On the role of mitochondria in cell injury caused by vanadate-induced Ca²⁺ overload

Incubation of isolated rat hepatocytes with vanadate (0.25, 0.5 and 1 mM) resulted in progressive accumulation of Ca²⁺ in the intracellular compartments. Vanadate- induced Ca²⁺ accumulation was related to inhibition of the plasma membrane Ca²⁺-extruding system, but did not involve either enhanced plasma membrane permeability to Ca²⁺ or the enhanced operation of a putative Na⁺/Ca²⁺ exchanger. After an initial rise in the cytosolic free Ca²⁺ concentration, as revealed by phosphorylase activation, Ca²⁺ was sequestered predominantly by the mitochondria with little contribution from the endoplasmic reticulum. As the amount of Ca²⁺ in the mitochondria increased, a progressive decrease in mitochondrial membrane potential occurred, together with an impairment of the ability of these organelles to further sequester Ca²⁺. Associated with this, there was a decrease in intracellular ATP level, formation of surface blebs and cytotoxicity. Addition of an uncoupler to vanadate-treated hepatocytes dramatically accelerated the appearance of plasma membrane blebs and toxicity. Our results demonstrate that under conditions in which the plasma membrane Ca²⁺ pump is inhibited, mitochondria play an important role in protecting hepatocytes against damage induced by Ca²⁺ overload.