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.
Li
J
J
Department of Surgery, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA.
Bombeck
C A
CA
Yang
S
S
Kim
Y M
YM
Billiar
T R
TR
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism*; D002478:Cells, Cultured; D003574:Cytochrome c Group / Q000378:metabolism; D003609:Dactinomycin / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D008099:Liver / Q000166:cytology / Q000187:drug effects*; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects; D008928:Mitochondria / Q000378:metabolism*; D009569:Nitric Oxide / Q000494:pharmacology*; D019001:Nitric Oxide Synthase / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D011499:Protein Processing, Post-Translational; D051381:Rats; D017207:Rats, Sprague-Dawley; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D003574:Cytochrome c Group; D014409:Tumor Necrosis Factor-alpha; D003609:Dactinomycin; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat; C505410:Casp3 protein, rat; C505528:Casp8 protein, rat; C506435:Casp9 protein, rat; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases