Bcl-xL AND Bcl-2 PROSURVIVAL PROTEINS PROTECT AGAINST NECROSIS IN PANCREATITIS

Abstract

Mitochondrial permeabilization is a common event in models of acute pancreatitis. It is associated with cytochrome c release, leading to apoptosis, and loss of the mitochondrial membrane potential (ΔΨm), mediating necrosis. Proteins of Bcl-2 family are key regulators of mitochondrial permeabilization. Here, we analyzed the changes in Bcl-2 proteins in models of pancreatitis and their effects on mitochondrial depolarization, cytochrome c release, and death responses of pancreatitis. Western blot analysis showed that the prosurvival Bcl-xL is greatly up-regulated in models of pancreatitis induced by cerulein, L-arginine, and choline-deficient ethionine supplemented diet. Increases in Bcl-xL were observed in both total tissue homogenates and mitochondrial fractions. With real time PCR we showed that Bcl-xL up-regulation occurred at the transcriptional level. We further measured the effects of recently introduced specific inhibitors of Bcl-xL/Bcl-2, HA14-I and BH3I-2', on death responses and the underlying signaling in the in vitro model of pancreatitis, i.e., acinar cells treated with supramaximal CCK-8. The Bcl-xL/Bcl-2 inhibitors dose-dependently decreased ΔΨm (as measured in both isolated pancreatic mitochondria and live acinar cells), promoting ATP depletion and necrosis in acinar cells treated with supramaximal CCK-8. These data indicate that Bcl-xL/Bcl-2 protect against necrosis in pancreatitis. The Bcl-xL/Bcl-2 inhibitors also stimulated CCK-induced cytochrome c release in acinar cells. However, both HA14-1 and BH3-I' inhibited CCK-induced apoptosis. The reason for this "paradoxical" effect is that ATP depletion caused by the Bcl-xL/Bcl-2 inhibitors prevented ATP-dependent caspase activation downstream of cytochrome c release. Thus, our data indicate that Bcl-xL/Bcl-2 inhibition stimulates necrosis and inhibits apoptosis, making pancreatitis more severe. The results suggest that up-regulation of Bcl-xL/Bcl-2 is a promising strategy to mitigate acinar cell death in pancreatitis.