Calegari F, Coco S, Taverna E, Bassetti M, Verderio C, Corradi N, Matteoli M, Rosa P
Consiglio Nazionale delle Ricerche, Center of Cellular and Molecular Pharmacology, Department of Medical Pharmacology, Via Vanvitelli 32, I-20129 Milan, Italy.
J Biol Chem. 1999 Aug 6;274(32):22539-47. doi: 10.1074/jbc.274.32.22539.
Glial cells have been reported to express molecules originally discovered in neuronal and neuroendocrine cells, such as neuropeptides, neuropeptide processing enzymes, and ionic channels. To verify whether astrocytes may have regulated secretory vesicles, the primary cultures prepared from hippocampi of embryonic and neonatal rats were used to investigate the subcellular localization and secretory pathway followed by secretogranin II, a well known marker for dense-core granules. By indirect immunofluorescence, SgII was detected in a large number of cultured hippocampal astrocytes. Immunoreactivity for the granin was detected in the Golgi complex and in a population of dense-core vesicles stored in the cells. Subcellular fractionation experiments revealed that SgII was stored in a vesicle population with a density identical to that of the dense-core secretory granules present in rat pheochromocytoma cells. In line with these data, biochemical results indicated that 40-50% of secretogranin II synthesized during 18-h labeling was retained intracellularly over a 4-h chase period and released after treatment with different secretagogues. The most effective stimulus appeared to be phorbol ester in combination with ionomycin in the presence of extracellular Ca(2+), a treatment that was found to produce a large and sustained increase in intracellular calcium Ca(2+) transients. Our findings indicate that a regulated secretory pathway characterized by (i) the expression and stimulated exocytosis of a typical marker for regulated secretory granules, (ii) the presence of dense-core vesicles, and (iii) the ability to undergo Ca(2+) increase upon specific stimuli is present in cultured hippocampal astrocytes.
据报道，神经胶质细胞可表达最初在神经元和神经内分泌细胞中发现的分子，如神经肽、神经肽加工酶和离子通道。为了验证星形胶质细胞是否可能具有调节性分泌囊泡，我们使用从胚胎和新生大鼠海马制备的原代培养物来研究分泌粒蛋白II（一种众所周知的致密核心颗粒标记物）的亚细胞定位和分泌途径。通过间接免疫荧光，在大量培养的海马星形胶质细胞中检测到了SgII。在高尔基体和细胞中储存的一群致密核心囊泡中检测到了颗粒蛋白的免疫反应性。亚细胞分级分离实验表明，SgII储存在与大鼠嗜铬细胞瘤细胞中存在的致密核心分泌颗粒密度相同的囊泡群体中。与这些数据一致，生化结果表明，在18小时标记期间合成的40-50%的分泌粒蛋白II在4小时的追踪期内保留在细胞内，并在用不同的促分泌剂处理后释放。最有效的刺激似乎是在细胞外Ca(2+)存在的情况下，佛波酯与离子霉素联合使用，这种处理被发现会导致细胞内钙Ca(2+)瞬变大幅持续增加。我们的研究结果表明，培养的海马星形胶质细胞中存在一种调节性分泌途径，其特征为：（i）调节性分泌颗粒典型标记物的表达和刺激后的胞吐作用；（ii）存在致密核心囊泡；（iii）在特定刺激下能够使Ca(2+)增加。
