Ethanol Prevents Oxidant-Induced Mitochondrial Permeability Transition Pore Opening in Cardiac Cells

Abstract

The purpose of this study was to determine if ethanol prevents the mitochondrial permeability transition pore (mPTP) opening via glycogen synthase kinase 3beta (GSK-3beta). Cardiac H9c2 cells were exposed to ethanol (10-1000 microM) for 20 min. GSK-3beta activity was determined by measuring its phosphorylation at Ser(9). Mitochondrial membrane potential (DeltaPsi(m)) was assessed by imaging (confocal microscopy) H9c2 cells loaded with tetramethylrhodamine ethyl ester (TMRE). To activate GSK-3beta, cells were transfected with constitutively active GSK-3beta (GSK-3beta-S9A-HA) mutant plasmid. Treatment of cardiac cells with low doses of ethanol (10-500 microM) significantly enhanced GSK-3beta phosphorylation, indicating that ethanol can inactivate GSK-3beta in H9c2 cells. The effect of ethanol on GSK-3beta activity was reversed by the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 and ethanol could enhance Akt phosphorylation, implying that the PI3K/Akt pathway accounts for the action of ethanol. Ethanol prevented oxidant (H(2)O(2))-induced loss DeltaPsi(m), an effect that was reversed by LY294002, indicating that ethanol can modulate the mPTP opening caused by oxidant stress through the PI3K/Akt pathway. Ethanol failed to preserve DeltaPsi(m) in cells transfected with the constitutively active GSK-3beta (GSK-3beta-S9A-HA) mutant, suggesting that ethanol prevents the mPTP opening by inactivating GSK-3beta. These data suggest that ethanol prevents the mPTP opening through inactivation of GSK-3beta. The PI3K/Akt signaling pathway is responsible for inactivation of GSK-3beta by ethanol.