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 interferonγ (IFNγ) (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 + IFNγ 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 NFκB transcription factors was determined at 2 and 24 hours. Results: LPS + IFNγ caused time-dependent increases in iNOS protein and activity. Increasing concentrations of DETA (up to 500 μM) 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 + IFNγ, 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 NFκB seemed to be involved in iNOS induction and its modulation by DETA and XO/X. Conclusions: LPS + IFNγ 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.