Evidence that zymogen granules are not a physiologically relevant calcium pool. Defining the distribution of inositol 1,4,5-trisphosphate receptors in pancreatic acinar cells

• Published in: The Journal of biological chemistry Vol. 272; no. 14; pp. 9093 - 9098
• Main Authors: Yule, D I, Ernst, S A, Ohnishi, H, Wojcikiewicz, R J
• Format: Journal Article
• Language: English
• Published: United States 04.04.1997
• Subjects: Animals; Calcium - metabolism; Calcium Channels - metabolism; Centrifugation; Cytoplasmic Granules - metabolism; Enzyme Precursors - metabolism; Fluorescent Antibody Technique, Indirect; Inositol 1,4,5-Trisphosphate Receptors; Microscopy, Electron; Pancreas - metabolism; Pancreas - ultrastructure; Rabbits; Receptors, Cytoplasmic and Nuclear - metabolism
• ISSN: 0021-9258
• DOI: 10.1074/jbc.272.14.9093

A key event leading to exocytosis of pancreatic acinar cell zymogen granules is the inositol 1,4,5-trisphosphate (InsP3)-mediated release of Ca2+ from intracellular stores. Studies using digital imaging microscopy and laser-scanning confocal microscopy have indicated that the initial release of Ca2+ is localized to the apical region of the acinar cell, an area of the cell dominated by secretory granules. Moreover, a recent study has shown that InsP3 is capable of releasing Ca2+ from a preparation enriched in secretory granules (Gerasimenko, O., Gerasimenko, J., Belan, P., and Petersen, O. H., (1996) Cell 84, 473-480). In the present study, we have investigated the possibility that zymogen granules express InsP3 receptors and are thus Ca2+ release sites. Immunofluorescence staining, obtained with antisera specific to types I, II, or III InsP3 receptors and analyzed by confocal fluorescence microscopy revealed that all InsP3 receptor types were present in acinar cells. The type II receptor localized exclusively to an area close to or at the luminal plasma membrane. While types I and III InsP3 receptors displayed a similar luminal distribution, these receptors were also present at low levels in nuclei. The localization of InsP3 receptor was in marked contrast to the distribution of amylase, a zymogen granule content protein. In a zymogen granule fraction prepared in an identical manner to the aforementioned report demonstrating InsP3-induced Ca2+ release, immunoblotting demonstrated the presence of types I, II, and III InsP3 receptors. Ca2+ release from this preparation in response to InsP3, but not thapsigargin, could also be demonstrated. In contrast, when the zymogen granules were further purified on a Percoll gradient, InsP3 receptors were undetectable, and InsP3 failed to release Ca2+. Transmission electron microscopy performed on both preparations showed that the Percoll-purified granule preparation consisted of essentially pure zymogen granules, whereas the granules prepared without this step were enriched in granules but also contained significant contamination by mitochondria, endoplasmic reticulum, and nuclei. It is concluded that zymogen granules do not express InsP3 receptors and thus are not a site of Ca2+ release relevant to the secretory process in the pancreatic acinar cell.