Nitric oxide attenuates but superoxide enhances iNOS expression in endotoxin- and IFNγ-stimulated skeletal muscle endothelial cells

Objective: Endothelial cells (ECs) in septic skeletal muscle may be exposed to large amounts of NO and superoxide generated by the skeletal muscle cells. We tested the hypothesis that inducible nitric oxide synthase (iNOS) induction in ECs (i.e., one of the steps in the septic process) is modulated by extravascularly generated nitric oxide (NO) and superoxide. Methods: To model sepsis in vitro, monolayers of microvascular ECs derived from rat skeletal muscle were incubated with a mixture of lipopolysaccharide (LPS) (25 ng/mL) and interferon gamma (IFN gamma) (100 U/mL) for up to 24 hours. Next, a long-term release NO donor (DETA NONOate), a superoxide-generating mixture (xanthine oxidase/xanthine; XO/X), or DETA + XO/X were added to the LPS + IFN gamma mixture. The iNOS protein and activity, as well as intracellular oxidative stress were measured at intervals up to 24 hours, whereas the activation of AP-1, IRF-1, and NF kappaB transcription factors was determined at 2 and 24 hours. Results: LPS + IFN gamma caused time-dependent increases in iNOS protein and activity. Increasing concentrations of DETA (up to 500 muM) decreased, whereas XO/X (10 mU per mL/0.1 mM, respectively) markedly enhanced, iNOS expression and activity. DETA attenuated the enhancement by XO/X. Although intracellular oxidative stress was not altered by LPS + IFN gamma, modulations of iNOS expression by DETA, XO/X, and DETA + XO/X correlated with changes in oxidative stress. Among the three transcription factors, only IRF-1 and NF kappaB seemed to be involved in iNOS induction and its modulation by DETA and XO/X. Conclusions: LPS + IFN gamma can induce iNOS expression in microvascular ECs from rat skeletal muscle, whereas NO and superoxide modulate this expression. On the basis of these observations we suggest that NO and superoxide from the extravascular tissue may play a key role in the inflammatory response of septic ECs.