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.

https://faseb.onlinelibrary.wiley.com/doi/pdf/10.1096/fasebj.6.12.1381691

Nitric oxide as a secretory product of mammalian cells.

NADPH diaphorase histochemistry selectively labels a number of discrete populations of neurons throughout the nervous system. This simple and robust technique has been used in a great many experimental and neuropathological studies; however, the function of this enzyme has remained a matter of speculation. We, therefore, undertook to characterize this enzyme biochemically. With biochemical and immunochemical assays, NADPH diaphorase was purified to apparent homogeneity from rat brain by affinity chromatography and anion-exchange HPLC. Western (immunoblot) transfer and immunostaining with an antibody specific for NADPH diaphorase labeled a single protein of 150 kDa. Nitric oxide synthase was recently shown to be a 150-kDa, NADPH-dependent enzyme in brain. It is responsible for the calcium/calmodulin-dependent synthesis of the guanylyl cyclase activator nitric oxide from L-arginine. We have found that nitric oxide synthase activity and NADPH diaphorase copurify to homogeneity and that both activities could be immunoprecipitated with an antibody recognizing neuronal NADPH diaphorase. Furthermore, nitric oxide synthase was competitively inhibited by the NADPH diaphorase substrate, nitro blue tetrazolium. Thus, neuronal NADPH diaphorase is a nitric oxide synthase, and NADPH diaphorase histochemistry, therefore, provides a specific histochemical marker for neurons producing nitric oxide.

https://www.pnas.org/content/pnas/88/7/2811.full.pdf

Neuronal NADPH diaphorase is a nitric oxide synthase.

https://www.cell.com/cell/pdf/0092-8674(94)90267-4.pdf

NO at work

Endothelium-derived relaxing factor (EDRF) activity has been attributed to the highly labile nitric oxide radical (NO). In view of the fact that the plasma and cellular milieux contain reactive species that can rapidly inactivate NO, it has been postulated that NO is stabilized by a carrier molecule that preserves its biological activity. Reduced thiol species are candidates for this role, reacting readily in the presence of NO to yield biologically active S-nitrosothiols that are more stable than NO itself. Because sulfhydryl groups in proteins represent an abundant source of reduced thiol in biologic systems, we examined the reaction of several sulfhydryl-containing proteins of diverse nature and function upon exposure to authentic NO and EDRF. We demonstrate that S-nitroso proteins form readily under physiologic conditions and possess EDRF-like effects of vasodilation and platelet inhibition. These observations suggest that S-nitrosothiol groups in proteins may serve as intermediates in the cellular metabolism of NO and raise the possibility of an additional type of cellular regulatory mechanism.

https://www.pnas.org/content/pnas/89/1/444.full.pdf

S-nitrosylation of proteins with nitric oxide: synthesis and characterization of biologically active compounds

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(R)-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 Ca2+ 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 vasodilation via peripheral nitrergic nerves. It has also been implicated in neuronal death in cerebrovascular stroke. Expression of isoform II of NO synthase can be induced with lipopolysaccharide and cytokines in a multitude of different cells. Based on sequencing data there is no evidence for more than one inducible isozyme at this time. NO synthase II is not regulated by Ca2+; it produces large amounts of NO that has cytostatic effects on parasitic target cells by inhibiting iron-containing enzymes and causing DNA fragmentation. Induced NO synthase II is involved in the pathophysiology of autoimmune diseases and septic shock. Isoform III of NO synthase has been found mostly in endothelial cells. It is constitutively expressed, but expression can be enhanced, eg, by shear stress. Its activity is regulated by Ca2+ and calmodulin. NO from endothelial cells keeps blood vessels dilated, prevents the adhesion of platelets and white cells, and probably inhibits vascular smooth muscle proliferation.

Nitric oxide synthase isozymes. Characterization, purification, molecular cloning, and functions.

Stimulation of soluble guanylyl cyclase in rat fetal lung fibroblasts (RFL-6 cells) was used as a sensitive assay for endothelium-derived relaxing factor/nitric oxide (EDRF/NO) formation. Intact N1E-115 cells released an EDRF/NO-like material that enhanced cyclic GMP levels in RFL-6 cells. The synthesis of this substance could be stimulated with the receptor agonist neurotensin (10 microM) or by addition of the EDRF/NO substrate L-arginine (100 microM). In Ca2(+)-free Locke9s solution, stimulation of EDRF/NO production by both neurotensin and L-arginine was abolished. The EDRF/NO-synthesizing activity was localized in the cytosol of N1E-115 cells. The activity was lost after boiling and it was highly sensitive to Ca2+ with the major increase in activity occurring between 100 and 500 nM Ca2+. L-Arginine and NADPH were required for maximal synthesis of EDRF/NO by the enzyme(s). The synthesis of EDRF/NO was inhibited by the following antagonists of calmodulin-regulated functions (with the approximate IC50 values given in parentheses): calmidazolium (7 microM), trifluoperazine (10 microM), fendiline (80 microM), W-7 (N-[6-aminohexyl]-5-chloro-1-naphthalenesulfonamide) (120 microM), and compound 48/80 (3 micrograms/ml). The EDRF/NO-synthesizing activity was partially purified from N1E-115 cytosol by DE 52 anion exchange chromatography. The activity was eluted with 0.1 M KCl. The enzyme(s) showed very little activity in the presence of L-arginine (100 microM) and NADPH (100 microM), but the activity could be fully restored by addition of exogenous calmodulin (EC50, approximately 2 units/ml). At 0.3 M KCl, a fraction eluted from the DE 52 column that was also able to fully restore the EDRF/NO-synthesizing activity. Thus, this fraction is likely to contain the endogenous Ca2(+)-binding protein. It is concluded that the activity of the EDRF/NO-synthesizing enzyme(s) in N1E-115 neuroblastoma cells is regulated by Ca2+ and calmodulin.

Hormone-induced biosynthesis of endothelium-derived relaxing factor/nitric oxide-like material in N1E-115 neuroblastoma cells requires calcium and calmodulin.

EDRF is an intracellular second messenger and autacoid to regulate cyclic GMP synthesis in many cells.

Quick stretch increases the production of inositol 1,4,5-trisphosphate(ip3) in porcine coronary artery

Specific attenuation of the pressure-induced contraction of rat cerebral artery by herbimycin A

Nitric oxide-cyclic GMP system potentiates glucose-induced rise in cytosolic Ca2+ concentration in rat pancreatic β-cells

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