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

A key event leading to exocytosis of pancreatic acinar cell zymogen gr anules is the inositol 1,4,5-trisphosphate (InsP(3))-mediated release of Ca2+ from intracellular stores. Studies using digital imaging micro scopy and laser-scanning confocal microscopy have indicated that the i nitial 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 InsP(3) is capable of releasing Ca2+ fro m a preparation enriched in secretory granules (Gerasimenko, O., Geras imenko, J., Belan, P., and Petersen, O. H., (1996) Cell 84, 473-480). In the present study, we have investigated the possibility that zymoge n granules express InsP(3) receptors and are thus Ca2+ release sites. Immunofluorescence staining, obtained with antisera specific to types I, II, or III InsP(3) receptors and analyzed by confocal fluorescence microscopy revealed that all InsP(3) receptor types were present in ac inar cells. The type II receptor localized exclusively to an area clos e to or at the luminal plasma membrane. While types I and III InsP(3) receptors displayed a similar luminal distribution, these receptors we re also present at low levels in nuclei. The localization of InsP(3) r eceptor was in marked contrast to the distribution of amylase, a zymog en granule content protein. In a zymogen granule fraction prepared in an identical manner to the aforementioned report demonstrating InsP(3) -induced Ca2+ release, immunoblotting demonstrated the presence of typ es I, II, and III InsP(3) receptors, Ca2+ release from this preparatio n in response to InsP(3), but not thapsigargin, could also be demonstr ated. In contrast, when the zymogen granules were further purified on a Percoll gradient, InsP(3) receptors were undetectable, and InsP(3) f ailed to release Ca2+. Transmission electron microscopy performed on b oth preparations showed that the Percoll-purified granule preparation consisted of essentially pure zymogen granules, whereas the granules p repared 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 InsP(3) receptors and thus are not a site of Ca2+ release relevant to the secr etory process in the pancreatic acinar cell.