Volume 120, Issue 7, Pages 1818-1827 (June 2001) Serine protease inhibitor causes F-actin redistribution and inhibition of calcium-mediated secretion in pancreatic acini  Vijay P. Singh, Ashok K. Saluja, Lakshmi Bhagat, Antti J. Hietaranta, Albert Song, Andreas Mykoniatis, Gijs J.D. van Acker, Michael L. Steer  Gastroenterology  Volume 120, Issue 7, Pages 1818-1827 (June 2001) DOI: 10.1053/gast.2001.24883 Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 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 then cerulein 0.1 nmol/L was added. Amylase secretion was measured as described in Materials and Methods, over the subsequent 30 minutes. Results shown are mean ± SEM for 3 independent experiments. Maximal secretion was defined as that observed in control acini exposed to cerulein (0.1 nmol/L) for 30 minutes. Gastroenterology 2001 120, 1818-1827DOI: (10.1053/gast.2001.24883) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 2 Effects of AEBSF on secretagogue-induced amylase secretion by pancreatic acini. Freshly prepared acini were incubated with AEBSF (4 mmol/L) (black bars) for 15 minutes and the various secretagogues (CER, cerulein 0.1 nmol/L; CCh, carbamylcholine 1 μmol/L; BOM, bombesin 0.1 μmol/L; IONO, ionomycin 1 μmol/L; SEC, secretin 10 nmol/L; and dBcAMP, dibutryl cAMP 2 mmol/L; VIP, vasoactive intestinal polypeptide 0.1 μmol/L) were added. Amylase secretion was measured as described in Materials and Methods, over the subsequent 30 minutes. Results shown represent means ± SEM for 3 independent experiments. Asterisks indicate significant differences when compared with the samples that were not incubated with AEBSF. Gastroenterology 2001 120, 1818-1827DOI: (10.1053/gast.2001.24883) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 3 Effect of AEBSF on time-dependence of cerulein stimulated amylase secretion. Freshly prepared acini were preincubated in the absence (○) or presence (●) of AEBSF (2 mmol/L) for 15 minutes and then cerulein (0.1 nmol/L) was added. Amylase secretion into the medium as a function of time after cerulein addition was measured as described in Materials and Methods. Results shown represent mean ± SEM values obtained from 3 independent experiments. Gastroenterology 2001 120, 1818-1827DOI: (10.1053/gast.2001.24883) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 4 Effect of AEBSF on calcineurin activity. Freshly prepared acini were incubated in HEPES buffer with 4 mmol/L AEBSF, 0.1 nmol/L cerulein, or 1 μmol/L cyclosporin A for 30 minutes. These acini were then lysed and calcineurin activity was measured as described in Materials and Methods. The activity in the presence of 4 mmol/L EGTA represents calcium-independent dephosphorylation of the substrate. Results shown are means ± SEM for 3 experiments. Gastroenterology 2001 120, 1818-1827DOI: (10.1053/gast.2001.24883) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 5 Effect of AEBSF on F-actin structure. (A and B) Freshly prepared acini were suspended in HEPES buffer or (C and D) incubated with AEBSF (4 mmol/L) for 30 minutes. The samples were fixed, stained with rhodamine phalloidin, and examined by (A and C) confocal microscopy and (B and D) differential interference contrast microscopy as described in Materials and Methods. Images are representative of those obtained from 3 independent experiments. Gastroenterology 2001 120, 1818-1827DOI: (10.1053/gast.2001.24883) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 6 Effects of AEBSF on ultrastructure of acini. (A and B) Freshly prepared acini were suspended in HEPES buffer or (C and D) incubated with AEBSF (4 mmol/L) for 30 minutes; (B and D) magnification of boxed region of interest from A and C, respectively. The samples were fixed, stained, and examined by electron microscopy as described in Materials and Methods. Note the very clear presence of microvilli containing F-actin filaments in the apical region of (B) control acini and (D) their ablation after incubation with AEBSF. (A and C) Magnification 13,000× and (B and D) magnification 50,000×. Images are representative of those obtained from 3 independent experiments. Gastroenterology 2001 120, 1818-1827DOI: (10.1053/gast.2001.24883) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 7 Reorganization of F-actin and its dependence on ATP. Freshly prepared acini were preincubated in the (A and D) absence and (E–H) presence of oligomycin (10 μg/mL) for 30 minutes. (B and F) AEBSF (4 mmol/L), (C and G) TPCK (250 μmol/L), or (D and H) cerulein (10 nmol/L) were then added. After an additional 30 minutes, the samples were fixed, stained, and examined by confocal microscopy as described in Materials and Methods. Images are representative of those obtained from 3 independent experiments. Arrows show apical surface and arrowheads show basal surface. Gastroenterology 2001 120, 1818-1827DOI: (10.1053/gast.2001.24883) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 8 Effects of dBcAMP on actin distribution. (A and D) Freshly prepared acini were preincubated with buffer alone or (B and E) with buffer containing AEBSF (4 mmol/L) or (C and F) cerulein (10 nmol/L) for 10 minutes. (D–F) dBcAMP (2 mmol/L) was then added to some samples. After an additional 30 minutes, the samples were fixed, stained, and examined by confocal microscopy as described in Materials and Methods. Images are representative of those obtained in 3 independent experiments. (E and F) Note the reapperance of focal F-actin aggregates in the apical region after incubation of acini with dBcAMP. Gastroenterology 2001 120, 1818-1827DOI: (10.1053/gast.2001.24883) Copyright © 2001 American Gastroenterological Association Terms and Conditions