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Nitric oxide suppresses apoptosis via interrupting caspase activation and mitochondrial dysfunction in cultured
1Department of Surgery, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA.
Abstract:
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.
Insights
Nitric oxide (NO) inhibits hepatocyte apoptosis by blocking caspase activation. This study shows NO prevents the processing and activation of key caspases, offering a novel therapeutic target for liver diseases.
Area of Science:
- Cell Biology
- Biochemistry
- Molecular Medicine
Background:
- Nitric oxide (NO) is known to inhibit apoptosis in hepatocytes.
- Previous studies indicated NO reduces caspase activity but did not clarify its effect on pro-caspase processing.
Purpose of the Study:
- To investigate whether nitric oxide (NO) disrupts the proteolytic processing and activation of pro-caspases in hepatocytes.
- To determine the role of NO in inhibiting tumor necrosis factor alpha (TNFalpha)-induced apoptosis.
Main Methods:
- Hepatocytes were treated with exogenous NO or endogenously produced NO via adenoviral inducible NO synthase gene transfer.
- Tumor necrosis factor alpha plus actinomycin D (TNFalpha/ActD) induced apoptosis.
- Affinity labeling with biotin-VAD-fmk and Western blot analysis were used to detect active caspases.
- Mitochondrial events and poly(ADP-ribose) polymerase cleavage were assessed.
Main Results:
- NO protected hepatocytes from TNFalpha/ActD-induced apoptosis.
- NO suppressed the proteolytic processing and activation of multiple pro-caspases, including caspase-3 and caspase-8.
- NO inhibited cytochrome c release, mitochondrial potential decrease, and PARP cleavage.
Conclusions:
- Nitric oxide inhibits hepatocyte apoptosis by preventing caspase activation, not just by inhibiting active caspases.
- Multiple caspases are involved in TNFalpha-mediated apoptosis in hepatocytes.
- NO represents a potential therapeutic strategy for modulating apoptosis in liver conditions.