O0093 THE RYANODINE RECEPTOR MODULATES SECRETAGOGUE STIMULATED PANCREATIC ZYMOGEN ACTIVATION

Abstract

Introduction: Premature activation of intra-acinar digestive proenzymes (zymogens) is an early and critical event in the pathogenesis of acute pancreatitis. A rise in cytosolic calcium is required for this activation. Two endoplasmic reticulum release channels contribute to the rise in cytosolic calcium, the 1,4,5-inositol trisphosphate receptor and the ryanodine receptor (RYR). In this study we examined the role of calcium release from the RYR in secretagogue stimulated zymogen activation using both in vitro and in vivo models. Methods: In the in vitro model, rat pancreatic acini were isolated by collagenase digestion and then preincubated with the RYR antagonist, dantrolene (100uM). Acini were subsequently stimulated with agonists that cause zymogen activation (chole-cystokinin (CCK) or carbachol +/− the cAMP analogue, 8-bromo-cAMP (100uM)). Chymotrypsin and trypsin activities were assayed in homogenates and percent amylase secretion was measured. To determine the relevance of the results, the effects of dantrolene on zymogen activation were examined in an in vivo model of pancreatitis using supraphysiologic CCK. Rats were pretreated (1 hr) with intravenous dantrolene (5mg/kg). Pancreatitis was induced by administering hourly intraperitoneal injections of the CCK analogue caerulein (40ug/kg/dose). Rats were sacrificed one hour after the first caerulein injection and pancreatic homogenates were used to assay chymotrypsin and trypsin activities. Results: Preincubation of isolated acinar cells with dan-trolene reduced supraphysiologic CCK (100nM)-induced and cAMP-enhanced zymogen activation by 30% and 70%, respectively. Dantrolene also reduced physiologic CCK (100pM) and cAMP-induced zymogen activation by 35%. Supraphysiologic carbachol (1mM)-induced and cAMP-enhanced activation were reduced by 30% and 50%, respectively. In contrast, amylase secretion was unaffected by the RYR antagonist in the above conditions. In the in vivo model, dantrolene pretreatment reduced supraphysiologic CCK-induced generation of chymotrypsin activity by 62% and trypsin activity by 45%. Conclusion: Together, these in vitro and in vivo studies suggest that initial zymogen activation, but not digestive enzyme secretion, is primarily mediated by calcium release from a pool that is regulated by the RYR. In addition, the RYR may be the major target of cAMP-dependent sensitization to secretagogue stimulated zymogen activation.