Oxidant-impaired intracellular Ca2+ signaling in pancreatic acinar cells:: role of the plasma membrane Ca2+-ATPase

Pancreatitis is an inflammatory disease of pancreatic acinar cells whereby intracellular calcium concentration ([Ca2+](i)) signaling and enzyme secretion are impaired. Increased oxidative stress has been suggested to mediate the associated cell injury. The present study tested the effects of the oxidant, hydrogen peroxide, on [Ca2+](i) signaling in rat pancreatic acinar cells by simultaneously imaging fura-2, to measure [Ca2+](i), and dichlorofluorescein, to measure oxidative stress. Millimolar concentrations of hydrogen peroxide increased cellular oxidative stress and irreversibly increased [ Ca2+](i), which was sensitive to antioxidants and removal of external Ca2+, and ultimately led to cell lysis. Responses were also abolished by pretreatment with ( sarco) endoplasmic reticulum Ca2+-ATPase inhibitors, unless cells were pre-stimulated with cholecystokinin to promote mitochondrial Ca2+ uptake. This suggests that hydrogen peroxide promotes Ca2+ release from the endoplasmic reticulum and the mitochondria and that it promotes Ca2+ influx. Lower concentrations of hydrogen peroxide ( 10-100 mu M) increased [ Ca2+](i) and altered cholecystokinin-evoked [ Ca2+](i) oscillations with marked heterogeneity, the severity of which was directly related to oxidative stress, suggesting differences in cellular antioxidant capacity. These changes in [ Ca2+](i) also upregulated the activity of the plasma membrane Ca2+-ATPase in a Ca2+-dependent manner, whereas higher concentrations ( 0.1-1 mM) inactivated the plasma membrane Ca2+-ATPase. This may be important in facilitating "Ca2+ overload," resulting in cell injury associated with pancreatitis.