Inhibition of phospholipase C attenuates liver mitochondrial calcium overload following cold ischemia.
BACKGROUND
Graft failure due to cold ischemia (CI) injury remains a significant problem during liver transplantation. During CI, the consumption of ATP and the increase in cellular Ca concentration may result in mitochondrial Ca (mCa) overload through the mCa uniporter, which can ultimately lead to apoptosis and graft nonfunction. We recently identified phospholipase C-dl (PLC-dl) as a novel regulator of mCa uptake in the liver, and now extend those studies to examine the role of mitochondrial PLC in liver CI injury.


METHODS
Rat livers were perfused with University of Wisconsin (UW) solution. Half was homogenized immediately; the other half was cold-stored for 24 hr in UW. Mitochondria were extracted by differential centrifugation and incubated in buffer containing ATP and 0.1 or 0.2 microM Ca. The selective PLC inhibitor, U-73122, was added to determine the effects of PLC inhibition on mCa uptake following CI. Western blots and densitometry quantified mitochondrial PLC expression. Mito Tracker Red fluorescence microscopy was used to verify mitochondrial transmembrane potential.


RESULTS
Twenty-four hour CI caused a significant increase in mCa uptake (P<0.001), and increasing extramitochondrial Ca potentiated this effect. The PLC inhibitor, U-73122, decreased mCa uptake in nonischemic mitochondria (P<0.001), and had a greater effect on CI mitochondria (P<0.001). Mitochondrial PLC-dl expression increased 175+/-75% following CI (P<0.05).


CONCLUSIONS
These data demonstrate that PLC-dl is essential for mCa uptake following CI, and that the PLC pathway may be sensitized by CI. The CI-induced increase in mitochondrial PLC-delta1 expression represents a novel mechanism whereby mCa uptake can increase independently of cytosolic conditions.
Department of Surgery, Vanderbilt University Medical Center, Nashville, TN 37232, USA.
Clayton D
CD
Knox
Janene M
JM
Pierce
Ian B
IB
Nicoud
Andrey E
AE
Belous
Christopher M
CM
Jones
Christopher D
CD
Anderson
Ravi S
RS
Chari
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D007511:Ischemia / Q000503:physiopathology*; D008099:Liver / Q000098:blood supply*; D008297:Male; D008930:Mitochondria, Liver / Q000187:drug effects / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors*
D000255:Adenosine Triphosphate; D010738:Type C Phospholipases; D002118:Calcium