Patel Y C, Galanopoulou A S, Rabbani S N, Liu J L, Ravazzola M, Amherdt M
McGill University, Department of Medicine, Royal Victoria Hospital and Montreal Neurological Institute, Quebec, Canada.
Mol Cell Endocrinol. 1997 Aug 8;131(2):183-94. doi: 10.1016/s0303-7207(97)00107-x.
We have characterized the biosynthetic origin of somatostatin-14 (SS-14), SS-28, and pro-SS[1-10] from pro-SS (PSS) in 1027B2 rat islet tumor cells. Because these cells lack regulated secretion and show unresponsiveness of the SS gene to cAMP, we have additionally carried out morphological and functional studies to elucidate the molecular defect in cAMP signalling and to localize the sites of PSS maturation along the secretory pathway. Cell extracts and secretion media were analysed by high performance liquid chromatography and specific C- and N-terminal radioimmunoassays. Electron microscopic sampling of 1027B2 cell cultures showed that most cells had very few dense core secretory granules for heterogeneous sizes. The cells expressed the endoproteases furin, PC1, and PC2 and contained large quantities of fully processed SS-14 and SS-28 with very little unprocessed PSS (ratio SS-14:SS-28:PSS = 39:51:10%). They secreted high concentrations of SS-14, SS-28, and PSS[1-10] constitutively along with PC1 and PC2. Pulse-chase studies demonstrated that PSS is rapidly (within 15 min), and efficiently processed to SS-14, SS-28, and PSS[1-10] via separate biosynthetic pathways: PSS --> SS-14 + 8 kDa; PSS --> SS-28 + 7 kDa; PSS --> PSS[1-10]. Monensin reduced intracellular SS-like immunoreactivity without altering processing efficiency. Transfection with the catalytic subunit of protein kinase A (PKA-C) activated SS promoter-CAT activating indicating that the defect in cAMP-dependent signaling in 1027B2 cells lies at the level of PKA-C. PKA-C overexpression failed to alter the ratio of processed SS-14 and SS-28. These results demonstrate that SS-14, SS-28, and PSS[1-10] are independently synthesized from PSS and that efficient precursor processing can occur within the constitutive secretory pathway in the relative absence of dense core secretory vesicles.
我们已经在1027B2大鼠胰岛肿瘤细胞中确定了生长抑素-14（SS-14）、SS-28和前体SS[1-10]源自前体SS（PSS）的生物合成起源。由于这些细胞缺乏调节性分泌，且SS基因对cAMP无反应，我们还进行了形态学和功能研究，以阐明cAMP信号传导中的分子缺陷，并确定PSS在分泌途径中的成熟位点。通过高效液相色谱以及特异性C端和N端放射免疫测定法对细胞提取物和分泌培养基进行了分析。对1027B2细胞培养物进行电子显微镜取样显示，大多数细胞的致密核心分泌颗粒数量很少，且大小不一。这些细胞表达了内肽酶弗林蛋白酶、PC1和PC2，并且含有大量完全加工的SS-14和SS-28，未加工的PSS很少（SS-14:SS-28:PSS的比例为39:51:10%）。它们持续分泌高浓度的SS-14、SS-28和PSS[1-10]，同时分泌PC1和PC2。脉冲追踪研究表明，PSS通过独立的生物合成途径迅速（在15分钟内）且有效地加工成SS-14、SS-28和PSS[1-10]：PSS→SS-14 + 8 kDa；PSS→SS-28 + 7 kDa；PSS→PSS[1-10]。莫能菌素降低了细胞内SS样免疫反应性，但未改变加工效率。用蛋白激酶A（PKA-C）的催化亚基转染激活了SS启动子-CAT活性，表明1027B2细胞中cAMP依赖性信号传导的缺陷在于PKA-C水平。PKA-C的过表达未能改变加工后的SS-14和SS-28的比例。这些结果表明，SS-14、SS-28和PSS[1-10]是从PSS独立合成的，并且在相对缺乏致密核心分泌小泡的情况下，有效的前体加工可在组成型分泌途径中发生。
