Gastroenterology

Gastroenterology

Volume 120, Issue 7, June 2001, Pages 1818-1827
Gastroenterology

Liver, Pancreas, and Biliary Tract
Serine protease inhibitor causes F-actin redistribution and inhibition of calcium-mediated secretion in pancreatic acini*,**

https://doi.org/10.1053/gast.2001.24883Get rights and content

Abstract

Background & Aims: The present study was undertaken to evaluate the role of serine proteases in regulating digestive enzyme secretion in pancreatic acinar cells. Methods: Isolated acini were stimulated by various secretagogues in the presence or absence of cell-permeant serine protease inhibitors 4-(2-aminoethyl)-benzenesulfonyl fluoride and Nα-p-tosyl-L-phenylalanine chloromethyl ketone. F-actin distribution was studied after staining with rhodamine phalloidin. Results: Both cell-permeant serine protease inhibitors blocked amylase secretion in response to secretagogues that use calcium as a second messenger (e.g., cerulein, carbamylcholine, and bombesin) but not to those that use adenosine 3',5'-cyclic monophosphate (cAMP) as a second messenger (e.g., secretin and vasoactive intestinal polypeptide). Incubation of the acini with these inhibitors also resulted in a dramatic redistribution of the F-actin cytoskeleton. This redistribution was 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 cerulein, which is known to inhibit secretion. Conclusions: These results suggest that a serine protease activity is essential for maintaining the normal apical F-actin distribution; its inhibition redistributes F-actin from the apical to the basolateral region and blocks secretion induced by secretagogues that act via calcium. cAMP reverses the F-actin redistribution and hence cAMP-mediated secretion is not affected.

GASTROENTEROLOGY 2001;120:1818-1827

Section snippets

Materials and methods

Male Wistar rats (90–120 g) were obtained from Charles River Laboratories (Wilmington, MA). Cerulein was purchased from Research Plus (Bayonne, NJ), collagenase from Worthington Biochemicals (Freehold, NJ), the trypsin substrate Boc-Glu-Ala-Arg-4-methylcoumaryl-7-amide (MCA) from Peptides International (Louisville, KY), and both Fura 2/AM and rhodamine phalloidin from Molecular Probes (Eugene, OR). The serine protease substrate tosyl-glycyl-prylyl-arginine-4-nitralide acetate (Chromozym TH) was

Cell-permeant serine protease inhibitors cause inhibition of Ca2+-mediated but not camp-mediated amylase secretion from intact acini

Cerulein-stimulated amylase secretion from freshly prepared acini is inhibited, in a concentration-dependent manner, by AEBSF, as well as the other cell-permeant serine protease inhibitors TPCK and TLCK (Figure 1).

. Concentration-dependent inhibition of cerulein-stimulated amylase secretion by pancreatic acini. Freshly prepared acini were incubated with varying concentrations of the serine protease inhibitors (TPCK, TLCK, and AEBSF) or AEBSF structural analogs (BSF, AEBSA, MSF) for 15 minutes and

Discussion

Pancreatic acinar cell stimulus-secretion coupling involves 2 parallel intracellular second messenger systems, 1 that results in an increase in [Ca2+]I and activation of protein kinase C, and another that results in generation of cAMP and activation of protein kinase A. In this article, we have shown that exposure to a cell-permeant serine protease inhibitor selectively interferes with secretion mediated via the Ca2+ pathway but does not alter secretion mediated via the cAMP pathway.

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*

Address for reprint requests to: Michael L. Steer, M. D., Department of Surgery, Beth Israel Deaconess Medical Center, Boston, Massachusetts 02215. fax: (617) 667-8679; e-mail: [email protected].

**

Supported by National Institutes of Health grant DK-31396.

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