Ethanol Enhances Carbachol-induced Protease Activation and Accelerates Ca2+ Waves in Isolated Rat Pancreatic Acini

Abstract

Alcohol abuse is a leading cause of pancreatitis, accounting for 30% of acute cases and 70 -90% of chronic cases, yet the mechanisms leading to alcohol-associated pancreatic injury are unclear. An early and critical feature of pancreatitis is the aberrant signaling of Ca 2 within the pancreatic acinar cell. An important conductor of this Ca 2 is the basolaterally localized, intracellular Ca 2 channel ryanodine receptor (RYR). In this study, we examined the effect of ethanol on mediating both pathologic intra-acinar protease activation, a precursor to pancreatitis, as well as RYR Ca 2 signals. We hypothesized that ethanol sensitizes the acinar cell to protease activation by modulating RYR Ca 2 . Acinar cells were freshly isolated from rat, pretreated with ethanol, and stimulated with the muscarinic agonist carbachol (1 M). Ethanol caused a doubling in the carbachol-induced activation of the proteases trypsin and chymotrypsin (p < 0.02). The RYR inhibitor dantrolene abrogated the enhancement of trypsin and chymotrypsin activity by ethanol (p < 0.005 for both proteases). Further, ethanol accelerated the speed of the apical to basolateral Ca 2 wave from 9 to 18 m/s (p < 0.0005; n 18 -22 cells/group); an increase in Ca 2 wave speed was also observed with a change from physiologic concentrations of carbachol (1 M) to a supraphysiologic concentration (1 mM) that leads to protease activation. Dantrolene abrogated the ethanol-induced acceleration of wave speed (p < 0.05; n 10 -16 cells/group). Our results suggest that the enhancement of pathologic protease activation by ethanol is dependent on the RYR and that a novel mechanism for this enhancement may involve RYR-mediated acceleration of Ca 2 waves.