Vitale N, Mukai H, Rouot B, Thiersé D, Aunis D, Bader M F
Institut National de la Santé et de la Recherche Médicale U-338 Biologie de la Communication Cellulaire, Strasbourg, France.
J Biol Chem. 1993 Jul 15;268(20):14715-23.
The use of non-hydrolyzable analogues of GTP in permeabilized secretory cells suggests that guanine nucleotide-binding regulatory proteins (G proteins) may be involved in regulated exocytosis. Because GTP analogues are known to modulate both monomeric low molecular mass G proteins and heterotrimeric G proteins, we have examined the effect of mastoparan, an activator of heterotrimeric G proteins, on secretion from intact and permeabilized chromaffin cells. In intact cells, mastoparan inhibited catecholamine secretion evoked by nicotine but had no effect on release induced by other secretagogues. In permeabilized cells, mastoparan inhibited calcium-dependent secretion providing that the pores created in the plasma membrane allow the penetration of the peptide into the cytoplasm. These results indicate that mastoparan blocks the exocytotic machinery through an intracellular target protein that may not be located just beneath the plasma membrane. Accordingly, mastoparan was able to stimulate G proteins associated with purified chromaffin granule membranes, in a range of concentration and Mg2+ requirement that was similar to its inhibitory effect on secretion. Mas 17, a mastoparan analogue inactive on purified G proteins, neither modified catecholamine secretion nor stimulated chromaffin granule G proteins. The substance P-related peptide, GPAnt-2, known to antagonize the effects of mastoparan on G(o), blocked both the inhibitory effect of mastoparan on secretion and the mastoparan-stimulated GTPase activity in chromaffin granule membranes. Moreover, specific antibodies raised against the carboxyl terminus of G(o) alpha reversed in a dose-dependent manner the inhibition by mastoparan on catecholamine release and the stimulation by mastoparan of chromaffin granule-associated G proteins. These results suggest that the secretory machinery in chromaffin cells can be blocked by activating a G(o) protein. Consistent with this finding, two other known activators of heterotrimeric G proteins, aluminum fluoride and benzalkonium chloride, inhibited calcium-evoked catecholamine secretion in streptolysin O-permeabilized chromaffin cells. We conclude that an inhibitory G(o) protein, possibly located on the membrane of secretory granules, is involved in the final stages of exocytosis in chromaffin cells.
在通透化的分泌细胞中使用不可水解的GTP类似物表明，鸟嘌呤核苷酸结合调节蛋白（G蛋白）可能参与调节性胞吐作用。由于已知GTP类似物可调节单体低分子量G蛋白和异源三聚体G蛋白，我们研究了异源三聚体G蛋白激活剂马斯托帕罗对完整和通透化嗜铬细胞分泌的影响。在完整细胞中，马斯托帕罗抑制尼古丁诱发的儿茶酚胺分泌，但对其他促分泌剂诱导的释放没有影响。在通透化细胞中，只要质膜上形成的孔允许该肽进入细胞质，马斯托帕罗就会抑制钙依赖性分泌。这些结果表明，马斯托帕罗通过一种可能不在质膜正下方的细胞内靶蛋白阻断胞吐机制。因此，马斯托帕罗能够在一系列浓度和镁离子需求范围内刺激与纯化的嗜铬颗粒膜相关的G蛋白，这与其对分泌的抑制作用相似。Mas 17是一种对纯化的G蛋白无活性的马斯托帕罗类似物，既不改变儿茶酚胺分泌，也不刺激嗜铬颗粒G蛋白。已知能拮抗马斯托帕罗对G(o)作用的P物质相关肽GPAnt-2，阻断了马斯托帕罗对分泌的抑制作用以及马斯托帕罗刺激的嗜铬颗粒膜中的GTP酶活性。此外，针对G(o)α羧基末端产生的特异性抗体以剂量依赖性方式逆转了马斯托帕罗对儿茶酚胺释放的抑制作用以及马斯托帕罗对嗜铬颗粒相关G蛋白的刺激作用。这些结果表明，嗜铬细胞中的分泌机制可通过激活G(o)蛋白来阻断。与此发现一致，另外两种已知的异源三聚体G蛋白激活剂氟化铝和苯扎氯铵，抑制了链球菌溶血素O通透化的嗜铬细胞中钙诱发的儿茶酚胺分泌。我们得出结论，一种可能位于分泌颗粒膜上的抑制性G(o)蛋白参与了嗜铬细胞胞吐作用的最后阶段。
