(20) GLOBAL CEREBRAL ISCHEMIA AND REPERFUSION DIFFERENTIALLY IMPAIR MITOCHONDRIAL CA2+ BUFFERING AND PROMOTE CA2+-INDUCED CYTOCHROME C RELEASE

G. Fiskum and A. Andreyev
Dept. of Anesthesiology, University of Maryland School of Medicine, Baltimore, MD 21201

Evidence indicates that mitochondrial Ca2+ overload contributes to excitotoxic neuronal death and that Ca2+-induced cytochrome c (Cyt c) release triggers apoptosis. Although excitotoxicity and apoptosis participate in ischemic brain injury, little is known regarding the effects of cerebral ischemia and reperfusion on the sensitivity of mitochondria to Ca2+-induced injury or to Ca2+-induced release of Cyt c. Brain mitochondria were isolated from control animals and animals subjected to 10 min global cerebral ischemia +/-24 h reperfusion. Mitochondria were suspended in a KCl-based medium at 37 degrees containing malate, glutamate, PI, Mg2+, and ATP. Following addition of CaCl2 and measurements of the medium [Ca2+] with the fluorometric indicator Calcium green 5N, the suspension was centrifuged and the release of Cyt c into the supernatant was measured with Western immunoblots. The maximum Ca2+ uptake capacity was reduced by 50% following 10 min ischemia but returned to normal after 24 h reperfusion. At saturating levels of added Ca2+, the amount of Cyt c released from ischemic or 24 h reperfused brain mitochondria was 300% greater than control mitochondria. Experiments are in progress to determine how oxidative modifications to mitochondrial proteins and lipids may cause these differential effects of cerebral ischemia and reperfusion on mitochondrial Ca2+ uptake and Cyt c release. (Supported by NS34152)