Induction of Nitric Oxide Synthase in the J774.7 Macrophage Cell Line

[摘要] When used alone, LPS and IFN-gamma were weak stimuli for inducing production of nitrite by J774.7 macrophages, but when used together, a powerful synergistic rise in nitrite accumulation was seen. Nitrite was derived from the L-arginine/NO pathway since its production was reduced by the NO synthase inhibitors, L-NMMA and L-NAME. L-NMMA was much more effective than L-NAME but the reason for this was obscure. The peptide polymyxin B also reduced nitrite accumulation by inhibiting the action of LPS on the cells. It is likely that NO was synthesised by the inducible form of NO synthase since nitrite production was low in the absence of stimuli and was reduced by dexamethasone in the presence of stimuli. Dexamethasone reduced nitrite accumulation, though whether via a direct action on the transcription process or through the formation of lipocortin 1 is unknown. Elevation of cyclic AMP levels reduced the production of nitrite by the cells, but only by a maximum of around 30%. Most agents had little effect on nitrite accumulation when added after LPS and IFN-gamma, and produced most of their effects when added before the induction of NO synthase by LPS and IFN-gamma. Thus, cyclic AMP can only regulate the induction process, probably at the level of transcription, to a slight degree. Elevation of cyclic GMP levels reduced accumulation of nitrite but only by around 30%. The effects of GTN may be mediated both via toxic effects on the NO synthase enzyme by NO and via a cyclic GMP-dependent mechanism. It is unknown if elevated cyclic GMP levels can induce phosphorylation of the NO synthase enzyme and so modulate its activity. It appears that both PKC and tyrosine kinase play a role in the induction of NO synthase by LPS and IFN-gamma, although evidence for the latter is more straightforward. The smooth muscle relaxant released from J774.7 cells following stimulation by LPS and EIFN-gamma was sensitive to attack from superoxide anions and blocked by haemoglobin. Furthermore, production of the relaxant was blocked following incubation with the inhibitor of NO synthase, L-NMMA. Thus, in all respects, this relaxant substance behaves like free NO. The mechanism by which macrophages protect themselves from the toxic effects of the high concentrations of NO they produce is unknown. The release of superoxide anions from J774.7 cells was undetectable, although the cells appear to release spontaneously a powerful oxidant. The nature of this oxidant is unknown, but it appears not to be hydrogen peroxide, peroxynitrite, hydroxyl radical or hypochlorous acid.

[发布机构] University:University of Glasgow

[关键词] Pharmacology