Activation of Soluble Adenylyl Cyclase Protects against Secretagogue Stimulated Zymogen Activation in Rat Pancreaic Acinar Cells

Thomas R Kolodecik,Edwin C Thrower,Lonny R Levin,Jochen Buck,Christine A Shugrue,Fred S Gorelick,Karl-Wilhelm Koch

doi:10.1371/journal.pone.0041320

Thomas R Kolodecik, Edwin C Thrower + Show 5 more

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https://doi.org/10.1371/journal.pone.0041320

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Abstract

An early feature of acute pancreatitis is activation of zymogens, such as trypsinogen, within the pancreatic acinar cell. Supraphysiologic concentrations of the hormone cholecystokinin (CCK; 100 nM), or its orthologue cerulein (CER), induce zymogen activation and elevate levels of cAMP in pancreatic acinar cells. The two classes of adenylyl cyclase, trans-membrane (tmAC) and soluble (sAC), are activated by distinct mechanisms, localize to specific subcellular domains, and can produce locally high concentrations of cAMP. We hypothesized that sAC activity might selectively modulate acinar cell zymogen activation. sAC was identified in acinar cells by PCR and immunoblot. It localized to the apical region of the cell under resting conditions and redistributed intracellularly after treatment with supraphysiologic concentrations of cerulein. In cerulein-treated cells, pre-incubation with a trans-membrane adenylyl cyclase inhibitor did not affect zymogen activation or amylase secretion. However, treatment with a sAC inhibitor (KH7), or inhibition of a downstream target of cAMP, protein kinase A (PKA), significantly enhanced secretagogue-stimulated zymogen activation and amylase secretion. Activation of sAC with bicarbonate significantly inhibited secretagogue-stimulated zymogen activation; this response was decreased by inhibition of sAC or PKA. Bicarbonate also enhanced secretagogue-stimulated cAMP accumulation; this effect was inhibited by KH7. Bicarbonate treatment reduced secretagogue-stimulated acinar cell vacuolization, an early marker of pancreatitis. These data suggest that activation of sAC in the pancreatic acinar cell has a protective effect and reduces the pathologic activation of proteases during pancreatitis.

Highlights

The exocrine pancreas responds to a meal by secreting digestive zymogens into the small intestine, where they are converted to active enzymes
Results soluble adenylyl cyclase (sAC) is Present in Pancreatic Acinar Cells The presence of sAC in pancreatic acinar cells was first determined by Polymerase Chain Reaction (PCR) and immunoblot (Fig. 1A, 1B)
These findings provide molecular evidence that sAC is present in pancreatic acinar cells

Summary

Introduction

The exocrine pancreas responds to a meal by secreting digestive zymogens ( proteases, such as trypsinogen and chymotrypsinogen) into the small intestine, where they are converted to active enzymes. The intracellular signaling molecule, cAMP, has been found to enhance secretagogue-sensitive zymogen activation when intracellular cAMP levels are increased using membrane permeable analogs of cAMP [1,2] This response was observed after treatment with secretin, vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating peptide (PACAP), whose receptors are linked to plasma membrane adenylyl cyclases [2]. Rap, a small GTP binding protein activated by cAMP stimulation of EPAC, modulates acinar cell amylase secretion [5] and is associated with zymogen granule membranes [6,7]. These studies demonstrated effects of cAMP on acinar cell responses, they did not examine the potential contribution by specific cAMP pools on pancreatitis responses

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