title: Fatty acid ethyl esters cause pancreatic calcium toxicity via inositol trisphosphate receptors and loss of ATP synthesis.
abstract: OBJECTIVE
  Fatty acid ethyl esters are ethanol metabolites inducing sustained, toxic elevations of the acinar cytosolic free calcium ion concentration ([Ca(2+)](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 [Ca(2+)](C) (Fluo 4, Fura Red), endoplasmic reticulum calcium ion concentration ([Ca(2+)](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 micromol/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 [Ca(2+)](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 [Ca(2+)](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 [Ca(2+)](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 [Ca(2+)](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.
authors.affiliation: Medical Research Council Secretory Control Research Group, Physiological Laboratory, University of Liverpool, Liverpool, United Kingdom.
authors.forename: David N
authors.initials: DN
authors.lastname: Criddle
authors.forename: John
authors.initials: J
authors.lastname: Murphy
authors.forename: Gregorio
authors.initials: G
authors.lastname: Fistetto
authors.forename: Stephanie
authors.initials: S
authors.lastname: Barrow
authors.forename: Alexei V
authors.initials: AV
authors.lastname: Tepikin
authors.forename: John P
authors.initials: JP
authors.lastname: Neoptolemos
authors.forename: Robert
authors.initials: R
authors.lastname: Sutton
authors.forename: Ole H
authors.initials: OH
authors.lastname: Petersen
mesh_terms: D000255:Adenosine Triphosphate / Q000096:biosynthesis*; D000818:Animals; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000502:physiology*; D020013:Calcium Signaling; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D000431:Ethanol / Q000378:metabolism* / Q000633:toxicity; D005227:Fatty Acids / Q000378:metabolism / Q000633:toxicity*; D053496:Inositol 1,4,5-Trisphosphate Receptors; D051379:Mice; D008928:Mitochondria / Q000378:metabolism; D010179:Pancreas / Q000187:drug effects*; D019512:Pancreatitis, Alcoholic / Q000378:metabolism / Q000517:prevention & control; D018160:Receptors, Cytoplasmic and Nuclear / Q000502:physiology*
chemical_list: D015220:Calcium Channels; D005227:Fatty Acids; D053496:Inositol 1,4,5-Trisphosphate Receptors; D018160:Receptors, Cytoplasmic and Nuclear; D002110:Caffeine; D000431:Ethanol; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D002118:Calcium