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 expression of NOS isoforms was studied in guinea pig skeletal muscle at the mRNA and protein level, and the effect of NO on contractile response was examined. Ribonuclease protection analyses demonstrated NOS I and NOS II mRNAs in diaphragm and gastrocnemius muscle. In Western blots, NOS I and NOS II immunoreactivities were found in the particulate but not the soluble fraction of skeletal muscle. NOS activity was found almost exclusively in the particulate fraction. About 50% of this activity was Ca2+ independent. In immunohistochemistry, the anti-NOS I antibody stained distinct membrane regions of muscle fibers. The most intense staining was seen in neuromuscular endplates identified by labeling with alpha-bungarotoxin. The anti-NOS II antibody labeled muscle fibers that contained alkali-labile myosin ATPase (type I fibers). NOS II was located to intracellular structures and was also seen in "specific pathogen-free" animals. Pretreatment of guinea pigs with bacterial lipopolysaccharide (LPS) markedly intensified NOS II staining. Significant NOS III immunoreactivity was detected only in vascular endothelium. In functional experiments, tetanic muscle contractions were induced in diaphragm and gastrocnemius muscle by electrical stimulation of the innervating nerves. Pretreatment of guinea pigs with LPS or addition of S-nitroso-N-acetyl-D,L-penicillamine to the organ bath markedly decreased tetanic contractions. N(G)-nitro-L-arginine, on the other hand, increased contractile force and reversed the effect of LPS. Our data indicate that NOS II and NOS I are expressed in different structures of skeletal muscle and are involved in the regulation of contractile response.

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SummaryUsing Western blot and fluorescent immunocytochemistry, NOS III (or ecNOS) and NOS II (or iNOS), but no NOS I (or ncNOS), were identified in preparations of human platelets. Reverse-transcription polymerase chain reactions (RT-PCR) demonstrated NOS III mRNA, but no NOS II mRNA (which is short-lived) and no NOS I mRNA in platelets. Immunofluorescent staining of human bone marrow smears showed the presence of NOS III, but not NOS I in megakaryocytes. A subpopulation of megakaryocytes also expressed NOS II. In preparations of human neutrophils, immunocytochemistry demonstrated NOS I in all cells, whereas no NOS III was detected. The few NOS II positive cells were characterized as contaminating eosinophils. Similarly, in RT-PCR, transcripts for NOS I and NOS II, but not for NOS III, were identified. Thus, the constitutive NOS isoform in megakaryocytes and platelets is NOS III, whereas neutrophils express NOS I. Some megakaryocytes and eosinophils also express NOS II.

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