Calcium ion-dependent signalling and mitochondrial dysfunction: mitochondrial calcium uptake during hormonal stimulation in intact liver cells and its implication for the mitochondrial permeability transition

Hormones that elevate cytosolic Ca²⁺ concentrations ([Ca²⁺]_cyt) often use Ca²⁺ as a messenger to activate intramitochondrial metabolic processes. However, the mitochondrial Ca²⁺ level also regulates the activation of the mitochondrial permeability transition (MPT), a process that involves the assembly of a high conductance proteinaceous pore across the inner and outer membrane. Studies on intact liver cells indicate that the MPT is a critical step in the cell killing induced by anoxia or respiratory inhibitors. In this study, we used freshly isolated hepatocytes to investigate to what extent the elevation of [Ca²⁺]_cyt by vasopressin or other agonists causes Ca²⁺ accumulation in the mitochondria and how this treatment affects the mitochondrial susceptibility to undergo the MPT. Hepatocytes were incubated with vasopressin, glucagon, or with thapsigargin (an inhibitor of the endoplasmic reticulum Ca²⁺ pump) prior to permeabilization with digitonin. Mitochondrial Ca²⁺ accumulation was determined by following the ionomycin-induced Ca²⁺ release in permeabilized cells and mitochondrial swelling was studied by following cyclosporin A-sensitive light scattering changes induced by phenylarsenoxide and rotenone. The results indicate that agents that elevate [Ca²⁺]_cyt cause a significant Ca²⁺ accumulation in the mitochondria. Excessive Ca²⁺ accumulation (> 10-fold increase over basal levels) was obtained with the combination of vasopressin and glucagon or with incubations containing thapsigargin. These conditions were also associated with a marked increase in rotenone-induced mitochondrial swelling. However, the more modest increase in mitochondrial Ca²⁺ content after treating cells with vasopressin alone did not enhance the swelling response; instead, vasopressin suppressed mitochondrial swelling compared to control incubations. Vasopressin also partly suppressed the swelling associated with thapsigargin treatment, although it did not significantly affect the Ca²⁺ accumulation under these conditions. This effect of vasopressin was mimicked by phorbol ester, suggesting a role for protein kinase C. The data indicate that mitochondrial Ca²⁺ accumulation following elevation of [Ca²⁺]_cyt enhances the susceptibility for activation of the MPT, a response that may increase cell injury during anoxia or in response to other challenges. However, hormones also activate protective responses in the cell that suppress the MPT.