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...

The endothelins comprise three structurally similar 21-amino acid peptides. Endothelin-1 and -2 activate two G-protein coupled receptors, ETA and ETB, with equal affinity, whereas endothelin-3 has a lower affinity for the ETA subtype. Genes encoding the peptides are present only among vertebrates. The ligand-receptor signaling pathway is a vertebrate innovation and may reflect the evolution of endothelin-1 as the most potent vasoconstrictor in the human cardiovascular system with remarkably long lasting action. Highly selective peptide ETA and ETB antagonists and ETB agonists together with radiolabeled analogs have accurately delineated endothelin pharmacology in humans and animal models, although surprisingly no ETA agonist has been discovered. ET antagonists (bosentan, ambrisentan) have revolutionized the treatment of pulmonary arterial hypertension, with the next generation of antagonists exhibiting improved efficacy (macitentan). Clinical trials continue to explore new applications, particularly in renal failure and for reducing proteinuria in diabetic nephropathy. Translational studies suggest a potential benefit of ETB agonists in chemotherapy and neuroprotection. However, demonstrating clinical efficacy of combined inhibitors of the endothelin converting enzyme and neutral endopeptidase has proved elusive. Over 28 genetic modifications have been made to the ET system in mice through global or cell-specific knockouts, knock ins, or alterations in gene expression of endothelin ligands or their target receptors. These studies have identified key roles for the endothelin isoforms and new therapeutic targets in development, fluid-electrolyte homeostasis, and cardiovascular and neuronal function. For the future, novel pharmacological strategies are emerging via small molecule epigenetic modulators, biologicals such as ETB monoclonal antibodies and the potential of signaling pathway biased agonists and antagonists.

The particulate enzyme responsible for the synthesis of endothelium-derived relaxing factor has been purified from cultured and native (noncultured) bovine aortic endothelial cells. Purification of the solubilized particulate enzyme preparation by affinity chromatography on adenosine 2',5'-bisphosphate coupled to Sepharose followed by Superose 6 gel filtration chromatography resulted in a single protein band after denaturing polyacrylamide gel electrophoresis that corresponded to -135 kDa. The enzyme activity in the various fractions was assayed by its stimulatory effect on soluble guanylyl cyclase of rat fetal lung fibroblasts , by the formation of L-citrulline from L-arginine, by measuring nitrite/nitrate formation, and by bioassay on endotheliumdenuded vascular strips. Endothelium-derived relaxing factor synthase was purified 3419-fold from the crude particulate fraction of cultured bovine aortic endothelial cells with a 12% recovery (RFL-6 assay). Purified endothelium-derived relaxing factor synthase required L-arginine, NADPH, Ca2 , calmodulin, and 5,6,7,8-tetrahydrobiopterin for full activity.Endothelial cells synthesize endothelium-derived relaxing factor (EDRF) from L-arginine (1, 2). The pharmacological and biochemical properties of EDRF are mimicked by nitric oxide (NO) (3) or NO-containing compounds (4). These agents activate soluble guanylyl cyclase (5, 6) thereby increasing cGMP and causing relaxation of vascular smooth muscle (7-9), inhibition of platelet aggregation (10), and other effects (9, 11). EDRF/NO synthase has been purified and characterized from brain (12-14), polymorphonuclear neutrophils (15), and endotoxin/cytokine-induced macrophages (16,17). The endothelial, brain, and neutrophil EDRF/NO synthases are constitutive enzymes whereas the macrophage activity is expressed only after induction with endotoxin and/or a cytokine. The constitutive enzyme from brain is soluble and Ca2+/calmodulin-regulated whereas the enzyme from neutrophils has been described as soluble and Ca2+-dependent, but not calmodulin-dependent. Analysis with denaturing gel electrophoresis revealed a single band corresponding to 155 kDa and 150 kDa for the brain (12) and neutrophil (15) In endothelial cells, a particulate Ca2+/calmodulin-regulated enzyme accounts for >95% of the total EDRF synthase activity (18,19). We now report the purification to homogeneity (denaturing molecular mass of 135 kDa) of this constitutive particulate EDRF synthase and characterize the enzyme as NADPH-and (6R)-5,6,7,8-tetrahydrobiopterin (BH4)-dependent. Some of these observations have been reported in abstract form (20,21).

Atherosclerosis is associated with reduced endothelium-derived relaxing factor bioactivity. To determine whether this is due to decreased synthesis of nitric oxide synthase (NOS), we examined normal and atherosclerotic human vessels by in situ hybridization and immunocytochemistry by using probes specific for endothelial (ecNOS), inducible (iNOS), and neuronal (nNOS) NOS isoforms, ecNOS was detected in endothelial cells overlying normal human aortas, fatty streaks, and advanced atherosclerotic lesions. A comparison of the relative expression of ecNOS to von Willebrand factor on serial sections of normal and atherosclerotic vessels indicated that there was a decrease in the number of endothelial cells expressing ecNOS in advanced lesions. iNOS and nNOS were not detected in normal vessels, but widespread production of these isoforms was found in early and advanced lesions associated with macrophages, endothelial cells, and mesenchymal-appearing intimal cells. These data suggest that there is (1) a loss of ecNOS expression by endothelial cells over advanced atherosclerotic lesions and (2) a significant increase in overall NOS synthesis by other cell types in advanced lesions composed of the ecNOS, nNOS, and iNOS isoforms. We hypothesize that the increased expression of NOS and presumably NO in atherosclerotic plaques may be related to cell death and necrosis in these tissues.

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Nitric oxide synthase (NOS Types I-III) generate nitric oxide (NO), which in turn activates soluble guanylyl cyclase (GC-S). The distribution of this NO-mediated (nitrinergic) signal transduction pathway in the body is unclear. A polyclonal monospecific antibody to rat cerebellum NOS-I and a monoclonal antibody to rat lung GC-S were employed to localize the protein components of this pathway in different rat organs and tissues. We confirmed the localization of NOS-I in neurons of the central and peripheral nervous system, where NO may regulate cerebral blood flow and mediate long-term potentiation. GC-S was located in NOSnegative neurons, indicating that NO acts as an intercellular signal molecule or neurotransmitter. However, NOS-I was not confined to neurons but was widely distributed over IntroductionThe intracellular formation of nitric oxide (NO) has been extensively studied in various mammalian tissues. NO (1,2), or a labile intermediate that is able to release NO, is generated from a terminal guanidino nitrogen of L-arginine (3-5) by a family of NO synthases (NOS; EC 1.14.23). L-Citrulline is the byproduct of this metabolic pathway. NO is the first messenger molecule of a signal transduction pathway (Figure 1). Although other targets for NO Supported by research grants DK 30787 and H I . 28474 several non-neural cell types and tissues. These included glia cells, m a d densa of kidney, epithelial cells of lung, uterus, and stomach, and islets of Langerhans. Our findings suggest that NOS-I is the most widely distributed isoform of NOS and, in addition to its neural functions, regulates secretion and non-vascular smooth muscle function. With the exception of bone tissue, NADPH-diaphorase (NADPH-d) activity was generally co-localized with NOS-I immunoreactivity in both neural and non-neural cells, and is a suitable histochemical marker for NOS-I but not a selective neuronal marker. (J Histochem Cytochem 40:1439-1456, 1992) KEY WORDS: Cyclic GMP; Brain; Pancreas; Kidney; Stomach; Lung; Uterus; Bone; Epithelium; Endometrium. exist (6), its main mechanism of action involves binding to and activation of soluble guanylyl cyclase (GC-S; GTP pyrophosphatelyase (cyclizing), EC 4.6.1.2) (7), which then forms the second messenger molecule guanosine 3',5'-cyclic monophosphate (cGMP).NOS have been purified and characterized from brain (8-14), macrophages (15,16), and endothelial cells (17), and recently also from human cerebellum (18). Molecular cloning has provided clear evidence that brain (19) and macrophage (20) NOS are products of different genes. The relation between brain and endothelial NOS is unclear. Another polydonal antibody raised against rat brain NOS (21) was reported to bind to endothelial cell matrix, and the authors suggested that brain and endothelial NOS are highly homologous if not identical (22). Conversely, endothelial NOS was reported by our laboratory to differ from brain NOS with respect to molecular mass and subcellular location (17) and we proposed a classification of at least three types...

Complementary DNA clones corresponding to human brain nitric oxide (NO) synthase have been isolated. The deduced amino acid sequence revealed an overall identity with rat brain NO synthase of about 93% and contained all suggested consensus sites for binding of the co-factors. The cDNA transfected COS-1 cells showed significant NO synthase activity with the typical co-factor requirements. Unexpectedly, messenger RNA levels of this isoform of NO synthase was more abundant in human skeletal muscle than human brain. Moreover, we detected high NO synthase activity and the expressed protein in human skeletal muscle by Western blot analysis, indicating a possible novel function of NO in skeletal muscle.

The particulate enzyme, endothelial nitric oxide synthase (eNOS), produces nitric oxide to maintain normal vasodilator tone in blood vessels. In this study, we demonstrate that eNOS is a Golgi-associated protein in cultured endothelial cells and intact blood vessels. Using a heterologous expression system in HEK 293 cells, we show that wild-type myristoylated and palmitoylated eNOS, but not mutant, non-acylated eNOS targets to the Golgi. More importantly, HEK 293 cells expressing wild-type eNOS release substantially more NO than cells expressing the mutant, non-acylated enzyme. Thus, eNOS is a novel Golgi-associated protein, and Golgi compartmentalization is necessary for the enzyme to respond to intracellular signals and produce NO.