Generation of ROS in response to CCK-8 stimulation in mouse pancreatic acinar cells

Abstract

In the present study we have studied the changes in the intracellular reduction–oxidation state in mouse pancreatic acinar cells following stimulation with cholecystokinin octapeptide (CCK-8) and its dependence on Ca 2+ mobilization. In our investigations cytosolic Ca 2+ concentration and reactive oxygen species (ROS) production were determined by loading of cells with fura-2 and CM-H 2DCF-DA, respectively. Changes in these parameters were determined by following changes in fluorescence in the cuvette of a spectrofluorimeter. The results show that stimulation of cells with CCK-8 and/or the sarco-endoplasmic reticulum Ca 2+ pump inhibitor, thapsigargin (Tps), both induced changes in cytosolic free Ca 2+ concentration and led to an increase in fluorescence of CM-H 2DCF-DA, reflecting an increase in oxidation. In the presence of Tps, addition of CCK-8 did not significantly increase fluorescence compared to that evoked by the SERCA inhibitor. Similar results were obtained in the absence of extracellular Ca 2+ and in the presence of EGTA. When the cells were challenged in the presence of the intracellular Ca 2+ chelator BAPTA and in the absence of extracellular Ca 2+ the responses to both CCK-8 and Tps were reduced although not completely inhibited. The mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxy-phenylhydrazone and the inhibitor of the electron transport chain, antimycin, evoked a marked increase in CM-H 2DCF-DA fluorescence and completely inhibited CCK-8 and Tps-evoked responses, indicating that ROS are generated in the mitochondria. In summary, stimulation of mouse pancreatic acinar cells with CCK-8 leads to generation of ROS, and this effect may be derived from Ca 2+ mobilization from intracellular stores and involves mitochondrial metabolism.