Reversed activity of mitochondrial adenine nucleotide translocator in ischemia-reperfusion

Background. Graft dysfunction as a result of preservation injury remains a major clinical problem in liver transplantation. This is related in part to accumulation of mitochondrial calcium. In an attempt to sustain cell and mitochondrial integrity during ischemia, intramitochondrial F,F, adenosine triphosphate (ATP) synthase reverses its activity and hydrolyzes ATP to maintain the mitochondrial transmembrane potential (mDeltapsi). It is not known how cytoplasmic ATP becomes available for hydrolysis by this enzyme. The authors hypothesized that mitochondrial adenine nucleotide translocator (ANT) reverses its activity during ischemia, making cytoplasmic ATP available for hydrolysis by FoF1 ATP synthase. Methods. Rat livers were perfused with cold University of Wisconsin solution at 4degreesC (39.2degreesF)through the portal vein and processed immediately or after 24 hr of cold storage. Mitochondria were separated by differential centrifugation. ATP-dependent mitochondrial calcium-45 (Ca-45)(2+) uptake was determined after incubation with ATP (5 mM) or adenosine diphosphate (ADP) (5 mM) with or without 15 muM of bongkrekic acid (BA), an ANT blocker; the nonhydrolyzable analog of ATP (adenosine 5'-beta,gamma-imidotriphosphate [AMP-PNP]) served as the negative control. All measurements were performed in triplicate. Student t test, P<0.05 was taken as significant. Results. Inhibition of ANT by BA prevents mitochondrial Ca2+ accumulation in the presence of ATP and high Ca-45(2+) concentrations, and increased extramitochondrial Ca-45(2+) stimulated mitochondrial Ca-45(2+) uptake in the presence of ATP but not ADP, AMP-PNP, or BA. Conclusions. These data demonstrate that ANT plays an important role in mitochondrial Ca2+ uptake under ischemic conditions by reversing its activity and allowing transport of extramitochondrial ATP into the matrix for hydrolysis by reversed FoF1 ATP synthase.