Fatty Acid Ethyl Esters Cause Pancreatic Calcium Toxicity via Inositol Trisphosphate Receptors and Loss of ATP Synthesis

Background & AimsFatty 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.MethodsIsolated 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 μmol/L). Whole-cell patch clamp was used to measure the calcium-activated chloride current and apply ethanol metabolites and/or ATP intracellularly.ResultsIntracellular 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.ConclusionsFatty 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.