Nitric oxide suppresses apoptosis via interrupting caspase activation and mitochondrial dysfunction in cultured hepatocytes

Nitric oxide (NO) is a potent inhibitor of apoptosis in many cell types, in cluding hepatocytes. We and others have described NO-dependent decreases in caspase activity in cells undergoing apoptosis. However, previous work has not determined whether NO disrupts the proteolytic processing and thus the activation of pro-caspases, Here we report that NO suppresses proteolytic processing and activation of multiple pro-caspases in intact cells, includi ng caspase-3 and caspase-8. We found that both exogenous NO as well as endo genously produced NO via adenoviral inducible NO synthase gene transfer pro tected hepatocytes from tumor necrosid factor (TNF) alpha plus actinomycin D (TNF alpha/ActD)-induced apoptosis. Affinity labeling with biotin-VAD-fmk of all active caspase species in TNF alpha-mediated apoptosis identified f our newly labeled spots (activated caspases) present exclusively in TNF alp ha/ActD-treated cells. Both NO and the caspase inhibitor, Ac-DEVD-CHO, prev ented the appearance of the four newly labeled spots or active caspases. Im munoanalysis of affinity labeled caspases demonstrated that caspase-3 was t he major effector caspase. Western blot analysis also identified the activa tion of caspase-8 in the TNF alpha/ActD-treated cells, and the activation w as suppressed by NO. Furthermore, NO inhibited several other events associa ted with caspase activation in cells, including release of cytochrome c fro m mitochondria, decrease in mitochondrial transmembrane potential, and clea vage of poly(ADP-ribose) polymerase in TNF alpha/ActD-treated cells. These findings indicate the involvement of multiple caspases in TNF alpha-mediate d apoptosis in hepatocytes and establish the capacity of NO to inhibit not only active caspases but also caspase activation.