Signaling mechanisms of interferon gamma induced apoptosis in chromaffin cells: involvement of nNOS, iNOS, and NFκB

Previous work of our group stated that exogenously added and endogenous nitric oxide (NO) generated by cytokines induce apoptosis in chromaffin cells. In this work, we investigate the specific regulation of the NO synthase (NOS) isoforms, inducible NOS (iNOS) and neuronal NOS (nNOS), and their particular participation in cell death induced by interferon gamma (IFN gamma). Lipopolysaccharide (LPS) and IFN gamma increase iNOS expression, with no effect on nNOS expression. On the other hand, dexamethasone increases basal nNOS expression but decreases LPS + IFN gamma-induced iNOS expression. IFN gamma-induced cell death was abolished by W-1400, a specific iNOS inhibitor, but only partially by nNOS inhibitors [N-omega-propyl-l-arginine (N-PLA), 3-Bromo-7-nitroindazol (7-NI), l-methyl thiocitrulline and N-methyl l-arginine], indicating the main iNOS participation in chromaffin cell death. IFN gamma and LPS induce nuclear factor kappa B (NF kappa B) translocation to the nucleus, a process implicated in activation of iNOS expression, as inhibition of NF kappa B translocation, by SN50, decreased iNOS expression. In addition, IFN gamma and LPS induce (847)Ser-nNOS phosphorylation, inhibiting nNOS activity. Both processes, nNOS phosphorylation and iNOS expression induced by LPS + IFN gamma, are regulated by Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) pathway, as IFN gamma increases (727)STAT-3 phosphorylation and specific inhibitors of JAK/STAT pathway, such as AG490, inhibited both processes. Taken together, these results support the hypothesis of an inactivating phosphorylation of nNOS by IFN gamma, via JAK/STAT, in bovine chromaffin cells. Low NO concentrations achieved by this event, would activate NF kappa B translocation, increasing iNOS expression and generating, this last, high apoptotic NO concentrations.