Hormonal stimulation, mitochondrial Ca2+ accumulation, and the control of the mitochondrial permeability transition in intact hepatocytes.
Ca2+ functions as an intracellular signal to transfer hormonal messages to different cellular compartments, including mitochondria, where it activates intramitochondrial Ca2(+)-dependent enzymes. However, excessive mitochondrial Ca2+ uptake can promote the mitochondrial permeability transition (MPT), a process known to be associated with cell injury. The factors controlling mitochondrial Ca2+ uptake and release in intact cells are poorly understood. In this paper, we investigate mitochondrial Ca2+ accumulation in intact hepatocytes in response to the elevation of cytosolic Ca2+ levels ([Ca2+]c) induced either by a hormonal stimulus (vasopressin), or by thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+ pump. After stimulation, cells were rapidly permeabilized for the determination of the mitochondrial Ca2+ content (Ca2+(m)) and to analyze the susceptibility of the mitochondria to undergo the MPT. Despite very similar levels of [Ca2+]c elevation, vasopressin and thapsigargin had markedly different effects on mitochondrial Ca2+ accumulation. Vasopressin caused a rapid (< 90 sec), but modest (< 2 fold) increase in Ca2+(m) that was not further increased during prolonged incubations, despite a sustained [Ca2+]c elevation. By contrast, thapsigargin induced a net Ca2+ accumulation in mitochondria that continued for up to 30 min and reached Ca2+(m) levels 10-20 fold over basal. Accumulation of mitochondrial Ca2+ was accompanied by a markedly increased susceptibility to undergo the MPT. Both mitochondrial Ca2+ accumulation and MPT activation were modulated by treatment of the cells with inhibitors of protein kinases and phosphatases. The results indicate that net mitochondrial Ca2+ uptake in response to hormonal stimulation is regulated by processes that depend on protein kinase activation. These controls are inoperative when the cytosol is flooded by Ca2+ through artificial means, enabling mitochondria to function as a Ca2+ sink under these conditions.
Hoek
J B
JB
Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, PA 19107, USA.
Walajtys-Rode
E
E
Wang
X
X
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007425:Intracellular Membranes / Q000187:drug effects; D008297:Male; D008930:Mitochondria, Liver / Q000187:drug effects / Q000378:metabolism*; D010539:Permeability / Q000187:drug effects; D051381:Rats; D017207:Rats, Sprague-Dawley; D019284:Thapsigargin / Q000494:pharmacology*; D014667:Vasopressins / Q000494:pharmacology*
D004791:Enzyme Inhibitors; D014667:Vasopressins; D019284:Thapsigargin; D002118:Calcium