Fatty acid ethyl esters cause pancreatic calcium toxicity via inositol trisphosphate receptors and loss of ATP synthesis

Background & Aims: Fatty acid ethyl esters are ethanol metabolites inducing sustained, toxic elevations of the acinar cytosolic free calcium ion concentration ([Ca2+](C)) implicated in pancreatitis. We sought to define the mechanisms of this elevation. Methods: Isolated mouse pancreatic acinar cells were loaded with fluorescent dyes for confocal microscopy to measure [Ca2+]C (Fluo 4, Fura Red), endoplasmic reticulum calcium ion concentration ([Ca2+](ER), Mg Fluo 4), mitochondrial membrane potential (TMRM), ADP:ATP ratio (Mg Green), and NADH autofluorescence in response to palmitoleic acid ethyl ester and palmitoleic acid (10-100 mu mol/L). Whole-cell patch clamp was used to measure the calcium-activated chloride current and apply ethanol metabolites and/or ATP intracellularly. Results: Intracellular delivery of ester induced oscillatory increases of [Ca2+](C) and calcium-activated currents, inhibited acutely by caffeine (20 mmol/L), but not atropine, indicating involvement of inositol trisphosphate receptor channels. The stronger effect of extracellular ester or acid caused depletion of [Ca2+](ER), not prevented by caffeine, but associated with depleted ATP, depleted NADH autofluorescence, and depolarized mitochondria, suggesting calcium-ATPase pump failure because of lack of ATP. Intracellular ATP abolished the sustained rise in [Ca2+](C), although oscillatory signals persisted that were prevented by caffeine. Inhibition of ester hydrolysis markedly reduced its calcium-releasing effect and consequent toxicity. Conclusions: Fatty acid ethyl ester increases [Ca2+](C) through inositol trisphosphate receptors and, following hydrolysis, through calcium-ATPase pump failure from impaired mitochondrial ATP production. Lowering cellular fatty acid substrate concentrations may reduce cell injury in pancreatitis.