CYTOSOLIC CATHEPSIN B INDUCES APOPTOTIC ACINAR CELL DEATH DURING PANCREATITIS

Abstract

Acinar cells are the primary site of injury as pancreatitis develops, which quickly leads to acinar cell death. The intra-acinar activation of trypsinogen is the initial trigger of acinar cell injury, and there is sufficient evidence in literature to suggest that cathepsin B is responsible for intra-acinar trypsinogen activation. It is believed that the activated trypsin causes further damage to acinar cells. No other role of cathepsin B besides that in trypsinogen activation has been documented as regards the pathophysiology of pancreatitis. The aim of this study is to demonstrate that, in addition to activating trypsinogen, cathepsin B plays an important role in acinar cell death initiation. Methods: Pancreatic acinar cells were prepared 60 minutes after either single i.p. injections of caerulein (10 μg/kg), or 4 h of l-arginine (5 g/kg); trypsinogen activation, cathepsin B distribution, and caspase 3 activation were compared with acinar cells from control animals. For in vitro studies, permeabilized acinar cells were stimulated with supra-maximal caerulein for 60 minutes; cytosolic and pellet fractions were prepared by centrifugation. Trypsin activation, cathepsin B distribution, and caspase 3 activation in response to supra-maximal caerulein stimulation were compared with control animals. Results: Cytosolic cathepsin B was significantly increased in acinar cells prepared from caerulein (caerulein: 13.91 ± 2.7 vs control: 2.41 ± 0.98% of total) or L-arginine-administered animals, compared to that prepared from control animals (L-arginine: 9.23 ± 0.98 vs control: 2.27 ± 0.23% of total). This was accompanied by a significant increase in caspase-3 activity (182.67 ± 13.2% of control). In vitro studies also showed a significant redistribution of cathepsin B to the cytosolic fraction (caerulein: 20.6 ± 2.13 vs control: 2.01 ± 0.12% of total) and caspase-3 activation (233 ± 5.56% of control). In the presence of cathepsin B-specific inhibitors, caspase-3 activation was inhibited (125 ± 5.6 vs. 233 ± 5.6). There was a significant increase in apoptosis of acinar cells stimulated with supramaximal caerulein as compared to control (158.0 ± 8.4%) and this was inhibited in the presence of cathepsin B-specific inhibitors. Conclusions: In addition to its known role in trypsinogen activation, cathepsin B also plays an important role in pancreatitis by activating caspase-3 and triggering acinar cell apoptosis.