Supramaximal cholecystokinin displaces Munc18c from the pancreatic acinar basal surface, redirecting apical exocytosis to the basal membrane
Herbert Y. Gaisano, Manfred P. Lutz, Juergen Leser, Laura Sheu, Grit Lynch, Lan Tang, Yoshikazu Tamori, William S. Trimble, Anne Marie F. Salapatek
Herbert Y. Gaisano, Manfred P. Lutz, Juergen Leser, Laura Sheu, Grit Lynch, Lan Tang, Yoshikazu Tamori, William S. Trimble, Anne Marie F. Salapatek
View: Text | PDF
Article

Supramaximal cholecystokinin displaces Munc18c from the pancreatic acinar basal surface, redirecting apical exocytosis to the basal membrane

Abstract

Exocytosis at the apical surface of pancreatic acinar cells occurs in the presence of physiological concentrations of cholecystokinin (CCK) but is inhibited at high concentrations. Here we show that Munc18c is localized predominantly to the basal membranes of acinar cells. Supramaximal but not submaximal CCK stimulation caused Munc18c to dissociate from the plasma membrane, and this displacement was blocked by protein kinase C (PKC) inhibitors. Conversely, whereas the CCK analog CCK-OPE alone failed to displace Munc18c from the membrane, this agent caused Munc18c displacement following minimal PKC activation. To determine the physiological significance of this displacement, we used the fluorescent dye FM1-43 to visualize individual exocytosis events in real-time from rat acinar cells in culture. We showed that supramaximal CCK inhibition of secretion resulted from impaired apical secretion and a redirection of exocytic events to restricted basal membrane sites. In contrast, CCK-OPE evoked apical exocytosis and could only induce basolateral exocytosis following activation of PKC. Infusion of supraphysiological concentrations of CCK in rats, a treatment that induced tissue changes reminiscent of mild acute pancreatitis, likewise resulted in rapid displacement of Munc18c from the basal membrane in vivo.

Authors

Herbert Y. Gaisano, Manfred P. Lutz, Juergen Leser, Laura Sheu, Grit Lynch, Lan Tang, Yoshikazu Tamori, William S. Trimble, Anne Marie F. Salapatek

×

Figure 2

Options: View larger image (or click on image) Download as PowerPoint
Effects of secretagogues on the cellular localization of Munc18c in disp...
Effects of secretagogues on the cellular localization of Munc18c in dispersed rat pancreatic acini. The dispersed acini were stimulated by the following protocols and were then all labeled with anti-Munc18c antibody except for h and i, which were labeled with anti–syntaxin-4 and anti–SNAP-23 antibodies as indicated in the figure. The cellular locations of these proteins were then visualized by confocal microscopy as described in Methods. (a) Submaximal CCK-8 (30 pM, 1 hour); (b, h, and i) supramaximal CCK-8 (10 nM, 15–30 minutes); (c) supramaximal CCK-OPE (1 μM, 1 hour); (d) 1 nM TPA (30 minutes); (e) 1 μM TPA (30 minutes); (f) 1 nM TPA (15 minutes) + 1 μM CCK-OPE (30 minutes); (g) calphostin C (500 nM, 40 minutes) + 10 nM CCK-8 (30 minutes). (j) Acini were stimulated with 10 nM CCK-8 at the indicated times and then fractionated into plasma membrane (PM) and cytosol (Cyt) fractions (see Methods). Thirty micrograms protein of the plasma membrane fractions and 40 μg of the cytosol fractions were separated on SDS-PAGE and immunoblotted with anti-Munc18c and –syntaxin-4 antibodies. This is a representative of three independent experiments.