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(0)F(1) 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 F(0)F(1) ATP synthase.


METHODS
Rat livers were perfused with cold University of Wisconsin solution at 4 degrees C (39.2 degrees F)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 (45Ca)2+ uptake was determined after incubation with ATP (5 mM) or adenosine diphosphate (ADP) (5 mM) with or without 15 microM 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 45Ca2+ concentrations, and increased extramitochondrial 45Ca2+ stimulated mitochondrial 45Ca2+ 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 F(0)F(1) ATP synthase.
Belous
Andrey
A
Department of Surgery, Vanderbilt University Medical Center, Nashville, TN 37232-4753, USA.
Knox
Clayton
C
Nicoud
Ian B
IB
Pierce
Janene
J
Anderson
Christopher
C
Pinson
C Wright
CW
Chari
Ravi S
RS
D000255:Adenosine Triphosphate / Q000502:physiology; D000818:Animals; D015153:Blotting, Western; D001865:Bongkrekic Acid / Q000494:pharmacology; D002118:Calcium / Q000493:pharmacokinetics; D015925:Cryopreservation; D007425:Intracellular Membranes / Q000378:metabolism; D008099:Liver / Q000378:metabolism*; D008297:Male; D008928:Mitochondria / Q000378:metabolism; D000226:Mitochondrial ADP, ATP Translocases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009994:Osmolar Concentration; D051381:Rats; D017207:Rats, Sprague-Dawley; D015427:Reperfusion Injury / Q000201:enzymology
D001865:Bongkrekic Acid; D000255:Adenosine Triphosphate; D000226:Mitochondrial ADP, ATP Translocases; D002118:Calcium