Yule D I, Ernst S A, Ohnishi H, Wojcikiewicz R J
Department of Physiology, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA.
J Biol Chem. 1997 Apr 4;272(14):9093-8. 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.
导致胰腺腺泡细胞酶原颗粒胞吐作用的一个关键事件是肌醇1,4,5 -三磷酸（InsP3）介导的细胞内钙库中Ca2+的释放。使用数字成像显微镜和激光扫描共聚焦显微镜进行的研究表明，Ca2+的初始释放定位于腺泡细胞的顶端区域，该区域是细胞中以分泌颗粒为主的区域。此外，最近的一项研究表明，InsP3能够从富含分泌颗粒的制剂中释放Ca2+（格拉西缅科，O.，格拉西缅科，J.，贝兰，P.，和彼得森，O. H.，（1996年）《细胞》84卷，473 - 480页）。在本研究中，我们研究了酶原颗粒表达InsP3受体并因此成为Ca2+释放位点的可能性。用针对I型、II型或III型InsP3受体的抗血清进行免疫荧光染色，并通过共聚焦荧光显微镜分析，结果显示所有类型的InsP3受体都存在于腺泡细胞中。II型受体仅定位于靠近或位于管腔质膜处的区域。虽然I型和III型InsP3受体显示出相似的管腔分布，但这些受体在细胞核中也有少量存在。InsP3受体的定位与淀粉酶（一种酶原颗粒内容蛋白）的分布形成了显著对比。在以与上述证明InsP3诱导Ca2+释放的报告相同的方式制备的酶原颗粒组分中，免疫印迹显示存在I型、II型和III型InsP3受体。也可以证明该制剂对InsP3而非毒胡萝卜素产生Ca2+释放反应。相比之下，当酶原颗粒在Percoll梯度上进一步纯化时，未检测到InsP3受体，并且InsP3未能释放Ca2+。对这两种制剂进行的透射电子显微镜检查表明，经Percoll纯化的颗粒制剂基本上由纯酶原颗粒组成，而未经过此步骤制备的颗粒富含颗粒，但也含有大量线粒体、内质网和细胞核的污染。得出的结论是，酶原颗粒不表达InsP3受体，因此不是胰腺腺泡细胞分泌过程中相关的Ca2+释放位点。
