Kinesin is involved in regulation of rat pancreatic amylase secretion Namiki Ueda, Hirohide Ohnishi, Chiho Kanamaru, Junko Suzuki, Tomohiro Tsuchida, Hirosato Mashima, Hiroshi Yasuda, Toshiro Fujita  Gastroenterology  Volume 119, Issue 4, Pages 1123-1131 (October 2000) DOI: 10.1053/gast.2000.18145 Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 1 Effect of recombinant kinesin on calcium-stimulated amylase secretion from streptolysin-O–permeabilized pancreatic acini. Enzymatically isolated acini were permeabilized with streptolysin-O and preincubated in the presence of indicated amounts of intact (●) or boiled (○) recombinant kinesin. 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 means ± SE for 3 independent experiments, each with duplicate determinations. Basal amylase release with intact (■) or boiled (2) recombinant kinesin was examined by incubating acini with calcium-free medium. *P < 0.01 by 2-way layout analysis of variance. Gastroenterology 2000 119, 1123-1131DOI: (10.1053/gast.2000.18145) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 2 Immunofluorescence localization of introduced hexahistidine-tagged recombinant kinesin to zymogen granules in rat pancreatic acini. (A) Immunofluorescence localization of zymogen granules stained with antiamylase antibody. (B) Introduced hexahistidine-tagged recombinant kinesin was stained with antihexahistidine tag antibody in the same field as A. (C) Combination of A and B. Yellow staining indicates the colocalization of recombinant kinesin and amylase to zymogen granules (original magnification 400×; bar = 10 μm). Gastroenterology 2000 119, 1123-1131DOI: (10.1053/gast.2000.18145) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 3 Confocal images of immunofluorescence localization of introduced hexahistidine-tagged recombinant kinesin to the microtubules network in rat pancreatic acini. (A) Immunofluorescence localization of microtubules stained with anti–α-tubulin antibody. (B) Introduced histidine-tagged recombinant kinesin was stained with antihexahistidine tag antibody. (C) Combination of A and B. Yellow staining indicates the localization of recombinant kinesin to microtubules (original magnification 800×; bar = 10 μm). Gastroenterology 2000 119, 1123-1131DOI: (10.1053/gast.2000.18145) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 4 Effect of nocodazole pretreatment on the recombinant kinesin-enhanced amylase secretion from streptolysin-O–permeabilized acini. Isolated acini were preincubated in the presence or absence of 20 μg/mL nocodazole for 90 minutes. After permeabilization, amylase secretion stimulated with 10 μmol/L calcium for 5 minutes at 30°C in the presence or absence of 4 nmol/L recombinant kinesin was determined. Results are expressed as amylase release as a percentage of total amylase content. Values are the means ± SE for 4 independent experiments, each with duplicate determinations. Statistics were calculated by the Student t test. Gastroenterology 2000 119, 1123-1131DOI: (10.1053/gast.2000.18145) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 5 Effect of antikinesin monoclonal antibody (SUK-4) on calcium-stimulated amylase secretion from streptolysin-O–permeabilized acini. Enzymatically isolated acini were permeabilized with streptolysin-O in the presence or absence of 200 μg/mL antikinesin monoclonal antibody (SUK-4). Amylase release was initiated by adding 10 μmol/L free calcium or a combination of 10 μmol/L free calcium and 100 μmol/L cAMP and incubated for 5 minutes at 30°C. Results are expressed as amylase release as a percentage of total amylase content. Values are the means ± SE for 4 independent experiments, each with duplicate determinations. Statistics were calculated by the Student t test. Gastroenterology 2000 119, 1123-1131DOI: (10.1053/gast.2000.18145) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 6 Immunoprecipitation of endogenous kinesin from rat pancreatic acini. To purify kinesin from pancreatic acinar cells, kinesin was immunoprecipitated from enzymatically isolated pancreatic acini using antikinesin monoclonal antibody (H-2) and visualized by Western blot using the antibody (lane 1). Negative control immunoprecipitation was performed with nonimmune mouse IgG (lane 2). Western blotting of 30 μg of rat brain crude lysate was performed as a positive control for the blots (lane 3). Gastroenterology 2000 119, 1123-1131DOI: (10.1053/gast.2000.18145) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 7 Effect of secretin and CCK on kinesin ATPase activity. Isolated pancreatic acini were stimulated with 1 nmol/L secretin, 100 pmol/L CCK, or a combination of 1 nmol/L secretin and 100 pmol/L CCK. Kinesin was then immunoprecipitated, and its ATPase activity was determined by the method described in Materials and Methods. Control experiments were performed in the same way but in the absence of secretagogues. Values are means ± SE obtained from 3 independent experiments in which ATPase activity was expressed as a percentage of the control. *P < 0.05 vs. control, **NS vs. secretin; Student t test. Gastroenterology 2000 119, 1123-1131DOI: (10.1053/gast.2000.18145) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 8 Effect of 8-bromo-cAMP and A23187 on kinesin ATPase activity. Isolated pancreatic acini were stimulated with 1 mmol/L 8-bromo-cAMP, 1 μmol/L A23187, or the combination of 1 mmol/L 8-bromo-cAMP and 1 μmol/L A23187. Kinesin was then immunoprecipitated, and its ATPase activity was determined by the method described in Materials and Methods. Control experiments were performed in the same way but in the absence of secretagogues. Values are means ± SE obtained from 3 independent experiments in which ATPase activity was expressed as a percentage of the control. *P < 0.05 vs. control, **NS vs. 8-bromo-cAMP; Student t test. Gastroenterology 2000 119, 1123-1131DOI: (10.1053/gast.2000.18145) Copyright © 2000 American Gastroenterological Association Terms and Conditions