Sontag J M, Aunis D, Bader M F
Centre de Neurochimie du CNRS, Unité INSERM U44, Strasbourg/France.
Eur J Cell Biol. 1988 Jun;46(2):316-26.
Adrenal medullary chromaffin cells were permeabilized by treatment with a streptococcal cytotoxin streptolysin O (SLO) which generates pores of macromolecular dimensions in the plasma membrane. SLO did not provoke spontaneous release of catecholamines or chromogranin A, a protein marker of the secretory granule, showing the integrity of the secretory vesicle membrane. However, the addition of micromolar free calcium concentration induced the corelease of noradrenaline and chromogranin A, indicating that secretory products are liberated by exocytosis. Calcium-dependent exocytosis from SLO-permeabilized cells required Mg-ATP and could not occur in the presence of other nucleotides. The pores generated by the toxin were large enough to introduce proteins, e.g., immunoglobulins, but also caused efflux of the cytosolic marker lactate dehydrogenase. Despite this, the cells remained responsive to calcium for up to 30 min after permeabilization, indicating that they retained their secretory machinery. In the search for a functional role of cytoskeletal proteins in the secretory process, we used SLO-permeabilized cells to examine the localization of filamentous actin, using rhodamine-phalloidin, and that of the actin-severing protein, gelsolin, using specific antibodies. It was found that both F-actin and gelsolin were exclusively localized in the subplasmalemmal region of the cell. We examined the relationship between actin disassembly, the elevation of intracellular calcium and secretion in SLO-treated cells. F-Actin destabilizing agents such as cytochalasin D or DNase I were found to potentiate calcium-stimulated release. The maximal effect was observed at low calcium concentrations (1-4 microM) and at the later stages of the secretory response (after 10 min stimulation). In addition, using rhodamine-phalloidin, we observed that calcium provoked simultaneously both cortical actin disassembly and catecholamine release in SLO-permeabilized cells. These results demonstrate that a close relationship exists between the secretory response and actin disassembly and provide further evidence that intracellular calcium controls the subplasmalemmal cytoskeletal actin organization and thereby the access of secretory granules to exocytotic sites.
用链球菌细胞毒素链球菌溶血素O（SLO）处理使肾上腺髓质嗜铬细胞通透化，该毒素在质膜上形成大分子尺寸的孔。SLO不会引发儿茶酚胺或嗜铬粒蛋白A（分泌颗粒的一种蛋白质标志物）的自发释放，表明分泌囊泡膜的完整性。然而，添加微摩尔浓度的游离钙会诱导去甲肾上腺素和嗜铬粒蛋白A的共同释放，表示分泌产物通过胞吐作用释放。SLO通透化细胞的钙依赖性胞吐作用需要Mg-ATP，且在存在其他核苷酸的情况下不会发生。毒素产生的孔足够大，可以引入蛋白质，如免疫球蛋白，但也会导致胞质标志物乳酸脱氢酶外流。尽管如此，细胞在通透化后长达30分钟内仍对钙有反应，表明它们保留了其分泌机制。在寻找细胞骨架蛋白在分泌过程中的功能作用时，我们使用SLO通透化细胞，用罗丹明-鬼笔环肽检测丝状肌动蛋白的定位，并用特异性抗体检测肌动蛋白切割蛋白凝溶胶蛋白的定位。发现F-肌动蛋白和凝溶胶蛋白都仅定位在细胞的质膜下区域。我们研究了SLO处理的细胞中肌动蛋白解聚、细胞内钙升高与分泌之间的关系。发现诸如细胞松弛素D或DNase I等F-肌动蛋白不稳定剂可增强钙刺激的释放。在低钙浓度（1 - 4 microM）和分泌反应的后期阶段（刺激10分钟后）观察到最大效应。此外，使用罗丹明-鬼笔环肽，我们观察到钙在SLO通透化细胞中同时引发皮质肌动蛋白解聚和儿茶酚胺释放。这些结果表明分泌反应与肌动蛋白解聚之间存在密切关系，并进一步证明细胞内钙控制质膜下细胞骨架肌动蛋白的组织，从而控制分泌颗粒到达胞吐位点。
