Three isozymes of nitric oxide (NO) synthase (EC 1.14.13.39) have been identified and the cDNAs for these enzymes isolated. In humans, isozymes I (in neuronal and epithelial cells), II (in cytokine-induced cells), and III (in endothelial cells) are encoded for by three different genes located on chromosomes 12, 17, and 7, respectively. The deduced amino acid sequences of the human isozymes show less than 59% identity. Across species, amino acid sequences for each isoform are well conserved (>90% for isoforms I and III, >80% for isoform II). All isoforms use L-arginine and molecular oxygen as substrates and require the cofactors NADPH, 6(7?)-5,6,7,8-tetrahydrobiopterin, flavin adenine dinucleotide, and flavin mononucleotide. They all bind calmodulin and contain heme. Isoform I is constitutively present in central and peripheral neuronal cells and certain epithelial cells. Its activity is regulated by Ca 2+ and calmodulin. Its functions include long-term regulation of synaptic transmission in the central nervous system, central regulation of blood pressure, smooth muscle relaxation, and vasodilatation via peripheral nitrergic nerves. It has also been implicated in T he initial evidence for the production of nitrogen oxides in mammals came from experiments demonstrating nitrate production in germ-free rats. 1This triggered the search for mammalian cells capable of synthesizing nitrogen oxides and in 1985 led to the discovery that macrophages could be induced with lipopolysaccharide to produce significant amounts of both nitrite and nitrate. 2 Further work demonstrated that L-arginine was the substrate for this pathway and that L-citrulline was formed as a coproduct.3 -4 One year later nitric oxide (NO) was identified as the initial product that is subsequently oxidized to nitrite and nitrate. 5 In parallel, Furchgott and coworkers* 7 had discovered endothelium-derived relaxing factor (EDRF). It had been established that, similar to nitrovasodilators, the EDRF-mediated vasodilatation was associated with increased levels of cyclic GMP and activation of cyclic GMP kinase activity in smooth muscle cells 810 and that the EDRF could directly stimulate purified soluble guanyh/1 cyclase. 1112 In 1987 it was concluded that NO can account for the biologic activity of EDRF, 1315 and, analogous to the macrophage system, L-arginine was established as a substrate for EDRF/NO synthesis in endothelial cells. physiological research demonstrated that stimulation of neuronal cells and brain slices with agonists leads to the release of a labile mediator that stimulates guanylyl cyclase and has the properties of NO.1820 During the past 4 years, significant progress has been made elucidating the mechanism of NO synthesis, the NO synthases involved, and the functions of NO in different biologic systems. The present review attempts to summarize this progress with some emphasis on the cardiovascular system. Isozymes of NO SynthaseMany cells are capable of synthesizing NO. Three isozymes of NO synthase (EC 1.14.13.39) have been...

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Nitric oxide synthase (NOS) exists in three established isoforms. NOS I (NOS1, ncNOS) was originally discovered in neurons. This enzyme and splice variants thereof have since been found in many other cells and tissues. NOS II (NOS2, iNOS) was first identified in murine macrophages, but can also be induced in many other cell types. NOS III (NOS3, ecNOS) is expressed mainly in endothelial cells. Whereas NOS II is a transcriptionally regulated enzyme, NOS I and NOS III are considered constitutively expressed proteins. However, evidence generated in recent years indicates that these two isoforms are also subject to expressional regulation. In view of the important biological functions of these isoforms, changes in their expression may have physiological and pathophysiological consequences. This review recapitulates compounds and conditions that modulate the expression of NOS I and NOS III, summarizes transcriptional and posttranscriptional effects that underlie these changes, and-where known-describes the molecular mechanisms leading to changes in transcription, RNA stability, or translation of these enzymes.

Nitric oxide (NO), generated by the inducible isoform of nitric oxide synthase (iNOS), has been described to have beneficial microbicidal, antiviral, antiparasital, immunomodulatory, and antitumoral effects. However, aberrant iNOS induction at the wrong place or at the wrong time has detrimental consequences and seems to be involved in the pathophysiology of several human diseases. iNOS is primarily regulated at the expression level by transcriptional and post-transcriptional mechanisms. iNOS expression can be induced in many cell types with suitable agents such as bacterial lipopolysaccharides (LPS), cytokines, and other compounds. Pathways resulting in the induction of iNOS expression may vary in different cells or different species. Activation of the transcription factors NF-kappaB and STAT-1alpha, and thereby activation of the iNOS promoter, seems to be an essential step for iNOS induction in most cells. However, at least in the human system, also post-transcriptional mechanism are critically involved in the regulation of iNOS expression. The induction of iNOS can be inhibited by a wide variety of immunomodulatory compounds acting at the transcriptional levels and/or post-transcriptionally.

Hypertension is a side effect of systemically administered glucocorticoids, but the underlying molecular mechanism remains poorly understood. Ingestion of dexamethasone by rats telemetrically instrumented increased blood pressure progressively over 7 days. Plasma concentrations of Na ؉ and K ؉ and urinary Na ؉ and K ؉ excretion remained constant, excluding a mineralocorticoid-mediated mechanism. Plasma NO 2 ؊ ͞NO3 ؊ (the oxidation products of NO) decreased to 40%, and the expression of endothelial NO synthase (NOS III) was found down-regulated in the aorta and several other tissues of glucocorticoid-treated rats. The vasodilator response of resistance arterioles was tested by intravital microscopy in the mouse dorsal skinfold chamber model. Dexamethasone treatment significantly attenuated the relaxation to the endothelium-dependent vasodilator acetylcholine, but not to the endothelium-independent vasodilator S-nitroso-N-acetyl-D,L-penicillamine. Incubation of human umbilical vein endothelial cells, EA.hy 926 cells, or bovine aortic endothelial cells with several glucocorticoids reduced NOS III mRNA and protein expression to 60 -70% of control, an effect that was prevented by the glucocorticoid receptor antagonist mifepristone. Glucocorticoids decreased NOS III mRNA stability and reduced the activity of the human NOS III promoter (3.5 kilobases) to Ϸ70% by decreasing the binding activity of the essential transcription factor GATA. The expressional down-regulation of endothelial NOS III may contribute to the hypertension caused by glucocorticoids.dexamethasone ͉ dihydrocortisol ͉ RNase protection assay ͉ Western blot ͉ Reporter gene assay ͉

Three isozymes of nitric oxide synthase (NOS) have been identified. Their cDNA- and protein structures as well as their genomic DNA structures have been described. NOS I (ncNOS, originally discovered in neurons) and NOS III (ecNOS, originally discovered in endothelial cells) are low output, Ca(2+)-activated enzymes whose physiological function is signal transduction. NOS II (iNOS, originally discovered in cytokine-induced macrophages) is a high output enzyme which produces toxic amounts of NO that represent an important component of the antimicrobial, antiparasitic and antineoplastic activity of these cells. Depending on the species, NOS II activity is largely (human) or completely (mouse and rat) Ca(2+)-independent. In the human species, the NOS isoforms I, II and III are encoded by three different genes located on chromosomes 12, 17 and 7, respectively. The amino acid sequences of the three human isozymes (deduced from the cloned cDNAs) show less than 59% identity. Across species, amino acid sequences are more than 90% conserved for NOS I and III, and greater 80% identical for NOS II. All NOS produce NO by oxidizing a guanidino nitrogen of L-arginine utilizing molecular oxygen and NADPH as co-substrates. All isoforms contain FAD, FMN and heme iron as prosthetic groups and require the cofactor BH4. NOS I and III are constitutively expressed in various cells. Nevertheless, expression of these isoforms is subject to regulation. Expression is enhanced by e.g. estrogens (for NOS I and III), shear stress, TGF-beta 1, and (in certain endothelial cells) high glucose (for NOS III). TNF-alpha reduces the expression of NOS III by a post-transcriptional mechanism destabilizing the mRNA. The regulation of the NOS I expression seems to be very complex as reflected by at least 8 different promoters transcribing 8 different exon 1 sequences which are expressed differently in different cell types. Expression of NOS II is mainly regulated at the transcriptional level and can be induced in many cell types with suitable agents such as LPS, cytokines, and other compounds. Whether some cells can express NOS II constitutively is still under debate. Pathways resulting in the induction of the NOS II promoter may vary in different cells. Activation of transcription factor NF-kappa B seems to be an essential step for NOS II induction in most cells. The induction of NOS II can be inhibited by a wide variety of immunomodulatory compounds acting at the transcriptional levels and/or post-transcriptionally.

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