MITOCHONDRIAL CA2+ OVERLOAD IS THE PRINCIPAL MECHANISM OF INJURY TO PANCREATIC ACINAR CELLS FROM BILE SALTS

Abstract

Background & Aims: Bile salts induce prolonged, global elevations of the free cytosolic ionised Ca2+ concentration ([Ca2+]C) that are toxic to pancreatic acinar cells, but the mechanism of injury has remained unclear. We sought to determine the contribution of mitochondrial Ca2+ overload to pancreatic acinar cell injury from the bile salt taurolithocholic-3-sulphate (TLCS). Methods: Isolated mouse pancreatic acinar cells were examined by confocal microscopy to measure changes in [Ca2+]C (Fluo4-AM), mitochondrial function (NADH autofluorescence), ATP concentration (Mg Green) and cell fate (propidium iodide, PI). TLCS (200 mM) was perfused externally while whole-cell recordings of Ca2+-dependent Cl− currents were made. All experiments were repeated at least six times, and in some experiments, supplementary ATP was added to the internal pipette solution. Results: TLCS induced prolonged (>30 s), global [Ca2+]C elevations accompanied by NADH and ATP depletion, inducing necrosis. Caffeine (20 mM), an inhibitor of inositol-trisphosphate-elicited Ca2+ release, inhibited these elevations. Supplementary ATP (4 mM) in the internal pipette solution reduced the period of globalisation associated with each [Ca2+]C elevation and prevented necrosis, as did removal of Ca2+ from the external medium. Control experiments demonstrated that neither pipette application nor absence of supplementary ATP (0 mM) in the internal pipette solution induced necrosis. Conclusion: Bile salts induce pancreatic acinar cell injury through mitochondrial inhibition, prevented by removal of Ca2+ from the cell exterior. These data indicate the principal mechanism of pancreatic acinar cell injury from bile salts is mitochondrial Ca2+ overload from excessive inositol-trisphosphate receptor Ca2+ channel release.