Thomas Diana D H, Weng Ning, Groblewski Guy E
Department of Nutritional Sciences, University of Wisconsin, 1415 Linden Drive, Madison, WI 53706, USA.
Am J Physiol Gastrointest Liver Physiol. 2004 Jul;287(1):G253-63. doi: 10.1152/ajpgi.00033.2004. Epub 2004 Feb 19.
Ca(2+)-regulated heat-stable protein (CRHSP-28) is a member of the TPD52 protein family that has been shown to regulate Ca(2+)-dependent secretory activity in pancreatic acinar cells. Immunofluorescence microscopy of isolated lobules demonstrated that CRHSP-28 is localized to a supranuclear apical compartment in acini and accumulates immediately below the apical membrane within 2 min of CCK octapeptide (CCK-8) stimulation. Dual-immunofluorescence microscopy demonstrated an endosomal localization of CRHSP-28 that strongly overlapped with early endosomal antigen-1 (EEA-1) on vesicular structures throughout the apical cytoplasm but showed only minimal overlap with the transferrin receptor, which is present in basolaterally derived endosomes. Significant overlapping of CRHSP-28 with the trans-Golgi network marker-38 was also noted in supranuclear regions of acini. Interestingly, treatment of lobules with brefeldin A reversibly disrupted the vesicular localization of CRHSP-28 and EEA-1 within the apical cytoplasm. The CCK-8-induced accumulation of CRHSP-28 in subapical regions of acini was not altered by inhibition of apical endocytosis with the actin filament-disrupting agent latrunculin B. Immunoelectron microscopy confirmed that CRHSP-28 is associated with the limiting membrane of irregularly shaped vesicular structures of low electron density in the apical cytoplasm that are positive for EEA-1 staining. Sparse, but significant, CRHSP-28 immunoreactivity was also observed along the limiting membrane of zymogen granules. Consistent with immunofluorescence data, CRHSP-28 was found to accumulate in clusters on endosomes and positioned between zymogen granules below the cell apex on CCK-8 stimulation. These data indicate that CRHSP-28 is present within endocytic and exocytic compartments of acinar cells and is acutely regulated by secretagogue stimulation.
钙调节热稳定蛋白（CRHSP-28）是TPD52蛋白家族的成员，已被证明可调节胰腺腺泡细胞中钙依赖性分泌活性。对分离的小叶进行免疫荧光显微镜检查显示，CRHSP-28定位于腺泡中核上的顶端区室，并在八肽胆囊收缩素（CCK-8）刺激后2分钟内立即聚集在顶端膜下方。双重免疫荧光显微镜检查显示，CRHSP-28在内体中定位，在整个顶端细胞质的囊泡结构上与早期内体抗原-1（EEA-1）强烈重叠，但与存在于基底外侧来源内体中的转铁蛋白受体仅有最小程度的重叠。在腺泡的核上区域也注意到CRHSP-28与反式高尔基体网络标记物-38有明显重叠。有趣的是，用布雷菲德菌素A处理小叶会可逆地破坏顶端细胞质中CRHSP-28和EEA-1的囊泡定位。用肌动蛋白丝破坏剂拉曲霉素B抑制顶端内吞作用不会改变CCK-8诱导的CRHSP-28在腺泡顶端区域的积累。免疫电子显微镜证实，CRHSP-28与顶端细胞质中低电子密度的不规则形状囊泡结构的限制膜相关，这些结构对EEA-1染色呈阳性。在酶原颗粒的限制膜上也观察到稀疏但明显的CRHSP-28免疫反应性。与免疫荧光数据一致，发现CRHSP-28在CCK-8刺激下在内体上成簇积累，并位于细胞顶端下方的酶原颗粒之间。这些数据表明，CRHSP-28存在于腺泡细胞的内吞和外排区室中，并受促分泌剂刺激的急性调节。
