(24) MITOCHONDRIAL Ca2+ AND REACTIVE OXYGEN SPECIES (ROS) TRIGGER THE MITOCHONDRIAL PERMEABILITY TRANSITION (MPT) AND CELL KILLING AFTER REPERFUSION OF CULTURED RAT HEPATOCYTES

J.J. Lemasters, J-S. Kim and T. Qian
Department of Cell Biology & Anatomy, University of North Carolina, Chapel Hill, NC

BACKGROUND:  The factors inducing the MPT after reperfusion remain incompletely understood.  The AIM of this study was to assess the role of mitochondrial free Ca2+ and ROS formation in MPT-dependent reperfusion injury to hepatocytes. 

METHODS: Rat hepatocytes were cultured overnight on collagen-coated 24-well microtiter plates or glass coverslips and then incubated in anoxic Krebs-Ringer-HEPES (KRH) buffer at pH 6.2 for 4 h to simulate ischemia. Cells were then reoxygenated at pH 7.4 to simulate reperfusion. At 45 min before reperfusion, some cells were anaerobically loaded with 10 µM of the Ca2+ chelators, BAPTA AM and Quin-2 AM, for 30 min at 0-1^oC to promote intramitochondrial loading or at 37^oC to promote cytosolic loading only. Cell killing was assessed by propidium iodide fluorometry. To monitor mitochondrial Ca2+ and membrane permeabilization, hepatocytes were loaded with 10 µM Rhod-2 AM for 1 h at 15-17^oC and subsequently with 1 µM calcein AM for 15 min at 37^oC. In some experiments, 1 µM dichlorofluorescin (DCF) diacetate was included during reperfusion to monitor ROS generation. Images of red (Rhod-2) and green (calcein and DCF) fluorescence were collected with a Zeiss LSM410 confocal microscope. 

RESULTS: After 2 h of reperfusion, cell killing increased to 61.4 ± 2.2% from 18.9 ± 3.7 % (S.E.) at the end of ischemia (n=6). Cell killing after reperfusion decreased to 37.3 ± 2.9 % and 33.4 ± 4.9 %, respectively, after cold-loading of BAPTA and Quin-2 (p<0.01, n=6). In contrast, warm-loading of Ca2+ chelators and sham cold-loading did not decrease cell killing. Mitochondrial Rhod-2 fluorescence increased within 1 min of reperfusion, indicating the rapid rise of mitochondrial free Ca2+.  DCF fluorescence inside mitochondria increased after 5 min of reperfusion, indicating mitochondrial ROS formation. The MPT then occurred after 7 min of reperfusion, as evidenced by permeation of calcein into mitochondria. Cold-loaded BAPTA and Quin-2 both suppressed mitochondrial ROS formation and the MPT after reperfusion. 

CONCLUSION:  In hepatocytes, reperfusion after simulated ischemia causes mitochondrial Ca2+ loading, which in turn stimulates a sequence of mitochondrial ROS production, onset of MPT, and cell death. Thus, Ca2+-dependent mitochondrial ROS formation appears to be the proximate trigger of the MPT after reperfusion.

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