Fig. 6.
Bcl-xL/Bcl-2 inhibitors induce mitochondrial depolarization, ATP depletion, and necrosis in pancreatic acinar cells. These effects are greater in CCK-hyperstimulated acinar cells than in unstimulated cells. (A–F) Rat pancreatic acinar cells were incubated for 3 h with and without 100 nM CCK-8 and indicated concentrations of HA14-1 (HA) or 15 µM BH3I-2′ (BH3). (G, H) Mouse pancreatic acinar cells were cultured for 24 h in DMEM medium, as described in Materials and methods, transfected with control or Bcl-xL siRNA, and then incubated for 3 h in 199 medium with and without 100 nM CCK-8. ΔΨm was measured in acinar cells loaded with the ΔΨm-sensitive dye JC-1 (A D); cellular ATP, by use of luciferin/luciferase-based ATP determination kit (B, E). Necrosis was measured (C,F) by the percentage of total cellular LDH released into the extracellular medium by LDH release or (G) as percentage of cells stained positively with trypan blue. (H). Bcl-xL levels in cells transfected with control and Bcl-xL siRNA were measured with Western blot. Blots were re-probed for ERK1/2 to confirm equal protein loading. ΔΨm and ATP values were normalized to those in cells incubated without CCK in the absence of the inhibitors (control). Values are means ± SE (n = 3). *p < 0.05 compared with cells incubated without CCK in the absence of the inhibitor, (control), or cells transfected with control siRNA. #p< 0.05 compared to cells incubated with CCK in the absence of the inhibitor.