Amylase release from streptolysin O-permeabilized pancreatic acini.
Intracellular mediators of exocytosis were investigated using isolated mouse pancreatic acini permeabilized with the bacterial toxin streptolysin O (SLO). Permeabilization was demonstrated by fluorescent staining with ethidium bromide and fluorescein diacetate and release of cytoplasmic lactate dehydrogenase. When SLO-permeabilized acini were incubated at 37 degrees C in Ca2(+)-EGTA buffers containing MgATP, amylase secretion was Ca2+ dependent with an EC50 of 0.40 microM Ca2+ and a maximally effective Ca2+ concentration of 1 microM. Maximal amylase secretion was 330% of that in Ca2(+)-free buffer (basal). The nonhydrolyzable GTP analogue guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S; 30 microM) increased the maximal secretion to 451% of basal in the presence of 1 microM Ca2+ and decreased the EC50 to 0.14 microM Ca2+. Removal of ATP plus addition of antimycin A and 2-deoxy-D-glucose inhibited Ca2(+)-dependent, GTP gamma S-enhanced amylase secretion by 56%. The phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA; 1 microM) also enhanced maximal secretion to 450% of basal and decreased the EC50 to 0.18 microM Ca2+. Enhancement of amylase secretion by submaximal concentrations of GTP gamma S or TPA was inhibited by the protein kinase C inhibitor staurosporine. These results suggest that Ca2+ stimulation of amylase secretion is potentiated by activation of protein kinase C. However, the enhancement of secretion by GTP gamma S and TPA was additive at their maximally effective concentrations, suggesting that another G protein(s) maybe involved in the terminal steps of exocytosis.
Department of Physiology, University of Michigan, Ann Arbor 48109-0622.
M
M
Kitagawa
J A
JA
Williams
R C
RC
De Lisle
D000470:Alkaloids / Q000494:pharmacology; D000681:Amylases / Q000378:metabolism*; D000818:Animals; D001426:Bacterial Proteins; D002118:Calcium / Q000494:pharmacology; D002463:Cell Membrane Permeability; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D066298:In Vitro Techniques; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008297:Male; D051379:Mice; D010179:Pancreas / Q000187:drug effects / Q000201:enzymology*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D019311:Staurosporine; D013301:Streptolysins / Q000494:pharmacology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013873:Thionucleotides / Q000494:pharmacology
D000470:Alkaloids; D001426:Bacterial Proteins; D013301:Streptolysins; D013873:Thionucleotides; C025964:streptolysin O; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D007770:L-Lactate Dehydrogenase; D011493:Protein Kinase C; D000681:Amylases; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium