative reactions or via indirect, radical-mediated mechanisms. These reactions trigger cellular responses ranging from subtle modulations of cell signaling to overwhelming oxidative injury, committing cells to necrosis or apoptosis. In vivo, peroxynitrite generation represents a crucial pathogenic mechanism in conditions such as stroke, myocardial infarction, chronic heart failure, diabetes, circulatory shock, chronic inflammatory diseases, cancer, and neurodegenerative disorders. Hence, novel pharmacological strategies aimed at removing peroxynitrite might represent powerful therapeutic tools in the future. Evidence supporting these novel roles of NO and peroxynitrite is presented in detail in this review.

5,514 citations

Journal ArticleDOI
Carl Nathan1
TL;DR: How different forms of nitric oxide synthase help confer specificity and diversity on the effects of this remarkable signaling molecule is reviewed.
Abstract: Evolution has resorted to nitric oxide (NO), a tiny, reactive radical gas, to mediate both servoregulatory and cytotoxic functions. This article reviews how different forms of nitric oxide synthase help confer specificity and diversity on the effects of this remarkable signaling molecule.

4,149 citations

Journal ArticleDOI
TL;DR: This review concentrates on advances in nitric oxide synthase (NOS) structure, function and inhibition made in the last seven years, during which time substantial advances have been made in the authors' understanding of this enzyme family.
Abstract: This review concentrates on advances in nitric oxide synthase (NOS) structure, function and inhibition made in the last seven years, during which time substantial advances have been made in our understanding of this enzyme family. There is now information on the enzyme structure at all levels from primary (amino acid sequence) to quaternary (dimerization, association with other proteins) structure. The crystal structures of the oxygenase domains of inducible NOS (iNOS) and vascular endothelial NOS (eNOS) allow us to interpret other information in the context of this important part of the enzyme, with its binding sites for iron protoporphyrin IX (haem), biopterin, L-arginine, and the many inhibitors which interact with them. The exact nature of the NOS reaction, its mechanism and its products continue to be sources of controversy. The role of the biopterin cofactor is now becoming clearer, with emerging data implicating one-electron redox cycling as well as the multiple allosteric effects on enzyme activity. Regulation of the NOSs has been described at all levels from gene transcription to covalent modification and allosteric regulation of the enzyme itself. A wide range of NOS inhibitors have been discussed, interacting with the enzyme in diverse ways in terms of site and mechanism of inhibition, time-dependence and selectivity for individual isoforms, although there are many pitfalls and misunderstandings of these aspects. Highly selective inhibitors of iNOS versus eNOS and neuronal NOS have been identified and some of these have potential in the treatment of a range of inflammatory and other conditions in which iNOS has been implicated.

3,418 citations

Journal ArticleDOI
25 Oct 1990-Nature
TL;DR: It is demonstrated that NO synthase in the brain to be exclusively associated with discrete neuronal populations, and prominent neural localizations provided the first conclusive evidence for a strong association of NO with neurons.
Abstract: Nitric oxide (NO), apparently identical to endothelium-derived relaxing factor in blood vessels, is also formed by cytotoxic macrophages, in adrenal gland and in brain tissue, where it mediates the stimulation by glutamate of cyclic GMP formation in the cerebellum Stimulation of intestinal or anococcygeal nerves liberates NO, and the resultant muscle relaxation is blocked by arginine derivatives that inhibit NO synthesis It is, however, unclear whether in brain or intestine, NO released following nerve stimulation is formed in neurons, glia, fibroblasts, muscle or blood cells, all of which occur in proximity to neurons and so could account for effects of nerve stimulation on cGMP and muscle tone We have now localized NO synthase protein immunohistochemically in the rat using antisera to the purified enzyme We demonstrate NO synthase in the brain to be exclusively associated with discrete neuronal populations NO synthase is also concentrated in the neural innervation of the posterior pituitary, in autonomic nerve fibres in the retina, in cell bodies and nerve fibres in the myenteric plexus of the intestine, in adrenal medulla, and in vascular endothelial cells These prominent neural localizations provide the first conclusive evidence for a strong association of NO with neurons

2,979 citations

Journal ArticleDOI
TL;DR: The regulation by gonadal and adrenal steroids is one of the most remarkable features of the OT system and is, unfortunately, the least understood.
Abstract: The neurohypophysial peptide oxytocin (OT) and OT-like hormones facilitate reproduction in all vertebrates at several levels. The major site of OT gene expression is the magnocellular neurons of the hypothalamic paraventricular and supraoptic nuclei. In response to a variety of stimuli such as suckling, parturition, or certain kinds of stress, the processed OT peptide is released from the posterior pituitary into the systemic circulation. Such stimuli also lead to an intranuclear release of OT. Moreover, oxytocinergic neurons display widespread projections throughout the central nervous system. However, OT is also synthesized in peripheral tissues, e.g., uterus, placenta, amnion, corpus luteum, testis, and heart. The OT receptor is a typical class I G protein-coupled receptor that is primarily coupled via Gq proteins to phospholipase C-β. The high-affinity receptor state requires both Mg2+ and cholesterol, which probably function as allosteric modulators. The agonist-binding region of the receptor has bee...

2,691 citations