Abstract

A genuine understanding of human exocrine pancreas biology and pathobiology has been hampered by a lack of suitable preparations and reliance on rodent models employing dispersed acini preparations. We have developed an organotypic slice preparation of the normal portions of human pancreas obtained from cancer resections. The preparation was assessed for physiologic and pathologic responses to the cholinergic agonist carbachol (Cch) and cholecystokinin (CCK-8), including 1) amylase secretion, 2) exocytosis, 3) intracellular Ca2+ responses, 4) cytoplasmic autophagic vacuole formation, and 5) protease activation. Cch and CCK-8 both dose-dependently stimulated secretory responses from human pancreas slices similar to those previously observed in dispersed rodent acini. Confocal microscopy imaging showed that these responses were accounted for by efficient apical exocytosis at physiologic doses of both agonists and by apical blockade and redirection of exocytosis to the basolateral plasma membrane at supramaximal doses. The secretory responses and exocytotic events evoked by CCK-8 were mediated by CCK-A and not CCK-B receptors. Physiologic agonist doses evoked oscillatory Ca2+ increases across the acini. Supraphysiologic doses induced formation of cytoplasmic autophagic vacuoles and activation of proteases (trypsin, chymotrypsin). Maximal atropine pretreatment that completely blocked all the Cch-evoked responses did not affect any of the CCK-8-evoked responses, indicating that rather than acting on the nerves within the pancreas slice, CCK cellular actions directly affected human acinar cells. Human pancreas slices represent excellent preparations to examine pancreatic cell biology and pathobiology and could help screen for potential treatments for human pancreatitis.

Highlights

  • Extensive studies on pancreatic acinar cell stimulus-secretion coupling have for more than three and a half decades employed dispersed acini preparations of rodent pancreas [1, 2]

  • As CCK receptors are of two subtypes, CCK-A and CCK-B, which CCK receptor subtype is in human pancreatic acinar cells has been a subject of continued debate

  • Supramaximal cholinergic or CCK-8 stimulation of rodent pancreatic acini causes inhibition of amylase secretion from maximal levels resulting from blockade of normal regulated apical exocytosis and diversion of exocytosis to the basolateral plasma membrane; the latter would result in interstitial pancreatitis [11, 12]

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Summary

Introduction

Extensive studies on pancreatic acinar cell stimulus-secretion coupling have for more than three and a half decades employed dispersed acini preparations of rodent pancreas [1, 2]. Pathologic basolateral exocytosis [11, 12] along with intracellular protease activation occurring initially within large cytoplasmic vacuoles [13,14,15] are the established cellular mechanisms of pancreatitis [16] Both of these events of acinar cell injury could be evoked by supramaximal CCK-8 or carbachol stimulation of rodent pancreas, it has yet to be proven whether in human pancreas these events are the cellular mechanisms of human pancreatitis. We show the human exocrine pancreas displayed a typical concentration-dependent amylase secretion, acinar exocytosis, and intracellular calcium ([Ca2ϩ]i) rise and supramaximal agonist stimulation-evoked cytoplasmic vacuole formation and protease activation Of note, all these events were evoked by carbachol and CCK-8 stimulation, the latter via pancreatic acinar CCK-A receptors and not mediated by neuronal input. The latter supports a direct CCK-8 signaling pathway for secretion and pancreatitis in the human pancreatic acinar cell

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