Albert M. Song

Phosphatidylinositol 3-kinase-dependent activation of trypsinogen modulates the severity of acute pancreatitis

Intra-acinar cell activation of digestive enzyme zymogens including trypsinogen is generally believed to be an early and critical event in acute pancreatitis. We have found that the phosphatidylinositol 3-kinase inhibitor wortmannin can reduce the intrapancreatic activation of trypsinogen that occurs during two dissimilar experimental models of rodent acute pancreatitis, secretagogue- and duct injection-induced pancreatitis. The severity of both models was also reduced by wortmannin administration. In contrast, the NF-kappa B activation that occurs during the early stages of secretagogue-induced pancreatitis is not altered by administration of wortmannin. Ex vivo, caerulein-induced trypsinogen activation is inhibited by wortmannin and LY294002. However, the cytoskeletal changes induced by caerulein were not affected by wortmannin. Concentrations of caerulein that induced ex vivo trypsinogen activation do not significantly increase phosphatidylinositol-3,4-bisphosphate or phosphatidylinositol 3,4,5-trisphosphate levels or induce phosphorylation of Akt/PKB, suggesting that class I phosphatidylinositol 3-kinases are not involved. The concentration of wortmannin that inhibits trypsinogen activation causes a 75% decrease in phosphatidylinositol 3-phosphate, which is implicated in vesicle trafficking and fusion. We conclude that a wortmannin-inhibitable phosphatidylinositol 3-kinase is necessary for intrapancreatic activation of trypsinogen and regulating the severity of acute pancreatitis. Our observations suggest that phosphatidylinositol 3-kinase inhibition might be of benefit in preventing acute pancreatitis.

Both thermal and non-thermal stress protect against caerulein induced pancreatitis and prevent trypsinogen activation in the pancreas

Background and aim: Recent studies have indicated that prior thermal stress causes upregulation of heat shock protein 70 (HSP70) expression in the pancreas and protects against secretagogue induced pancreatitis. The mechanisms responsible for the protective effect are not known. Similarly, the effects of prior non-thermal stress on HSP70 expression and pancreatitis are not known. The current studies were designed to specifically address these issues. Methods: In the current studies pancreatitis was induced by administration of a supramaximally stimulating dose of caerulein 12 hours after thermal stress and 24 hours after non-thermal (that is, β adrenergic stimulation) stress. Results: Both thermal and non-thermal stresses caused pancreatic HSP70 levels to rise and resulted in increased expression of HSP70 in acinar cells. Both forms of stresses protected against caerulein induced pancreatitis and prevented the early intrapancreatic activation of trypsinogen which occurs in this model of pancreatitis. Conclusions: These results suggest that both thermal and non-thermal stresses protect against pancreatitis by preventing intrapancreatic digestive enzyme activation and that HSP70 may mediate this protective effect.

Serine proteases regulate exocytosis and F-actin distribution in rat pancreatic acinar cells

Background: The polarized pancreatic acinar cell secretes digestive enzymes from its apical pole in response to various secretagogues. However not much is known about the steps that occur in this process subsequent to the increase of cytosolic calcium. We have recently shown that a serine protease activity is essential for the exocytosis of digestive enzymes from pancreatic acinar cells in response to calcium-mediated secretagogues. However the mechanism by which this protease regulates exocytosis is not known. In the present study we have evaluated the effect of serine protease inhibition on the acinar cell cytoskeleton. Methods: Acini were prepared by collagenase digestion and were stimulated by various secretagogues in the presence or absence of cell permeant serine protease inhibitors 4-(2aminoethyl)-benzenesulfonyl flouride (AEBSF) and TPCK. F-Actin distribution was studied after staining with rhodamine phalloidin and examination under the confocal microscope. Results: Both cell permeant serine protease inhibitors (AEBSF and TPCK) blocked exocytosis in response to secretagogues that use calcium as a second messenger (e.g. caerulein, carbamylcholine and bombesin). The non-cell permeant inhibitor SBTI did not affect exocytosis. Incubation of the acini with the cell permeant serine protease inhibitors resulted in a dramatic redistribution of the F-actin cytoskeleton. Under basal conditions F-actin was primarily localized at the apical pole of the acinar cell. After exposure to the cell permeant serine protease inhibitors, F-actin was redistributed to the basolateral region of the cell. This redistribution was blocked by oligomycin, showing that the phenomenon is energy dependent. Similar redistribution of F-actin from the apical to the basolateral region was also observed when acini were incubated with a supramaximally stimulating concentration of caerulein which is known to inhibit secretion via its low affinity receptor. Conclusion: These results suggest that a serine protease activity is essential for maintaining the normal apical F-actin distribution and hence apical exocytosis in the rat pancreatic acinar cell. Blockage of this activity redistributes F-actin from the apical to the basolateral region and blocks exocytosis in response to agents acting via calcium.