Inhibition of ATP-dependent microsomal Ca2+ sequestration during oxidative stress and its prevention by glutathione

The metallochromic indicator arsenazo III was used to study the effect of oxidative stress on ATP-dependent Ca2+ uptake by rat liver microsomes. Addition of ATP caused a rapid increase in ionophore A23187-releasable Ca2+ which stabilized in 2-3 min and provided a rapid and very sensitive assay for ATP-dependent Ca2+ sequestration. Quantitatively, this fraction was sufficient to account for virtually all of the nonmitochondrial ionophore-releasable Ca2+ of rat hepatocytes. Incubation with t-butyl hydroperoxide caused a rapid loss in the ability of microsomes to sequester Ca2+ in the presence of ATP. Addition of dithiothreitol or a physiological concentration of GSH to these incubations provided effective protection against the oxidative damage. ATP-dependent microsomal Ca2+ sequestration is therefore sensitive to oxidative damage and may be a primary site of injury leading to disturbed Ca2+ homeostasis during the early stages of drug hepatotoxicity. Intracellular thiols, notably GSH, may prevent these changes by protecting the microsomal Ca2+ pump from oxidative damage.