Involvement of Rab4 in regulated exocytosis of rat pancreatic acini Hirohide Ohnishi, Tetsuya Mine, Hiroshi Shibata, Namiki Ueda, Tomohiro Tsuchida, Toshiro Fujita  Gastroenterology  Volume 116, Issue 4, Pages 943-952 (April 1999) DOI: 10.1016/S0016-5085(99)70078-8 Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 1 Characterization of anti-Rab4 antibody. Various G proteins including Rab5, H-Ras, Gq/11, and Gβ were immunoprecipitated from rat pancreatic acini using rabbit polyclonal antibodies against each G protein. Hemagglutinin-tagged Rab3D (HA-Rab3D) was immunoprecipitated using anti–hemagglutinin tag monoclonal antibody from pancreatic acini of transgenic mice overexpressing hemagglutinin-tagged Rab3D. Total homogenate of rat adipocytes (50 μg protein/lane, lanes 1 and 2) and immunoprecipitated hemagglutinin-tagged Rab3D (lanes 3 and 4), Rab5 (lanes 5 and 6), H-Ras (lanes 7 and 8), Gq/11 (lanes 9 and 10), and Gβ (lanes 11 and 12) were fractionated on 12.5% (lanes 1–8) or 10% (lanes 9–12) sodium dodecyl sulfate–polyacrylamide gels and transferred to nitrocellulose membranes. Membranes were then probed with anti-Rab4 antibody (lanes 1, 4, 6, 8, 10, and 12), anti-hemagglutinin tag antibody (lane 3), anti-Rab5 antibody (lane 5), anti–H-Ras antibody (lane 7), anti-Gq/11 antibody (lane 9), and anti-Gβ antibody (lane 11). The anti-Rab4 antibody preincubated with the peptide for immunization was used for lane 2. The bands were visualized by enhanced chemiluminescence. The molecular mass standards are indicated on the left. Gastroenterology 1999 116, 943-952DOI: (10.1016/S0016-5085(99)70078-8) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 2 Western blotting of Rab4 in zymogen granule membranes, a cytosol fraction, and an endosome-rich microsome fraction. Indicated amounts of protein of adipocytes homogenate (AC), pancreatic total homogenate (TH), purified zymogen granule membranes (ZGM), microsome fraction (MF), and cytosol fraction (CF) were separated by 12.5% sodium dodecyl sulfate minigels and transferred to nitrocellulose membranes. Membranes were then probed with anti-Rab4 guinea pig antibody, followed by detection with Vectastain ABC kit, and visualized by enhanced chemiluminescence. The molecular mass standards are indicated on the left. Gastroenterology 1999 116, 943-952DOI: (10.1016/S0016-5085(99)70078-8) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 3 Confocal immunofluorescence micrographs of rat pancreatic acini double-stained with anti-Rab4 and antiamylase antibodies. (A) Immunofluorescence localization of Rab4 in rat pancreatic acini. (B) Immunofluorescence localization of amylase in the same field of A. (C) Combination of A and B Yellow staining indicates the colocalization of Rab4 and amylase in the apical zymogen granule region (bar = 10 μm). Gastroenterology 1999 116, 943-952DOI: (10.1016/S0016-5085(99)70078-8) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 4 Effect of Rab4, Rab3C, and Rab3D peptides on calcium-stimulated amylase secretion from streptolysin O–permeabilized pancreatic acini. Enzymatically isolated pancreatic acini were permeabilized with streptolysin O and preincubated in the presence of indicated amounts of Rab4 peptide (●), Rab3C peptide (○), and Rab3D peptide (). Amylase release was then initiated by adding 10 μmol/L free calcium and incubated for 5 minutes at 30°C. Results are expressed as amylase release as a percentage of total amylase content. Values are the mean ± SE for 3 independent experiments each with triplicate determinations. Basal amylase release with or without 400 μmol/L each peptide (Rab4 peptide, ■; Rab3C peptide, □; Rab3D peptide, ●) was examined by incubating acini with calcium free medium. *P < 0.01; **P < 0.05. Gastroenterology 1999 116, 943-952DOI: (10.1016/S0016-5085(99)70078-8) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 5 Effect of anti-Rab4 IgG and nonimmune IgG on calcium-stimulated amylase secretion from streptolysin O–permeabilized pancreatic acini. Enzymatically isolated pancreatic acini were permeabilized with streptolysin O and preincubated in the presence of indicated amounts of anti-Rab4 guinea pig IgG (●) or nonimmune guinea pig IgG (○). Amylase release was then initiated by adding 10 μmol/L free calcium and incubated for 5 minutes at 30°C. Results are expressed as amylase release as a percentage of total amylase content. Values are the mean ± SE for 3 independent experiments each with triplicate determinations. Basal amylase release with or without 200 μg/mL of each IgG (anti-Rab4 IgG, ■; nonimmune IgG, □) was examined by incubating acini with calcium free medium. *P < 0.01. Gastroenterology 1999 116, 943-952DOI: (10.1016/S0016-5085(99)70078-8) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 6 Immunoprecipitation of Rab4 from rat pancreatic acini. To purify Rab4 protein from pancreatic acini, Rab4 was immunoprecipitated from enzymatically isolated pancreatic acini using anti-Rab4 guinea pig IgG and visualized by Western blotting using the antibody (lane 2). Control immunoprecipitation was performed with nonimmune guinea pig IgG (lane 1). Gastroenterology 1999 116, 943-952DOI: (10.1016/S0016-5085(99)70078-8) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 7 Effect of CCK on GTP binding to Rab4. Isolated pancreatic acini were permeabilized with streptolysin O and stimulated with 100 pmol/L CCK for various times in presence of [α-32P]GTP. Rab4 was then immunoprecipitated, and radioactivity incorporated into Rab4 was counted by liquid scintillation count. Control experiments were performed in the same way but in the absence of CCK. (A) Representative of specific cpm bound. (B) Means + SE obtained from 3 independent experiments in which bound nucleotide is expressed as a percentage of the control. Gastroenterology 1999 116, 943-952DOI: (10.1016/S0016-5085(99)70078-8) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 8 Effect of calphostin C on CCK-stimulated GTP-binding to Rab4. Isolated pancreatic acini were preincubated with 1 μmol/L calphostin C as described in Materials and Methods and permeabilized with streptolysin O and stimulated with 100 pmol/L CCK for various times in the presence of α-[32P]GTP. Rab4 was then immunoprecipitated, and radioactivity incorporated into Rab4 was counted by liquid scintillation count. Control experiments were performed in the same way but in the absence of CCK. (A) Representative of specific cpm bound. (B) Means + SE obtained from 3 independent experiments in which bound nucleotide is expressed as a percentage of the control. Gastroenterology 1999 116, 943-952DOI: (10.1016/S0016-5085(99)70078-8) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 9 Effect of TPA on GTP binding to Rab4. Isolated pancreatic acini were permeabilized with streptolysin O and stimulated with 1 μmol/L TPA for various times in presence of α-[32P]GTP. Rab4 was then immunoprecipitated, and radioactivity incorporated into Rab4 was counted by liquid scintillation count. Control experiments were performed in the same way but in the absence of TPA. (A) Representative of specific cpm bound. (B) Means + SE obtained from 3 independent experiments in which bound nucleotide is expressed as a percentage of the control. Gastroenterology 1999 116, 943-952DOI: (10.1016/S0016-5085(99)70078-8) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 10 Effect of incubation on amount of Rab4 protein in streptolysin O–permeabilized acini. Streptolysin O–permeabilized acini were incubated in the same condition as that of control studies of GTP binding shown in Figures 7–9. At times specified, acini were spun down, lysed, and prepared for Western blotting using 90 μg of acinar protein per lane. Blots were probed with anti-Rab4 antibody and visualized by enhanced chemiluminescence. Gastroenterology 1999 116, 943-952DOI: (10.1016/S0016-5085(99)70078-8) Copyright © 1999 American Gastroenterological Association Terms and Conditions