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, including 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, including Caspase-3 and Caspase-8. We found that both exogenous NO as well as endogenously produced NO via adenoviral inducible NO Synthase gene transfer protected hepatocytes from tumor necrosid factor (TNF) alpha plus actinomycin D (TNFalpha/ActD)-induced Apoptosis. Affinity labeling with biotin-VAD-fmk of all active Caspase species in TNFalpha-mediated Apoptosis identified four newly labeled spots (activated caspases) present exclusively in TNFalpha/ActD-treated cells. Both NO and the Caspase Inhibitor, Ac-DEVD-CHO, prevented the appearance of the four newly labeled spots or active caspases. Immunoanalysis of affinity labeled caspases demonstrated that Caspase-3 was the major effector Caspase. Western blot analysis also identified the activation of Caspase-8 in the TNFalpha/ActD-treated cells, and the activation was suppressed by NO. Furthermore, NO inhibited several other events associated with Caspase activation in cells, including release of cytochrome c from mitochondria, decrease in mitochondrial transmembrane potential, and cleavage of poly(ADP-ribose) polymerase in TNFalpha/ActD-treated cells. These findings indicate the involvement of multiple caspases in TNFalpha-mediated Apoptosis in hepatocytes and establish the capacity of NO to inhibit not only active caspases but also Caspase activation.