Evidence for mitochondrial localization of the hormone-responsive pool of Ca2+ in isolated hepatocytes

Conditions are described that allow chlortetracycline, a fluorescent probe of membrane-associated Ca2+, to monitor the content of the major exchangeable pool of intracellular Ca2+ present in the isolated rat hepatocyte. Chlortetracycline fluorescence is decreased in cells whose Ca2+ content is diminished by treatment either with carbonylcyanide-m-chlorophenylhydrazone or with ionophore A23187. Norepinephrine releases Ca2+ from this exchangeable pool and decreases both the fluorescence signal and its subsequent response to A23187. Previous suggestions that chlortetracycline fluorescence is localized in the mitochondria of liver and other cells is supported by comparison of the fluorescence that follows the addition of chlortetracycline to intact hepatocytes and to isolated hepatic microsomes and mitochondria. Identification of the hormone-responsive pool of Ca2+ with the mitochondria is strengthened by comparison of the total calcium content of mitochondria isolated from control and hormone-treated animals. The uptake and release of Ca2+ in control and hormone-treated hepatocytes rendered permeable by treatment with digitonin is also consistent with this interpretation.