Dose-dependent effect of hydrogen peroxide on calcium mobilization in mouse pancreatic acinar cells

Abstract

We have employed confocal laser scanning microscopy to investigate how intracellular free calcium concentration ([Ca²⁺]_i) is influenced by hydrogen peroxide (H₂O₂) in collagenase-dispersed mouse pancreatic acinar cells. In the absence of extracellular calcium, treatment of cells with increasing concentrations of H₂O₂resulted in an increase in [Ca²⁺]_i, indicating the release of calcium from intracellular stores. Micromolar concentrations of H₂O₂induced an oscillatory pattern, whereas 1 mmol H₂O₂/L caused a slow and sustained increase in [Ca²⁺]_i. H₂O₂abolished the typical calcium release stimulated by thapsigargin or by the physiological agonist cholecystokinin octapeptide (CCK-8). Depletion of either agonist-sensitive or mitochondrial calcium pools was unable to prevent calcium release induced by 1 mmol H₂O₂/L, but depletion of both stores abolished it. Additionally, lower H₂O₂concentrations were able to release calcium only after depletion of mitochondrial calcium stores. Treatment with either the phospholipase C inhibitor U-73122 or the inhibitor of the inositol 1,4,5-trisphosphate (IP3) receptor xestospongin C did not modify calcium release from the agonist-sensitive pool induced by 100 µmol H₂O₂/L, suggesting the involvement of a mechanism independent of IP3 generation. In addition, H₂O₂reduced amylase release stimulated by CCK-8. Finally, either the H₂O₂-induced calcium mobilization or the inhibitory effect of H₂O₂on CCK-8-induced amylase secretion was abolished by dithiothreitol, a sulphydryl reducing agent. We conclude that H₂O₂at micromolar concentrations induces calcium release from agonist- sensitive stores, and at millimolar concentrations H₂O₂can also evoke calcium release from the mitochondria. The action of H₂O₂is mediated by oxidation of sulphydryl groups of calcium ATPases independently of IP3 generation.Key words: hydrogen peroxide, pancreatic acinar cells, intracellular calcium stores, amylase secretion.