Inducible NO synthase: role in cellular signalling.
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The discovery of endothelium-derived relaxing factor and its identification as nitric oxide (NO) was one of the most exciting discoveries of biomedical research in the 1980s. Besides its potent vasodilatory effects, NO was found under certain circumstances to be responsible for the killing of microorganisms and tumour cells by activated macrophages and to act as a novel, unconventional type of neurotransmitter. In 1992, Science picked NO as the 'Molecule of the Year', and over the past years NO has become established as a universal intercellular messenger that acutely affects important signalling pathways and, on a more long-term scale, modulates gene expression in target cells. These actions will form the focus of the present review. (+info)
Expression of nitric oxide synthase in inflammatory bowel disease is not affected by corticosteroid treatment.
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AIM: To examine the effect of corticosteroid treatment on the expression of inducible nitric oxide synthase (iNOS) in the colon of patients with inflammatory bowel disease. METHODS: Four groups of patients were studied: (1) ulcerative colitis treated with high dose corticosteroids (six patients, 10 blocks); (2) ulcerative colitis patients who had never received corticosteroids (10 patients, 16 blocks); (3) Crohn's disease treated with high dose corticosteroids (12 patients, 24 blocks); (4) Non-inflammatory, non-neoplastic controls (four patients, six blocks). Full thickness paraffin sections of colons removed at surgery were immunostained with an antibody raised against the C terminal end of iNOS. Sections were assessed semiquantitatively for the presence and degree of inflammation and immunoreactivity for nitric oxide synthase. RESULTS: Cases of ulcerative colitis and Crohn's disease with active inflammation showed strong staining for nitric oxide synthase. The staining was diffuse in ulcerative colitis and patchy in Crohn's disease, in accordance with the distribution of active inflammation. Staining was seen in epithelial cells and was most intense near areas of inflammation such as crypt abscesses. Non-inflamed epithelium showed no immunoreactivity. Treatment with corticosteroids made no difference to the amount of nitric oxide synthase. CONCLUSIONS: Expression of nitric oxide synthase is increased in both ulcerative colitis and Crohn's disease and appears to be unaffected by treatment with corticosteroids. Disease severity necessitated surgery in all the cases included in this study, regardless of whether or not the patients had received long term corticosteroid treatment. It seems therefore that a high level of iNOS expression and, presumably, production of nitric oxide characterise cases which are refractory to clinical treatment; this suggests that specific inhibition of the enzyme may be a useful therapeutic adjunct. (+info)
Phenotype of mice and macrophages deficient in both phagocyte oxidase and inducible nitric oxide synthase.
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The two genetically established antimicrobial mechanisms of macrophages are production of reactive oxygen intermediates by phagocyte oxidase (phox) and reactive nitrogen intermediates by inducible nitric oxide synthase (NOS2). Mice doubly deficient in both enzymes (gp91(phox-/-)/NOS2(-/-)) formed massive abscesses containing commensal organisms, mostly enteric bacteria, even when reared under specific pathogen-free conditions with antibiotics. Neither parental strain showed such infections. Thus, phox and NOS2 appear to compensate for each other's deficiency in providing resistance to indigenous bacteria, and no other pathway does so fully. Macrophages from gp91(phox-/-)/NOS2(-/-) mice could not kill virulent Listeria. Their killing of S. typhimurium, E. coli, and attenuated Listeria was markedly diminished but demonstrable, establishing the existence of a mechanism of macrophage antibacterial activity independent of phox and NOS2. (+info)
Role of nitric oxide in lipopolysaccharide-induced hepatic injury in D-galactosamine-sensitized mice as an experimental endotoxic shock model.
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The role of nitric oxide (NO) in lipopolysaccharide (LPS)-induced hepatic injury was studied in D-galactosamine (D-GalN)-sensitized mice. The inducible isoform of NO synthase (iNOS) was immunohistochemically detected on hepatocytes around blood vessels in livers of mice injected with D-GalN and LPS not on hepatocytes in mice injected with D-GalN or LPS alone, although mRNA for iNOS was found in those mice. Nitrotyrosine (NT) was also found in livers of mice injected with D-GalN and LPS. The localization of NT was consistent with that of iNOS, and the time courses of NT and iNOS expression were almost the same. Expression of iNOS and NT was detected exclusively in the hepatic lesions of mice injected with D-GalN and LPS. Anti-tumor necrosis factor alpha neutralizing antibody inhibited iNOS and NT expression and hepatic injury. The results suggested that NO from iNOS may play a role in LPS-induced hepatic injury on D-GalN-sensitized mice as an experimental endotoxic shock model. (+info)
Gamma interferon stimulates rat alveolar macrophages to kill Pneumocystis carinii by L-arginine- and tumor necrosis factor-dependent mechanisms.
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Pneumocystis carinii pneumonia remains a serious complication for immunocompromised patients. In the present study, P. carinii organisms interacted with gamma interferon (IFN-gamma)-stimulated alveolar macrophages (AMs) to activate the L-arginine-dependent cytocidal pathway involving reactive nitrogen intermediates (RNI) that were assayed as nitrite (NO2-). Unstimulated cultures of AMs produced negligible quantities of RNI. Addition of P. carinii organisms to IFN-gamma-primed AMs resulted in greatly enhanced production of RNI. NO2- levels increased from 0.8 +/- 0.4 to 11.1 +/- 3.8 microM as early as 6 h after P. carinii organisms were incubated with IFN-gamma-stimulated AMs and to 35.1 +/- 8.9 microM after a 24-h incubation, a near-maximum level. High levels of NO2- were produced by AMs primed with as little as 10 U of IFN-gamma per ml in the presence of P. carinii, and a 20-fold increase in IFN-gamma concentration resulted in only a further 65% increase in NO2- production. RNI-dependent killing of P. carinii was demonstrated by both a 51Cr release assay and a [35S]methionine pulse immunoprecipitation assay. Addition of either monoclonal tumor necrosis factor alpha (TNF-alpha) neutralizing antibody or 200 microM NG-monomethyl-L-arginine (L-NGMMA), a competitive inhibitor of the L-arginine-dependent pathway, significantly decreased NO2- production and reduced P. carinii killing. TNF-alpha alone had no effect on P. carinii viability. These results suggest that (i) the specific interaction of P. carinii organisms with IFN-gamma-primed AMs triggers the production of RNI, (ii) RNI are toxic to P. carinii, and (iii) TNF-alpha likely plays a central role in mediating P. carinii killing by IFN-gamma-stimulated AMs. (+info)
Effect of transforming growth factor beta on experimental Salmonella typhimurium infection in mice.
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We have investigated the effect of the in vivo administration of recombinant transforming growth factor beta (rTGF-beta) on the pathogenic mechanisms involved in Salmonella typhimurium experimental infection in mice. The protective response elicited by macrophages was induced by rTGF-beta1 by 2 days after experimental infection, as demonstrated by an increased NO production, while the humoral protective effect began with cytokine mRNA expression 2 days after the challenge and continued after 5 days with cytokine release and lymphocyte activation. We demonstrated that all mice who received rTGF-beta1 survived 7 days after infection. The number of bacteria recovered in the spleens and in the livers of rTGF-beta1-treated mice 2 and 5 days after infection was significantly smaller than that found in the same organs after phosphate-buffered saline (PBS) inoculation. Furthermore, 2 and 5 days after infection, splenic macrophages from rTGF-beta1-treated mice showed a greater NO production than did those from PBS-treated mice. The effect of rTGF-beta1 on S. typhimurium infection in mice was correlated with the expression of cell costimulatory CD28 molecules. Five days after S. typhimurium infection, the percentage of CD28(+)-expressing T cells in splenic lymphocytes from rTGF-beta1-treated mice increased with respect to that from control mice. Gamma interferon (IFN-gamma) mRNA was present in a greater amount in spleen cells from rTGF-beta1-treated mice after 2 days, although the intensity of the band decreased 5 days after the challenge. A similar pattern was obtained with the mRNAs for interleukin-1alpha (IL-1alpha), IL-6, TGF-beta, and inducible nitric oxide synthase, which showed greater expression in cells obtained from rTGF-beta1-treated and S. typhimurium-infected mice 2 days after challenge. The treatment with rTGF-beta1 induced an increase in IL-1alpha and IFN-gamma release in the supernatant of splenocyte cultures 5 days after the experimental infection with S. typhimurium. Moreover, we demonstrated that 5 days after infection, the IFN-gamma titer was significantly greater in the sera of rTGF-beta-treated mice than in those of PBS-treated mice. Also, hsp60 showed greater expression 2 days after the challenge in splenocytes from rTGF-beta1-treated mice. The role played by proinflammatory and immunoregulatory cytokines and by CD28 is discussed. (+info)
AMP-activated protein kinase phosphorylation of endothelial NO synthase.
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The AMP-activated protein kinase (AMPK) in rat skeletal and cardiac muscle is activated by vigorous exercise and ischaemic stress. Under these conditions AMPK phosphorylates and inhibits acetyl-coenzyme A carboxylase causing increased oxidation of fatty acids. Here we show that AMPK co-immunoprecipitates with cardiac endothelial NO synthase (eNOS) and phosphorylates Ser-1177 in the presence of Ca2+-calmodulin (CaM) to activate eNOS both in vitro and during ischaemia in rat hearts. In the absence of Ca2+-calmodulin, AMPK also phosphorylates eNOS at Thr-495 in the CaM-binding sequence, resulting in inhibition of eNOS activity but Thr-495 phosphorylation is unchanged during ischaemia. Phosphorylation of eNOS by the AMPK in endothelial cells and myocytes provides a further regulatory link between metabolic stress and cardiovascular function. (+info)
The stimulatory effects of Hofmeister ions on the activities of neuronal nitric-oxide synthase. Apparent substrate inhibition by l-arginine is overcome in the presence of protein-destabilizing agents.
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A variety of monovalent anions and cations were effective in stimulating both calcium ion/calmodulin (Ca2+/CaM)-independent NADPH-cytochrome c reductase activity of, and Ca2+/CaM-dependent nitric oxide (NO.) synthesis by, neuronal nitric oxide synthase (nNOS). The efficacy of the ions in stimulating both activities could be correlated, in general, with their efficacy in precipitating or stabilizing certain proteins, an order referred to as the Hofmeister ion series. In the hemoglobin capture assay, used for measurement of NO. production, apparent substrate inhibition by L-arginine was almost completely reversed by the addition of sodium perchlorate (NaClO4), one of the more effective protein-destabilizing agents tested. Examination of this phenomenon by the assay of L-arginine conversion to L-citrulline revealed that the stimulatory effect of NaClO4 on the reaction was observed only in the presence of oxyhemoglobin or superoxide anion (generated by xanthine and xanthine oxidase), both scavengers of NO. Spectrophotometric examination of nNOS revealed that the addition of NaClO4 and a superoxide-generating system, but neither alone, prevented the increase of heme absorption at 436 nm, which has been attributed to the nitrosyl complex. The data are consistent with the release of autoinhibitory NO. coordinated to the prosthetic group of nNOS, which, in conjunction with an NO. scavenger, causes stimulation of the reaction. (+info)