Regulation of inducible nitric oxide synthase

One of the major functions of macrophages is to provide the body with an immediate innate defence against pathogenic micro-organisms. This defence is largely dependent on the generation of nitric oxide (NO) and superoxide by macrophages which leads to the killing of these pathogens. NO is also important in many other biological functions. It is derived from L-arginine and molecular oxygen by the enzyme NO synthase (NOS). There are a number of classes of NOS including neuronal NOS (nNOS), endothelial NOS (eNOS) and cytokine-inducible NOS (iNOS). iNOS is upregulated and activated by several immunological stimuli including interferon gamma (IFN-gamma; lipopolysaccharide (LPS) and tumor necrosis factor-alpha (TNF-alpha), such activation leads to the production of large quantities of NO which can be cytotoxic. The potential toxicity of NO makes it important to understand the regulation of its production. In the study presented here, J774 cells, a murine macrophage cell line, were used as a model system for studying the induction and regulation of iNOS activation. These cells and murine peritoneal macrophages produce large amounts of NO in response to the T cell-derived lymphokine, IFN- and/or the potent macrophage activator, LPS in a dose-dependent manner. Northern and Western blotting revealed the process of induction of NO synthesis in J774 cells: The maximal induction of NO synthase mRNA was at 4 h while the maximum levels of NOS protein was observed at 8 to 12 h after treatment with IFN-gamma and LPS. IFN-? LPS-induced NO2- accumulation was abolished in the culture supernatants of samples that were pre-treated with cycloheximide. These suggest that iNOS is regulated transcriptionally in a manner that requires de novo protein synthesis. Protein phosphorylation plays a crucial role in regulating the signal transduction cascades leading to many biological responses in eukaryotes. Signals that are reversibly controlled by protein phosphorylation are modulated not only by a protein kinase but also by a protein phosphatase. In this project, I have shown that the induction of iNOS activity in J774 cells by IFN-gamma and LPS was reduced by more than 50% if the cells were pretreated with protein tyrosine kinase (PTK) inhibitors such as Tyrphostin 25, Tyrphostin AG126, and Herbimycin A. In contrast, iNOS was unaffected by pre-incubation with Tyrphostin 1, an inert analogue of these PTK inhibitors. Consistent with these findings, IFN-gamma and LPS-induced iNOS activity was enhanced by 30% in the presence of vanadate, a protein tyrosine phosphatase inhibitor. These results suggested that the activation of tyrosine kinase(s) plays a role in induction of NO synthesis. (Abstract shortened by ProQuest.)