376 Enterra® Gastric Electrical Stimulation for Diabetic Gastroparesis: Results from a Multicenter Randomized Study

Abstract

During initial stages of development of pancreatitis, co-localization of zymogens and lysosomal enzymes is observed in experimental models of pancreatitis. This leads to activation of trypsinogen to trypsin by cathepsin B. We have recently shown that these co-localized organelles are leaky thus resulting in the release of active trypsin and cathepsin B into cytosol. Whether and how active trypsin lead to acinar cell death during pancreatitis is not understood. The aim of the present study was to elucidate if trypsin or cathepsin B released into cytosol contributes to apoptotic cell death. Methods: Rat pancreatic acinar cells were prepared by collagenase digestion and stimulated with supramaximal concentrations of caerulein. These were permeabilzed using streptolysin O and separated into membrane and cytosolic fraction. In another set of experiments acinar cells were incubated with different concentrations of sphingosine (5-20μM), a lysosomotropic agent. Caspase 3 activation was measured by luminescence based assay. Trypsin and Cathepsin B activities were measured by fluorometric assays. Cytochrome c was detected by Western blotting. Results: Supramaximal stimulation of pancreatic acinar cells with caerulein resulted in significant increase in trypsin (12.3±2.1% of total) and cathepsin B (21.6±3.4% of total) activity in the cytosol. There was also significant increase in cytochrome c and caspase 3 (233.5±4.6 % of control) activities in the cytosolic fraction. Inhibition of cathepsin B resulted in significant decrease in caspase 3 activity. Addition of cathepsin B but not that of active trypsin to unstimulated permeabilzed acinar cells resulted in caspase 3 activation. Incubation of intact unstimulated pancreatic acinar cells with sphingosine resulted in permeability of lysosomes as measured by acridine orange fluorescence and release of cathepsin B into cytosol. This release again resulted in caspase 3 activation (203±4.4% over control) which was inhibited when cells were pretreated with cathepsin B inhibitor CA074me. Conclusion: Our results clearly show that release of cathepsin B into the cytosol of pancreatic acinar cells is sufficient for their apoptotic cell death throughmitochondrial pathway and trypsin doesn't directly participate in apoptosis observed during pancreatitis.