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The interface between calmodulin (CaM) and the NO synthase (NOS) heme domain is the least characterized interprotein interface that the NOS isoforms must traverse through during catalysis. Our previous molecular dynamics simulations predicted a salt bridge between K497 in human inducible NOS (iNOS) heme domain and D118(CaM). Herein, the FMN - heme interdomain electron transfer (IET) rate was found to be notably decreased by charge-reversal mutation, while the IET in the iNOS K497D mutant is significantly restored by the CaM D118K mutation. The results of wild-type protein with added synthetic peptides further demonstrate the critical nature of K497 relative to the rest of the peptide sequence in modulating the IET. These data provide definitive evidence supporting the regulatory role of the isoform-specific K497 residue.

A decrease in constitutive NO synthase (cNOS) activity and an increase in inducible NO synthase (iNOS) activity occurred with an increase in myeloperoxidase (MPO) activity, an index of neutrophil infiltration, in the gastric mucosa of rats with water immersion restraint (WIR) stress. This increase in gastric mucosal MPO activity was enhanced by pretreatment with NG-monomethyl l-arginine, a non-selective NOS inhibitor, but was prevented with maintenance of gastric mucosal cNOS activity by pretreatment with aminoguanidine, a selective iNOS inhibitor. The MPO activity was negatively correlated with the cNOS activity in all WIR-stressed rats used (r=−0.723). These results suggest that a decrease in cNOS activity could contribute to an increase in neutrophil infiltration in the gastric mucosa of WIR-stressed rats.

The inducible NO synthase (iNOS) was found to be expressed in pancreatic lesions of adult diabetes-prone BB rats. Pancreatic iNOS mRNA was detected by reverse transcriptase PCR in pancreatic RNA of adult diabetes-prone BB rats but not in normal Wistar rats, young diabetes-prone BB rats without insulitis or in diabetes-resistant BB rats. Immunohistochemistry of pancreatic sections using an iNOS-specific antiserum labeled the pancreas of adult diabetes-prone BB rats but not Wistar rats. Parallel staining for EDI-positive macrophages showed restriction of iNOS expression to areas of islet infiltration by macrophages. In conclusion, the data provide direct evidence for enhanced expression of inducible NO synthase in tissue lesions during the development of autoimmune diabetes.

We investigated whether sodium diethyldithiocarbamate (DETC), an inhibitor of the nuclear transcription factor kappa B (NFkappaB), modulates induction of NO synthase (NOS) in murine bone marrow-derived macrophages. A short exposure (between 1 and 16 h) of L929-cell medium-preconditioned macrophages to E. coli lipopolysaccharide (LPS) significantly increased the level of NOS mRNA, and elicited NO formation as detected by electron spin resonance spectroscopy and by the release of nitrite. DETC (0.1–1 mM) present during stimulation with LPS prevented the increase in NOS mRNA and the expression of NOS activity. These findings suggest that NFkappaB is involved in the signal transduction pathway linking stimulation of macrophages by LPS with transcription of, the gene encoding inducible NOS.

Macrophages contain arginase and an inducible NO synthase, demonstrated by using l-arginine, the common substrate, for production of both nitric oxide and urea. Arginase was inhibited by nitrite, the stable end product of NO. This inhibition was non-competitive, and could not be explained by the reaction of nitrite with arginine, or by the irreversible covalent modification of arginase, or by the removal of Mn2+, a cofactor of arginase.

The effect of NO on phosphotyrosine protein phosphatases (PTPases) has been investigated in vivo. NO production is induced in interferon-γ and lipopolyaccharide stimulated RAW-264.7 macrophages as indicated by the increase of NO2− in the medium. Our results demonstrate an inhibition of p-nitrophenylphosphatase activity as a consequence of macrophages activation. Under the described experimental conditions, most of the hydrolysis of p-nitrophenylphosphate can be ascribed to the action of cellular PTPases. The presence of N G - monomethyl- l -arginine , a specific inhibitor of NO synthase decreases the inactivation rate of both membrane-bound and soluble PTPases. This evidence further confirms the ability of NO to inactivate PTPases and suggests a possible role of NO in the regulation of cellular processes involving this class of phosphatases.

In this study, the role of cyclic GMP, the end product of the NO-cyclic GMP signalling pathway, in the regulation of ecNOS was investigated. Bovine pulmonary endothelial cells were exposed to 8-bromo-cyclic GMP and its effect on NO production, ecNOS protein, and mRNA levels was analyzed. Endothelial cells on exposure to 8-bromo-cyclic GMP produced significantly increased amounts of NO, detected as increased cyclic GMP in cocultures with vascular smooth muscle cells both under basal conditions and with agonist stimulation. 8-Bromo-cyclic GMP significantly increased the ecNOS protein and mRNA levels as detected on Western and Northern blots respectively. This 8-bromo-cyclic GMP mediated increase of NO production, ecNOS protein and mRNA levels suggests that cyclic GMP up-regulates the expression of ecNOS. Thus, there may be an intercellular feedback mechanism involved at the molecular level in the expression of the NO-cyclic GMP signalling pathway in blood vessels.

We have examined the effects of sex hormones on calcium-dependent NO production and protein levels of NO synthase in cultured human aortic endothelial cells, which were treated with various doses of 17 beta-estradiol and testosterone for 8-48 h. Treatment with 17 beta-estradiol enhanced calcium-dependent NO production, but testosterone had exerted no effect. Western blot using monoclonal anti-human endothelial NO synthase antibody clarified that increased NO production by 17 beta-estradiol treatment was accompanied by increased NO synthase protein. Our results provide evidence that human endothelial NO synthase can be regulated by estrogens.

Ca2+/calmodulin-dependent nitric oxide synthase was found to be induced during rat liver necrosis caused by administration of Propionibacterium acnes and E. coli lipopolysaccharide to rats. Examination of the specific induction of Ca2+/calmodulin-dependent NO synthase showed that the enzyme was induced in the lung, spleen and colon as well as the liver. Northern blot analysis revealed that the induction occurred at the transcriptional level.

The measurement of nitric oxide (NO) is important for direct examination of the regulatory roles of NO in various biological systems. Diaminofluoresceins (DAFs), new fluorescence indicators for NO, were applied to detect the release of NO from bovine aortic endothelial cells (ECs). DAFs react with NO to yield the corresponding green-fluorescent triazolofluoresceins, which provide the advantages of specificity, sensitivity and a simple protocol for the direct detection of NO. Using these DAFs, we could detect the generation of NO not only from inducible NO synthase expressed in macrophages, but also from constitutive NO synthase expressed in ECs.

To assess peroxynitrite formation in lipopolysaccharide (LPS)-stimulated human blood, we have measured nitric oxide (NO)-dependent intracellular oxidation of dihydrorhodamine 123 (DHR 123) to rhodamine. LPS increased DHR 123 oxidation in neutrophil granulocytes, monocytes and lymphocytes in a time-dependent fashion. Greater extent of DHR 123 oxidation was detected in neutrophils and monocytes than in lymphocytes. These changes were accompanied by accumulation of rhodamine in the plasma. While intracellular DHR 123 oxidation and rhodamine accumulation in the plasma were not affected by inhibition of constitutive NO synthase at 30 and 60 min after addition of LPS, they were markedly attenuated by inhibition of inducible NO synthase at 4, 8, 16 and 24 h after addition of LPS. These results demonstrate that human leukocytes can produce high amounts of peroxynitrite in response to LPS, and may contribute to the elevated plasma peroxynitrite levels observed during endotoxic shock.

NOSTRIN, an NO synthase binding protein, belongs to the PCH family of proteins, exposing a typical domain structure. While its SH3 domain and the C-terminal coiled-coil region cc2 have been studied earlier, the function of the N-terminal half comprising a Cdc15 domain with an FCH (Fes/CIP homology) region followed by a coiled-coil stretch cc1 is unknown. Here, we show that the FCH region is necessary and sufficient for membrane association of NOSTRIN, whereas the Cdc15 domain further specifies subcellular distribution of the protein. Thus, the FCH region and the Cdc15 domain fulfill complementary functions in subcellular targeting of NOSTRIN.

The hypothesis was investigated that the nitric oxide (NO) synthase intermediate, NG-hydroxy-l-arginine (HOArg), is an arginase inhibitor in rabbit or rat alveolar macrophages. Exogenously applied HOArg strongly inhibited the arginase activity present in these cells (IC50 ≥ 15 μM), and attenuated l-[3H]arginine transport (IC50 ≥ 500 μM) in rabbit alveolar macrophages. Moreover, up to 37 μM HOArg were detected in the conditioned medium, but not in the lysate, of rat alveolar macrophages exposed to bacterial lipopolysaccharide for 18 h. HOArg may thus be a potent endogenous arginase inhibitor in these cells which increases the availability of l-arginine for NO biosynthesis.