Abstract
Alcohol abuse is a leading cause of pancreatitis, accounting for 30% of acute and up to 90% of chronic cases, yet the mechanisms leading to alcohol-induced injury remain elusive. An early and critical feature of this disease is the aberrant signaling of Ca2+ within the pancreatic acinar cell. An important conductor of this Ca2+ release is the basolaterally localized, intracellular Ca2+ channel the ryanodine receptor (RYR). In this study, we examined the role of the RYR in mediating Ca2+ signals during alcohol exposure. We hypothesized that alcohol triggers the release of Ca2+ from pathologically leaky, hyper-phosphorylated RYRs. Acinar cells were freshly isolated from rat, loaded with the Ca2+ dye fluo-4, and imaged by time lapse confocal microscopy. Spatial and temporal changes in Ca2+ release were examined after perfusion with the Ca2+-activating agonist carbachol (1 uM) with or without an ethanol concentration clinically achievable after heavy intoxication (100 mM). Ethanol accelerated the speed of the apical to basolateral Ca2+ wave from 9 to 18 um/sec (P<0.0005; n=18-22 cells/group). Ethanol also caused a similar doubling in intra-cellular cAMP levels from 1.5 to 3 fmoles. Acceleration of the acinar cell Ca2+ wave by alcohol was abrogated by the PKA inhibitor PKI (1 uM; P<0.05; n=10-16 cells/group). Using an epitope-specific phospho-antibody that recognizes the PKA site on the RYR, we found that ethanol increased RYR phosphorylation by nearly five-fold (P<0.05). Finally, the alcohol-induced acceleration of Ca2+ wave speed was completely reduced by the RYR inhibitor dantrolene (100 uM; P<0.05; n=10-16 cells/group). In summary, our results have for the first time implicated a pathological role for RYR-Ca2+ release in the predisposition to alcohol-induced pancreatitis via cAMP/PKA hyper-phosphorylation of the RYR.
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