Dunn L A, Holz R W
J Biol Chem. 1983 Apr 25;258(8):4989-93.
The plasma membrane of cultured chromaffin cells from bovine adrenal medulla was rendered leaky by incubation in low concentrations of digitonin. Digitonin (20 microM) induced Ca2+-dependent release of 10-20% of the catecholamine in the presence of 10 microM Ca2+ without addition of secretagogue. Half-maximal catecholamine release occurred at approximately 1 microM Ca2+ x Mg2+ could not substitute for Ca2+. Cells incubated with digitonin rapidly lost their ability to exclude trypan blue. Digitonin caused release of the cytosolic markers lactic dehydrogenase and phenylethanolamine-N-methyltransferase, but, in contrast to release of catecholamine, the release of the cytosolic proteins was inhibited by Ca2+. Soluble dopamine-beta-hydroxylase, a protein marker of granule contents, was released proportionally with catecholamine in a Ca2+ dependent manner. Catecholamine release was optimal in solutions containing MgATP. Hence, digitonin-treated cells, although they lose soluble cytosolic proteins and presumably low molecular weight cytosolic constituents, maintain the Ca2+-dependent reactions of exocytosis in the presence of MgATP. Digitonin-treated chromaffin cells may be a powerful system in which to study the biochemical mechanisms underlying exocytosis.
通过在低浓度洋地黄皂苷中孵育，使来自牛肾上腺髓质的培养嗜铬细胞的质膜变得渗漏。在存在10μM Ca²⁺且未添加促分泌剂的情况下，洋地黄皂苷（20μM）诱导了10 - 20%的儿茶酚胺的Ca²⁺依赖性释放。儿茶酚胺释放量达到半数最大值时，Ca²⁺浓度约为1μM，Mg²⁺不能替代Ca²⁺。用洋地黄皂苷孵育的细胞迅速丧失了排斥台盼蓝的能力。洋地黄皂苷导致胞质标记物乳酸脱氢酶和苯乙醇胺 - N - 甲基转移酶的释放，但与儿茶酚胺的释放相反，胞质蛋白的释放受到Ca²⁺的抑制。可溶性多巴胺 - β - 羟化酶是颗粒内容物的一种蛋白质标记物，它以Ca²⁺依赖性方式与儿茶酚胺成比例地释放。在含有MgATP的溶液中，儿茶酚胺释放最为理想。因此，经洋地黄皂苷处理的细胞，尽管它们失去了可溶性胞质蛋白以及可能的低分子量胞质成分，但在MgATP存在的情况下仍维持胞吐作用的Ca²⁺依赖性反应。经洋地黄皂苷处理的嗜铬细胞可能是研究胞吐作用潜在生化机制的一个强大系统。
