Ethanol impairs CCK-8-evoked amylase secretion through Ca 2+-mediated ROS generation in mouse pancreatic acinar cells

Abstract

In the present study, we have investigated the effect of ethanol on amylase release in response to cholecystokinin octapeptide (CCK-8). We have also studied the effect of ethanol on cytosolic free Ca 2+ concentration ([Ca 2+] c) and reactive oxygen species (ROS) production by loading of cells with fura-2 and 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H 2DCFDA), respectively. Our results show that stimulation of pancreatic acinar cells with CCK-8 induced a dose-dependent amylase secretion, resulting in a maximum at 0.3 nM of 19.39 ± 2.73% of the total content of amylase. Treatment of pancreatic acini with ethanol did not induce any significant effect on amylase release at a wide range of concentrations (1–50 mM). In contrast, incubation of cells with 50 mM ethanol clearly reduced amylase release stimulated by CCK-8. The inhibitory effect of ethanol on CCK-8-induced amylase secretion was abolished by dithiothreitol, a sulfhydryl reducing agent. Ethanol induced an increase in [Ca 2+] c resulting in a level higher than the prestimulation level both in the presence and in the absence of extracellular Ca 2+. Additionally, ethanol led to an increase in fluorescence of CM-H 2DCFDA, reflecting an increase in oxidation. A decrease in oxidation was observed in the absence of extracellular Ca 2+ and in the presence of ethylene glycol-bis(2-aminoethylether)- N, N, N′, N′-tetraacetic acid. Similarly, when the cells were challenged in the presence of the intracellular Ca 2+ chelator 1,2-Bis(2-amino-5-methylphenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) and in the absence of extracellular Ca 2+, the responses to ethanol were reduced, although not completely inhibited. Taken together, our results suggest that ethanol induces generation of ROS by a Ca 2+-dependent mechanism and reduces CCK-8-evoked amylase secretion in exocrine pancreatic cells.