Chronic inhibition of nitric oxide synthase activity by N(G)-nitro-L-arginine induces nitric oxide synthase expression in the developing rat cerebellum

Studies on chronic inhibition of nitric oxide synthase (NOS) in the CNS suggest a plastic change in nitric oxide (NO) synthesis in areas related to motor control, which might protect the animal from the functional and behavioral consequences of NO deficiency. In the present study, the acute and chronic effect of the substrate analogue inhibitor N(G)-nitro-l-arginine (l-NNA) was examined on NO production, NO-sensitive cyclic guanosine monophosphate (cGMP) levels and the expression of NOS isoforms in the developing rat cerebellum. Acute intraperitoneal administration of the inhibitor (5-200mg/kg) to 21-day-old rats reduced NOS activity and NO concentration dose dependently by 70-90% and the tissue cGMP level by 60-80%. By contrast, chronic application of l-NNA between postnatal days 4-21 diminished the total NOS activity and NO concentration only by 30%, and the tissue cGMP level by 10-50%. Chronic treatment of 10mg/kg l-NNA induced neuronal (n)NOS expression in granule cells, as revealed by in situ hybridization, NADPH-diaphorase histochemistry and Western-blot, but it had no significant influence on tissue cGMP level or on layer formation of the cerebellum. However, a higher concentration (50mg/kg) of l-NNA decreased the intensity of the NADPH-diaphorase reaction in granule cells, significantly reduced cGMP production, and retarded layer formation and induced inducible (i)NOS expression & activity in glial cells. Treatments did not affect endothelial (e)NOS expression. The administration of the biologically inactive isomer D-NNA (50mg/kg) or saline was ineffective. The present findings suggest the existence of a concentration-dependent compensatory mechanism against experimentally-induced cronich inhibition of NOS, including nNOS or iNOS up-regulation, which might maintain a steady-state NO level in the developing cerebellum.     

Effects of arginase isoforms on NO Production by nNOS.

Both arginase isoforms (AI and AII) regulate high-level NO production by the inducible NOS, but whether the arginase isoforms also regulate low-level NO production by neuronal NOS (nNOS) is not known. In this study, 293 cells that stably overexpress nNOS gene (293nNOS cells) were transfected with rat AI (pEGFP-AI) or AII (pcDNA-AII) plasmids, and nitrite production was measured with or without supplemental L-arginine. Transfection with pEGFP-AI increased AI expression and activity 10-fold and decreased intracellular l-arginine by 50%. Nitrite production was inhibited by >80% when no l-arginine was supplemented but not when 1 mM L-arginine was present. The inhibition was reversed by an arginase inhibitor, N(omega)-hydroxy-L-arginine. Transfection with pcDNA-AII increased AII expression and activity but had little effect on nitrite production even if no l-arginine was added. These results suggest that, in 293nNOS cells, AI was more effective in regulating NO production by nNOS, most likely by competing for L-arginine.     

Selective inhibition of inducible nitric oxide synthase exacerbates erosive joint disease

NO is an essential cytotoxic agent in host defense, yet can be autotoxic if overproduced, as evidenced in inflammatory lesions and tissue destruction in experimental arthritis models. Treatment of streptococcal cell wal1-induced arthritis in rats with N:(G)-monomethyl-L-arginine (L-NMMA), a competitive nonspecific inhibitor of both constitutive and inducible isoforms of NO synthase (NOS), prevents intraarticular accumulation of leukocytes, joint swelling, and bone erosion. Because increased inducible NOS (iNOS) expression and NO generation are associated with pathogenesis of chronic inflammation, we investigated whether a selective inhibitor of iNOS, N:-iminoethyl-L-lysine (L-NIL), would have more directed anti-arthritic properties. Whereas both L-NMMA and L-NIL inhibited nitrite production by streptococcal cell wall-stimulated rat mononuclear cells in vitro and systemic treatment of arthritic rats with L-NMMA ablated synovitis, surprisingly L-NIL did not mediate resolution of inflammatory joint lesions. On the contrary, daily administration of L-NIL failed to reduce the acute response and exacerbated the chronic inflammatory response, as reflected by profound tissue destruction and loss of bone and cartilage. Although the number of iNOS-positive cells within the synovium decreased after treatment with L-NIL, immunohistochemical analyses revealed a distinct pattern of endothelial and neuronal NOS expression in the arthritic synovium that was unaffected by the isoform-specific L-NIL treatment. These studies uncover a contribution of the constitutive isoforms of NOS to the evolution of acute and chronic inflammation pathology which may be important in the design of therapeutic agents.     

Neuronal nitric oxide synthase: expression in rat parietal cells

Nitric oxide synthases (NOS) are enzymes that catalyze the generation of nitric oxide (NO) from L-arginine and require nicotinamide adenine dinucleotide phosphate (NADPH) as a cofactor. At least three isoforms of NOS have been identified: neuronal NOS (nNOS or NOS I), inducible NOS (iNOS or NOS II), and endothelial NOS (eNOS or NOS II). Recent studies implicate NO in the regulation of gastric acid secretion. The aim of the present study was to localize the cellular distribution and characterize the isoform of NOS present in oxyntic mucosa. Oxyntic mucosal segments from rat stomach were stained by the NADPH-diaphorase reaction and with isoform-specific NOS antibodies. The expression of NOS in isolated, highly enriched (>98%) rat parietal cells was examined by immunohistochemistry, Western blot analysis, and RT-PCR. In oxyntic mucosa, histochemical staining revealed NADPH-diaphorase and nNOS immunoreactivity in cells in the midportion of the glands, which were identified as parietal cells in hematoxylin and eosin-stained step sections. In isolated parietal cells, decisive evidence for nNOS expression was obtained by specific immunohistochemistry, Western blotting, and RT-PCR. Cloning and sequence analysis of the PCR product confirmed it to be nNOS (100% identity). Expression of nNOS in parietal cells suggests that endogenous NO, acting as an intracellular signaling molecule, may participate in the regulation of gastric acid secretion.     

The protective effect of aminoguanidine on cerebral ischemic damage in the rat brain

The NADPH-diaphorase (NADPH-d) histochemical technique is commonly used to localize the nitric oxide (NO) produced by the enzyme nitric oxide synthase (NOS) in neural tissue. The expression of inducible nitric oxide synthase (iNOS) is induced in the late stage of cerebral ischemia, and NO produced by iNOS contributes to the delay in recovery from brain neuronal damage. The present study was performed to investigate whether the increase in nitric oxide production via inducible nitric oxide synthase was suppressed by the administration of aminoguanidine, a selective iNOS inhibitor, as it follows a decrease of NADPH-diaphorase activity (a marker for NOS) after four-vessel occlusion used as an ischemic model. The administration of aminoguanidine (100 mg/kg i.p., twice per day up to 3 days immediately after the ischemic insult) reduced the number of NADPH-diaphorase positive cells to control levels. Our results indicated that aminoguanidine suppressed NADPH-diaphorase activity, and also decreased the number of NADPH-diaphorase positive cells in the CA1 region of the hippocampus following ischemic brain injury.     

Requirement of nitric oxide for murine oocyte maturation, embryo development, and trophoblast outgrowth in vitro

We investigated the extent to which NO participates in the developmental competence (oocyte maturation, fertilization and embryo development to blastocyst) using an in vitro culture system adding sodium nitroprusside (SNP), NO donor, and NOS inhibitor (N-omega-nitro-L-arginine methyl ester, L-NAME). We also assessed the effects of NO/NOS system on blastocyst implantation using an in vitro trophoblast outgrowth assay. The treatment of low concentrations of SNP (10(-7) M) significantly stimulated meiotic maturation to metaphase II stages in cumulus enclosed oocytes. In contrast, 10(-3) and 10(-5) M L-NAME demonstrated a significant suppression in resumption of meiosis. This inhibition was reversed by the addition of SNP. No development beyond the four-cell stage was observed by the addition of high concentration of SNP (10(-3) M). Inhibition of embryo development, especially the conversion of morulae to blastocysts, was also observed in the treatment of lower doses of SNP (10(-5) and 10(-7) M). Similarly, inhibition of NO by NOS inhibitor resulted in the dose-dependent inhibition of embryo development and hatching rates, but the concomitant addition of SNP with L-NAME reversed the inhibitory effect by each SNP or L-NAME treatment. Furthermore, low concentration of SNP (10(-7) M) but not high concentration of SNP (10(-3) M) significantly stimulated trophoblast outgrowth, whereas the addition of L-NAME suppressed the spreading of blastocysts in a dose-dependent manner. These results suggest that NO may have crucial roles in oocyte maturation and embryogenesis including the process of implantation. The observed differences in required amount of NO and the sensitivity to cytotoxicity of NO in each developmental stage embryos may also suggest that NO/NOS system is tightly regulated in developmental stage specific manner.     

Proinflammatory agents,IL-8 and IL-10, upregulate inducible nitric oxide synthase expression and nitric oxide production in avian osteoclast-like cells

Nitric oxide synthase (NOS) isoenzymes generate nitric oxide (NO), a sensitive multifunctional intercellular signal molecule. High NO levels are produced by an inducible NOS (iNOS) in activated macrophages in response to proinflammatory agents, many of which also regulate local bone metabolism. NO is a potent inhibitor of osteoclast bone resorption, whereas inhibitors of NOS promote bone resorption both in vitro and in vivo. The possibility that osteoclasts, like macrophages, express a regulated iNOS and produce NO as a potential autocrine signal following inflammatory stimulation was investigated in well-characterized avian marrow-derived osteoclast-like cells. NO production (reflected by medium nitrite levels) was markedly elevated in these cells by the proinflammatory agents lipopolysaccharide (LPS) and the synergistic action of IL-1α, TNFα, and IFNγ. Inhibitors of NOS activity (aminoguanidine, L-NAME) or iNOS induction (dexamethasone, TGFβ) reduced LPS-stimulated nitrite production. LPS also increased the NOS-associated diaphorase activity of these cells and their reactivity with anti-iNOS antibodies. RT-PCR cloning, using avian osteoclast-like cell RNA and human iNOS primers, yielded a novel 900 bp cDNA with high sequence homology (76%) to human, rat, and mouse iNOS genes. In probing osteoclast-like cell RNA with the PCR-derived iNOS cDNA, a 4.8 kb mRNA species was detected whose levels were greatly increased by LPS. Induction of iNOS mRNA by LPS, or by proinflammatory cytokines, occurred prior to the rise of medium nitrite in time course studies and was diminished by dexamethasone. Moreover, osteoclast-like cells demonstrated an upregulation of NO production and iNOS mRNA by IL-8 and IL-10, regulatory mechanism's not previously described. It is concluded that osteoclast-like cells express a novel iNOS that is upregulated by inflammatory mediators, leading to NO production. Therefore, NO may serve as both a paracrine and autocrine signal for modulating osteoclast bone resorption. © 1996 Wiley-Liss, Inc.     

Dynamin activates NO production in rat renal inner medullary collecting ducts via protein-protein interaction with NOS1

We hypothesized that nitric oxide synthase (NOS) isoforms may be regulated by dynamin (DNM) in the inner medullary collecting duct (IMCD). The aims of this study were to determine which DNM isoforms (DNM1, DNM2, DNM3) are expressed in renal IMCDs, whether DNM interacts with NOS, whether a high-salt diet alters the interaction of DNM and NOS, and whether DNM activates NO production. DNM2 and DNM3 are highly expressed in the rat IMCD, while DNM1 is localized outside of the IMCD. We found that DNM1 interacts with NOS1α, NOS1β, and NOS3 in the inner medulla of male Sprague-Dawley rats on a 0.4% salt diet. DNM2 interacts with NOS1α, while DNM3 interacts with both NOS1α and NOS1β. DNM2 and DNM3 do not interact with NOS3 in the rat inner medulla. We did not observe any change in the DNM/NOS interactions with rats on a 4% salt diet after 7 days. Furthermore, NOS1α interacts with DNM2 in mIMCD3 and COS7 cells transfected with NOS1α and DNM2-GFP constructs and the NOS1 reductase domain is necessary for the interaction. Finally, COS7 cells expressing NOS1α or NOS1α/DNM2-GFP had significantly higher nitrite production compared with DNM2-GFP only. Nitrite production was blocked by the DNM inhibitor dynasore or the dominant negative DNM2K44A. Ionomycin stimulation further increased nitrite production in the NOS1α/DNM2-GFP cells compared with NOS1α only. In conclusion, DNM and NOS1 interact in the rat renal IMCD and this interaction leads to increased NO production, which may influence NO production in the renal medulla.     

Differential NOS expression in freshwater and aestivating Protopterus dolloi (lungfish): heart vs kidney readjustments.

African lungfish Protopterus dolloi is an obligatory air-breather, which aestivates in a cocoon during the dry season. Aestivation associates with functional modifications in many tissues and organs, including heart and kidney. Due to its pleiotropic modulatory effects, nitric oxide (NO), generated by nitric oxide synthases (NOSs), may coordinate organ rearrangement, allowing adaptive adjustments under stressful environmental conditions. By immunofluorescence, Western blotting and NADPH-diaphorase, we examined cardiac and renal localization and activity of NOSs isoforms in both freshwater (FW) and aestivating [6 days (6DA) and 40 days (40DA) of estivation] P. dolloi. In heart and kidney endothelial NOS (eNOS) is the major isoform with respect to inducible and neuronal NOS (iNOS and nNOS, respectively). Cardiac eNOS locates in the epicardium, the trabecular endothelial endocardium, and myocardiocytes of both FW and aestivating fish. Western blotting revealed that cardiac eNOS expression increases in 6DA, but decreases in 40DA fish. In FW fish kidney eNOS is present in vascular endothelial cells and in podocytes of renal corpuscles. In tubular epithelial cells it is restricted to the apical pole. With aestivation, both renal localization and expression of eNOS increase. NADPH-diaphorase revealed an enhancement of cardiac and renal NOS activities during aestivation. Results suggest that in P. dolloi NO contributes, in an autocrine-paracrine fashion, to cardiac and renal readjustments during aestivation. Our findings are of evolutionary interest, since they document for the first time the presence of a NOS system in a ancestral fish, indicative of deep phylogenetic roots of NO bio-synthesis.     

Activation of the mitogen-activated protein kinase cascade through nitric oxide synthesis as a mechanism of neuritogenic effect of genipin in PC12h cells

Prominent neurite outgrowth induced by genipin, a plant-derived iridoid, was substantially inhibited by addition of NG-nitro-L-arginine methyl ester (L-NAME), a nitric oxide (NO) synthase (NOS) inhibitor, and carboxy-PTIO, an NO scavenger, in PC12h cells. Increases of the NADPH-diaphorase activity and neuronal and inducible NOS proteins in cells preceded the neurite outgrowth after addition of genipin to medium. NO donors could induce the neurite outgrowth dose-dependently in the cells. On the other hand, an inhibitor of soluble guanylate cyclase (SGC), which is known to be a stimulatory target of NO, abolished greatly the genipin-induced neurite outgrowth. Addition of extracellular signal-regulated kinase (ERK) kinase inhibitors could almost completely abolish the neurite induction. L-NAME remarkably depressed genipin-stimulated phosphorylation of ERK-1 and -2. A neuritogenic effect of nerve growth factor (NGF) in PC12h cells was also remarkably inhibited by the NOS inhibitor, NO scavenger and SGC inhibitor. These findings suggest that induced NO production followed by cyclic GMP-mediated stimulation of the mitogen-activated protein kinase (MAPK) cascade is implicated in the neuritogenesis by genipin and NGF in PC12h cells.     

Plasminogen activators in tissue remodeling and invasion: mRNA localization in mouse ovaries and implanting embryos

To assess in vivo the postulated participation of urokinase-type (u-PA) and tissue-type (t-PA) plasminogen activators in processes involving tissue remodeling and cell migration, we have studied the cellular distribution of u-PA and t-PA mRNAs during mouse oogenesis and embryo implantation. By in situ hybridizations, we detected t-PA mRNA in oocytes and u-PA mRNA in granulosa and thecal cells from preovulatory follicles. These findings are compatible with a role for plasminogen activators in oogenesis and follicular disruption. We demonstrated the presence of u-PA mRNA in the invasive and migrating trophoblast cells of 5.5- and 6.5-d-old embryos. At 7.5 days, u-PA mRNA was predominantly localized to trophoblast cells that had reached the deep layers of the uterine wall, while the peripheral trophoblast cells surrounding the presomite stage embryo were devoid of specific signal. In 8.5-d-old embryos abundant u-PA mRNA expression resumed transiently in the giant trophoblast cells at the periphery of the embryo and in the trophoblast cells of the ectoplacental cone, to become undetectable in 10.5-d-old embryos. These observations establish the in vivo expression of the u-PA gene by invading and migrating trophoblast cells in a biphasic time pattern; they are in agreement with the proposed involvement of the enzyme in the extracellular proteolysis accompanying embryo implantation.     

Nitric oxide involvement in pancreatic beta cell apoptosis by glibenclamide.

Glibenclamide as a second-generation compound of sulfonylurea has widely been used in the treatment of type 2 diabetes patients. It has been shown that it induces apoptosis in beta cells, which is partially mediated by Ca(2+) influx. Here, we investigated the role of nitric oxide (NO) and nitric oxide synthase (NOS) isoforms on glibenclamide-induced apoptosis in rat insulinoma cells. Our results showed that glibenclamide induces NO generation (measured as nitrite) that is accompanied with decrease of cell viability in a defined concentration of glibenclamide. The effects of glibenclamide on cell viability were partially inhibited after treatment with N(G)-nitro-L-arginine methyl ester (L-NAME), inhibitor more selective for constitutive nitric oxide synthase, and in the presence of D600--a blocker of voltage-gated L-type Ca(2+) channels inhibited Ca(2+) influx into beta cells, whereas aminoguanidine (AG), a preferential inhibitor of inducible NOS, was significantly less effective. Analysis of DNA fragmentation by electrophoresis and staining with Hoechest 33342 and propidium iodide showed that L-NAME, but not AG, prevented DNA fragmentation and decreased the number of cells with condensed and fragmented nuclei. It revealed that the effects of glibenclamide on apoptosis were partially inhibited by treatment with L-NAME. In conclusion, we have shown that NO production in glibenclamide treated cells may be involved in the induction of apoptotic cell death in pure beta cell line and it may be due to Ca(2+) dependent activation of constitutive NOS isoforms.     

A review and discussion of platelet nitric oxide and nitric oxide synthase: do blood platelets produce nitric oxide from <Emphasis Type="SmallCaps">l</Emphasis>-arginine or nitrite?

The NO/sGC/cGMP/PKG system is one of the most powerful mechanisms responsible for platelet inhibition. In numerous publications, expression of functional NO synthase (NOS) in human and mouse platelets has been reported. Constitutive and inducible NOS isoforms convert l-arginine to NO and l-citrulline. The importance of this pathway in platelets and in endothelial cells for the regulation of platelet function is discussed since decades. However, there are serious doubts in the literature concerning both expression and functionality of NOS in platelets. In this review, we aim to present and critically evaluate recent data concerning NOS expression and function in platelets, and to especially emphasise potential pitfalls of detection of NOS proteins and measurement of NOS activity. Prevailing analytical problems are probably the main sources of contradictory data on occurrence, activity and function of NOS in platelets. In this review we also address issues of how these problems can be resolved. NO donors including organic nitrites (RONO) and organic nitrate (RONO₂) are inhibitors of platelet activation. Endogenous inorganic nitrite (NO₂ ^?), the product of NO autoxidation, and exogenous inorganic nitrite are increasingly investigated as NO donors in the circulation. The role of platelets in the generation of NO from nitrite is also discussed.     

Evidence for nitrite reductase activity in intact mouse Leydig tumor cells

Nitric oxide (NO) supposedly derived via L-arginine-NO synthase (NOS) pathway has been implicated in inhibiting steroidogenesis by binding the heme moiety of steroidogenic enzymes. Previously, nitrite, and to a lesser extent nitrate ions inhibited steroidogenesis via NO by hitherto unknown reduction mechanism. Recently, a putative mammalian nitrite reductase activity ascribed to complex III of mitochondrial respiratory chain complexes (MRCC) has been reported, where MRCC inhibitors reduced NO production from nitrite variably. We thus studied the effects of MRCC inhibitors on testosterone production in mouse Leydig tumor cells (MLTC-1) without (basal) or with human chorionic gonadotropin (hCG) stimulation. In stimulated MLTC-1, MRCC inhibitors decreased testosterone production, order being: complex III (antimycin A and myxothiazol) > complex I (rotenone) > complex II (thenoyltrifluoroacetone), while cAMP production increased inversely. In unstimulated MLTC-1, MRCC inhibitors in same order, increased basal testosterone production, which correlated inversely with the percentage inhibition of NO production, with one exception; while antimycin A did not inhibit NO production in the nitrite reductase study mentioned above, it increased basal testosterone production in the present study. While MLTC-1 expressed mRNA for endothelial and neuronal, but not inducible NOS, various stimulators and inhibitors of L-arginine-NOS pathway had no effect on basal testosterone production in MLTC-1 or fresh Balb/c Leydig cells. Moreover, hCG increased nitrate uptake into MLTC-1, which suggests the gonadotropin aids nitrite and nitrate ions in their steroidogenesis inhibitory activity. In conclusion, this study supports the existence of a surrogate mammalian nitrite reductase and the dormancy of L-arginine-NOS pathway in MLTC-1.     

The role of inducible nitric oxide synthase in gamete interaction and fertilization: a comparative study on knockout mice of three NOS isoforms

Nitric oxide (NO), which is produced from l-arginine by three isoforms of NO synthase (NOS), has been implicated in reproductive functions. However, the specific role of NOS isoforms in gamete function and fertilization is not clear. Three types of NOS knockout mice were super ovulated and fertilized in vitro and in vivo. The sperm count and motility, in vivo and in vitro fertilization rate as indicated by two-cell embryos and blastocyst rate were examined. The sperm count and motility from all three knockout mice were not significantly different from that of the wild type. Inducible NOS (iNOS) knockout mice were found to have the largest number of two-cell embryos/mouse collected after fertilization in vivo (P<0.01), but the rate of blastocyst formation from two-cell embryos in vitro was similar for all three knockouts. The rate of in vitro fertilization using either iNOS-deficient sperm or oocytes, but not those deficient in the other two NOS isoforms, was significantly elevated when compared to that in the wild type (P<0.001). While all three types of NOS do not seem to play a significant role in pre-ejaculated sperm function, iNOS may play an inhibitory role in sperm and oocyte functions affecting the process of fertilization and early embryo development.     

Chronic stimulation of D1 dopamine receptors in human SK-N-MC neuroblastoma cells induces nitric-oxide synthase activation and cytotoxicity

Elevated synaptic levels of dopamine may induce striatal neurodegeneration in l-DOPA-unresponsive parkinsonism subtype of multiple system atrophy (MSA-P subtype), multiple system atrophy, and methamphetamine addiction. We examined the participation of dopamine and D1 dopamine receptors in the genesis of postsynaptic neurodegeneration. Chronic treatment of human SK-N-MC neuroblastoma cells with dopamine or H2O2 increased NO production and accelerated cytotoxicity, as indexed by enhanced nitrite levels and cell death. The antioxidant sodium metabisulfite or SCH 23390, a D1 dopamine receptor-selective antagonist, partially blocked dopamine effects but together ablated dopamine-mediated cytotoxicity, indicating the participation of both autoxidation and D1 receptor stimulation. Direct activation of D1 dopamine receptors with SKF R-38393 caused cytotoxicity, which was refractory to sodium metabisulfite. Dopamine and SKF R-38393 induced overexpression of the nitric-oxide synthase (NOS) isoforms neuronal NOS, inducible NOS (iNOS), and endothelial NOS in a protein kinase A-dependent manner. Functional studies showed that approximately 60% of total NOS activity was due to activation of iNOS. The NOS inhibitor N(G)-nitro-l-arginine methyl ester and genistein, wortmannin, or NF-kappaB SN50, inhibitors of protein tyrosine kinases phosphatidylinositol 3-kinase and NF-kappaB, respectively, reduced nitrite production by dopamine and SKF R-38393 but were less effective in attenuating H2O2-mediated effects. In rat striatal neurons, dopamine and SKF R-38393, but not H2O2, accelerated cell death through increased expression of neuronal NOS and iNOS but not endothelial NOS. These data demonstrate a novel pathway of dopamine-mediated postsynaptic oxidative stress and cell death through direct activation of NOS enzymes by D1 dopamine receptors and its associated signaling pathways.     

Ca2+/calmodulin-regulated nitric oxide synthases.

NO synthase (NOS) catalyzes the oxidation of L-arginine to L-citrulline and nitric oxide (NO) or a NO-releasing compound. At least three isoforms of NOS exist (types I-III). The activities of the type I isoform purified from brain and the type III isoform purified from endothelial cells are regulated by the intracellular free calcium concentration ([Ca2+]i) and the Ca(2+)-binding protein calmodulin. At resting [Ca2+]i, both isozymes are inactive; they become fully active at [Ca2+]i greater than or equal to 500 nM Ca2+. Longer lasting increases in [Ca2+]i may downregulate NO formation, for in vitro phosphorylation by Ca2+/calmodulin protein kinase II decreases the Vmax of NOS. Besides the conversion of L-arginine, type I NOS, Ca2+/calmodulin dependently, generates H2O2 and reduces cytochrome c/P450. Other redox activities, i.e. the reduction of nitroblue tetrazolium to diformazan (NADPH-diaphorase) or of quinoid-dihydrobiopterin to tetrahydrobiopterin, by NOS appear to be Ca2+/calmodulin-independent.