title: Ethanol impairs CCK-8-evoked amylase secretion through Ca2+-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(2)DCFDA), respectively. Our results show that stimulation of pancreatic acinar cells with CCK-8 induced a dose-dependent amylase secretion, resulting in a maximum at 0.3nM 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 (1-50mM). In contrast, incubation of cells with 50mM 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(2)DCFDA, 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.
authors.affiliation: Department of Physiology, Faculty of Veterinary Sciences, University of Extremadura, Avenida Universidad s/n, E-10071, Cáceres, Spain. agmateos@unex.es
authors.forename: Antonio
authors.initials: A
authors.lastname: González
authors.forename: Ana M
authors.initials: AM
authors.lastname: Núñez
authors.forename: María P
authors.initials: MP
authors.lastname: Granados
authors.forename: José A
authors.initials: JA
authors.lastname: Pariente
authors.forename: Ginés M
authors.initials: GM
authors.lastname: Salido
mesh_terms: D000681:Amylases / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D000431:Ethanol / Q000494:pharmacology*; D008297:Male; D051379:Mice; D010179:Pancreas / Q000166:cytology / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism*; D012844:Sincalide / Q000494:pharmacology*
chemical_list: D017382:Reactive Oxygen Species; D000431:Ethanol; D000681:Amylases; D012844:Sincalide; D002118:Calcium