Release of calcium from mitochondrial and nonmitochondrial intracellular stores in mouse pancreatic acinar cells by hydrogen peroxide

In the present study we have studied how [Ca2+](i) is influenced by H2O2 in collagenase-dispersed mouse pancreatic acinar cells and the mechanism unde rlying this effect by using a digital microspectrofluorimetric system. In t he presence of normal extracellular calcium concentration, perfusion of pan creatic acinar cells with 1 mM H2O2 caused a slow sustained [Ca2+](i) incre ase, reaching a stable plateau after 10-15 min of perfusion. This increase induced by H2O2 was also observed in a nominally calcium-free medium, refle cting the release of calcium from intracellular store(s). Application of 1 mM H2O2 to acinar cells, in which nonmitochondrial agonist-releasable calci um pools had been previously depleted by a maximal concentration of CCK-8 ( 1 nM) or thapsigargin (0.5 muM) was still able to induce calcium release. S imilar results were observed when thapsigargin was substituted for the mito chondrial uncoupler FCCP (0.5 muM). By contrast, simultaneous addition of t hapsigargin and FCCP clearly abolished the H2O2-induced calcium increase. I nterestingly, coincubation of intact pancreatic acinar cells with CCK-8 plu s thapsigargin and FCCP in the presence of H2O2 did not significantly affec t the transient calcium spike induced by the depletion of nonmitochondrial and mitochondrial agonist-releasable calcium pools, but was followed by a s ustained increase of [Ca2+](i). In addition, H2O2 was able to block calcium efflux evoked by CCK and thapsigargin. Finally, the transient increase in [Ca2+](i) induced by H2O2 was abolished by an addition of 2 mM dithiothreit ol (DTT), a sulfhydryl reducing agent. Our results show that H2O2 releases calcium from CCK-8- and thapsigargin-sensitive intracellular stores and fro m mitochondria. The action of H2O2 is likely mediated by oxidation of sulfh ydryl groups of calcium-ATPases.