Figure 2.

Genetic ablation of cyclophilin D (Ppif^−/−) protects pancreatic acinar cells from pancreatitis toxins (fluorescence mean±SEM, F/F₀). (A) Cholecystokinin-8 (CCK-8) (10 nM) induced cytosolic calcium elevations (Fluo-4, left) in Wt (C57BL/6) and Ppif^−/− cells, with faster clearance in Ppif^−/−; Δψ_m (TMRM, middle) and NAD(P)H (right) were preserved in Ppif^−/− not wild type (Wt) cells. (B) Taurolithocholic acid sulfate (TLCS) (500 µM) induced similar calcium changes, clearing faster in Ppif^−/−; whereas Δψ_m and NAD(P)H were preserved in Ppif^−/− not Wt. (C) TLCS (500 μM) induced similar mitochondrial calcium elevations (Rhod-2, left) in Ppif^−/− and Wt cells, as well as similar reactive oxygen species (ROS) elevations (DCFDA, middle) in Ppif^−/− and Wt cells (menadione, MEN oxidant control); insets show ROS-sensitive DCFDA cell fluorescence (white bars=10 μm); ethanol (ETOH, 10 mM) and palmitoleic acid (POA, 20 μM) induced falls of Δψ_m (right) in Wt not Ppif^−/− cells. (D) Significantly increased propidium iodide (PI) uptake in Wt not Ppif^−/− cells after CCK-8 (10 nM) or TLCS (500 µM) (top, *p<0.05 toxin in Wt versus no toxin or toxin in Ppif^−/−), but similar general caspase activation (bottom, *p<0.05 no toxin vs each toxin group). (E) Cyclophilin absence in Ppif^−/− pancreas (immunoblot, left) and cytochrome c (Cyt c) cytosolic fraction immunoblots (densitometry normalised to lactate dehydrogenase (LDH), Cox IV to rule out mitochondrial contamination, right) showed Cyt c release after CCK-8 by Wt and less by Ppif^−/− mitochondria. (F) Necrotic cell death pathway activation (Sytox Orange; SO) from TLCS (500 µM) was delayed in Ppif^−/− vs Wt pancreas lobules (*p<0.05).