Fatty acid ethyl esters cause pancreatic calcium toxicity via inositol trisphosphate receptors and loss of ATP synthesis.
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.
Criddle
David N
DN
Medical Research Council Secretory Control Research Group, Physiological Laboratory, University of Liverpool, Liverpool, United Kingdom.
Murphy
John
J
Fistetto
Gregorio
G
Barrow
Stephanie
S
Tepikin
Alexei V
AV
Neoptolemos
John P
JP
Sutton
Robert
R
Petersen
Ole H
OH
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*
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