A23187 and related Ca2+ ionophores are widely used to study Ca2+- dependent cell injury. Here, using laser scanning confocal microscopy and parameter-indicating fluorophores, we investigated the role of the mitochondrial permeability transition (MPT) in Br-A23187 toxicity to cultured rat hepatocytes. After 10 μM Br-A23187, over 60% of hepatocytes lost viability within 1 h. This necrotic cell killing was preceded by increased mitochondrial free Ca2+, mitochondrial depolarization, and onset of the MPT. Cyclosporin A (CsA), a blocker of the permeability transition pore, prevented the MPT and cell killing but had no effect on increased mitochondrial free Ca2+ and depolarization after Br-A23187. To determine whether Br-A23187-induced cell killing was linked to loss of cellular ATP supply, hepatocytes were incubated with fructose and oligomycin, a source of glycolytic ATP and an inhibitor of the uncoupler-stimulated mitochondrial ATPase, respectively. Fructose plus oligomycin prevented cell killing after Br-A23187 but not the MPT. When fructose plus oligomycin prevented necrotic cell killing, apoptosis developed after 10 h. When cells were treated additionally with CsA, these apoptotic changes were prevented. In conclusion, the MPT mediates Br-A23187 cytotoxicity. Acutely, the MPT causes mitochondrial uncoupling and profound ATP depletion, which leads to necrotic cell death. However, when glycolytic ATP generation is available, the MPT induces apoptosis. CsA blocks the MPT and prevents both necrotic and apoptotic cell killing after Br-A23187.
Bibliographical noteFunding Information:
1This work was supported, in part, by Grants AG60721, DK37034, and AA11606 from National Institutes of Health. 2Present address: Confocal Facility, Box 248, The Rockefeller University, 1230 York Ave., New York, NY 10021. 3Present address: Department of Cellular and Structural Biology, The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Dr., San Antonio, Texas 78284-7762. 4To whom correspondence should be addressed.
- Calcium ionophore
- Confocal microscopy
- Mitochondrial permeability transition