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

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 Ca2+ mobilization. In our investigations cytosolic Ca2+ concentration and reactive oxygen species (ROS) production were determined by loading of cells with fura-2 and CM-H2DCF-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 Ca2+ Pump inhibitor, thapsigargin (Tps), both induced changes in cytosolic free Ca2+ concentration and led to an increase in fluorescence of CM-H2DCF-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 Ca2+ and in the presence of EGTA. When the cells were challenged in the presence of the intracellular Ca2+ chelator BAPTA and in the absence of extracellular Ca2+ 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-H2DCF-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 Ca2+ mobilization from intracellular stores and involves mitochondrial metabolism. (C) 2004 Elsevier B.V. and Mitochondria Research Society. All rights reserved.