FATTY ACID ETHYL ESTERS INDUCE PANCREATIC CA2+ TOXICITY THROUGH INOSITOL TRISPHOSPHATE RECEPTORS AND LOSS OF MITOCHONDRIAL ATP PRODUCTION

Abstract

Background & Aims: Fatty acid ethyl esters are ethanol metabolites that induce sustained, toxic elevations of acinar cytosolic Ca2+ concentrations ([Ca2+]c) implicated in pancreatitis. We sought to determine the mechanisms of this elevation. Methods: Isolated mouse pancreatic acinar cells were loaded with fluorescent dyes for confocal microscopy to measure [Ca2+]c (Fluo4, Fura Red), endoplasmic reticulum Ca2+ concentrations ([Ca2+]ER, Mg Fluo4), mitochondrial membrane potential (TMRM) and NADH autofluorescence in response to palmitoleic acid ethyl ester (POAEE) and palmitoleic acid (POA) (10-100 μM). Whole-cell patch clamp was used to measure the Ca2+-activated Cl− current and apply ethanol metabolites and/or ATP intracellularly. Results: Intracellular delivery of ester induced oscillatory increases of [Ca2+]c and Ca2+-activated currents, inhibited acutely by caffeine (20 mM, n = 11), but not atropine (n = 4), indicating involvement of inositol trisphosphate receptor channels. The stronger effect of extracellular application of ester or acid induced depletion of [Ca2+]ER (n = 15-22), which was not prevented by caffeine (n = 10-11), but was associated with decreased NADH autofluorescence and depolarised mitochondria (n = 9-16), suggesting Ca2+-ATPase pump failure due to lack of ATP. Intracellular ATP abolished the sustained rise in [Ca2+]c (10 of 11 cells), although oscillatory signals persisted that were prevented by caffeine. Inhibition of ester hydrolysis by BNPP (200 μM) markedly reduced its Ca2+ releasing effect (6 of 8 cells) and consequent toxicity (assessed by propidium iodide staining, n = 6). Conclusions: Fatty acid ethyl ester increases [Ca2+]c through inositol trisphosphate receptors and, following hydrolysis, through Ca2+-ATPase pump failure from impaired mitochondrial ATP production. Lowering intracellular fatty acid substrate concentrations may reduce cell injury in pancreatitis.