Chemically-induced perturbations of intracellular Ca2+ homeostasis appear to be fundamental to the toxicological mechanism of a wide variety of chemical agents. While increased cytosolic free Ca2+ is believed to be cytotoxic, it is not known what primary event(s) links Ca2+ increases to cell injury and death. A likely possibility would involve an irreversible process normally regulated by Ca2+ ion that becomes lethal to the cell during an increased or prolonged Ca2+ signal. Our preliminary data and a recent report of Nicotera et al. (7) indicate one such irreversible process that may contribute to cell injury is Ca2+- activated proteolysis. Three sources of intracellular proteolysis may be affected by increased Ca2+: 1) Ca2+-activated neutral proteases (CANP), 2) increased lysosomal autophagy and 3) lysis of the lysosomal membrane and the subsequent release of lysosomal proteases into the cytosol. Of the three, the CANP is currently regarded as the most probable source. The involvement of Ca2+ ion in the activation of proteolysis and the consequences of these events with regard to cell injury will be evaluated directly in saponin permeabilized cultured myocytes. To gain a clearer understanding of the biochemical processes involved, cultured beating myocytes will be challenged with several toxic insults, each designed to raise or lower intracellular free Ca2+ levels. To characterize the response of the myocytes a number of key biochemical processes will be monitored, where appropriate, during toxic challenges. These include myocyte Ca2+ homeostasis, energy charge, thiol redox status, and NAD(P)H/NAD(P) redox state. Cell function, based on beating frequency, and viability, based on plasma membrane integrity, will also be monitored. Inhibitor studies are planned to determine which of the three sources of proteolysis is linked to the onset of cell injury. The conversion of xanthine dehydrogenase to xanthine oxidase and the aberrant expression of beta-adrenergic receptors are two possible mechanisms by which Ca2+-activated proteolysis may affect cell function and viability. The toxicological significance of these events will be examined. Perturbations in Ca2+ homeostasis may represent a fundamental mechanism by which many different chemicals exert a toxic response in cells. These events may have broad implications for a wide variety of toxic chemical insults. A clearer understanding of these processes is expected to lead to the development of pharmacological interventions aimed at reducing or eliminating the toxicity of many chemical agents.
