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Nitric oxide protects rat hepatocytes against reperfusion injury mediated by the mitochondrial permeability transition

Authors

  • Jae-Sung Kim,

    1. Department of Cell and Developmental Biology, School of Medicine, University of North Carolina, Chapel Hill, NC
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    • J.-S. K. and S. O. contributed equally to this work.

  • Shigetoshi Ohshima,

    1. Department of Cell and Developmental Biology, School of Medicine, University of North Carolina, Chapel Hill, NC
    Current affiliation:
    1. The First Department of Internal Medicine, Akita University School of Medicine, 1-1-1, Hondo, Akita 010-8543, Japan
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    • J.-S. K. and S. O. contributed equally to this work.

  • Peter Pediaditakis,

    1. Department of Cell and Developmental Biology, School of Medicine, University of North Carolina, Chapel Hill, NC
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  • John J. Lemasters

    Corresponding author
    1. Department of Cell and Developmental Biology, School of Medicine, University of North Carolina, Chapel Hill, NC
    • Department of Cell and Developmental Biology, University of North Carolina at Chapel Hill, Campus Box 7090, 236 Taylor Hall, Chapel Hill, NC 27599-7090
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    • fax: 919-966-7197


Abstract

We investigated the effects of nitric oxide (NO) on hepatocellular killing after simulated ischemia/reperfusion and characterized signaling factors triggering cytoprotection by NO. Cultured rat hepatocytes were incubated in anoxic Krebs-Ringer–HEPES buffer at pH 6.2 for 4 hours and reoxygenated at pH 7.4 for 2 hours. During reoxygenation, some hepatocytes were exposed to combinations of NO donors (S-nitroso-N-acetylpenicillamine [SNAP] and others), a cGMP analogue (8-bromoguanosine-3,5-cGMP [8-Br-cGMP]), and a cGMP-dependent protein kinase inhibitor (KT5823). Cell viability was determined by way of propidium iodide fluorometry. Inner membrane permeabilization and mitochondrial depolarization were monitored by confocal microscopy. SNAP, but not oxidized SNAP, increased cGMP during reperfusion and decreased cell killing. Other NO donors and 8-Br-cGMP also prevented cell killing. Both guanylyl cyclase and cGMP-dependent kinase inhibition blocked the cytoprotection of NO. However, 5-hydroxydecanoate and diazoxide— mitochondrial KATP channel modulators—did not affect NO-dependent cytoprotection or reperfusion injury. During reoxygenation, confocal microscopy showed mitochondrial repolarization, followed by depolarization, inner membrane permeabilization, and cell death. In the presence of either SNAP or 8-Br-cGMP, mitochondrial repolarization was sustained after reperfusion preventing inner membrane permeabilization and cell death. In isolated rat liver mitochondria, a cGMP analogue in the presence of a cytosolic extract and adenosine triphosphate blocked the Ca2+-induced mitochondrial permeability transition (MPT), an effect that was reversed by KT5823. In conclusion, NO prevents MPT-dependent necrotic killing of ischemic hepatocytes after reperfusion through a guanylyl cyclase and cGMP-dependent kinase signaling pathway, events that may represent the target of NO cytoprotection in preconditioning. (HEPATOLOGY 2004;39:1533–1543.)

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