Tachikawa E, Takahashi S, Furumachi K, Kashimoto T, Iida A, Nagaoka Y, Fujita T, Takaishi Y
Department of Pharmacology, School of Medicine, Iwate Medical University, Japan.
Mol Pharmacol. 1991 Nov;40(5):790-7.
We examined the effect of trichosporin-B-III, an alpha-aminoisobutyric acid-containing antibiotic peptide consisting of 19 amino acid residues and a phenylalaninol, on catecholamine secretion from cultured bovine adrenal chromaffin cells. Incubation of the cells with trichosporin-B-III (3-20 microM) caused an increase in the secretion of catecholamines. The secretion induced by trichosporin-B-III at low concentrations (3 and 5 microM) was completely dependent on external Ca2+, whereas that induced by higher concentrations (10 and 20 microM) was partly independent of Ca2+. Trichosporin-B-III at low concentration (5 microM) did not increase the release of lactate dehydrogenase, a marker enzyme of cytoplasm, from the cells. In contrast, the peptide at higher concentration (10 microM) increased the release of the enzyme. Trichosporin-B-III also caused both 45Ca2+ influx into the cells and an increase in the intracellular free Ca2+ concentration. The increases in catecholamine secretion and 45Ca2+ influx behaved similarly in relation to trichosporin-B-III concentration (3-10 microM). The time courses of the increases in secretion, 45Ca2+ influx, and intracellular free Ca2+ concentration induced by trichosporin-B-III were also quite similar. Trichosporin-B-III-induced (at 5 microM) secretion was not affected by the elimination of Na+ from the incubation medium or by the addition of tetrodotoxin, a blocker of highly selective voltage-dependent Na+ channels, or hexamethonium, a blocker of nicotinic acetylcholine receptors. On the other hand, both diltiazem (2-200 microM) and nicardipine (1-200 microM), blockers of voltage-dependent Ca2+ channels, inhibited the secretion induced by trichosporin-B-III (5 microM) in a concentration-dependent manner. Trichosporin-B-III-induced (at 5 microM) secretion also was suppressed by the addition of Mn2+ (5 mM) to the medium. The diltiazem (20 microM) inhibition of trichosporin-B-III-induced (at 5 microM) secretion was reversed by increasing the external Ca2+ concentration. These results indicate that trichosporin-B-III causes the secretion of catecholamines from bovine adrenal chromaffin cells by two mechanisms, Ca2+ dependent and Ca2+ independent (only at high concentrations of trichosporin-B-III). Furthermore, these results strongly suggest that trichosporin-B-III, in Ca(2+)-dependent secretion, activates endogenous voltage-dependent Ca2+ channels, or itself forms the channels in the membranes, and induces Ca2+ influx into the cells.
我们研究了曲古抑菌素 -B-III（一种由19个氨基酸残基和一个苯丙氨醇组成的含α-氨基异丁酸的抗生素肽）对培养的牛肾上腺嗜铬细胞儿茶酚胺分泌的影响。用曲古抑菌素 -B-III（3 - 20微摩尔）孵育细胞会导致儿茶酚胺分泌增加。低浓度（3和5微摩尔）的曲古抑菌素 -B-III诱导的分泌完全依赖于细胞外Ca2+，而高浓度（10和20微摩尔）诱导的分泌部分不依赖于Ca2+。低浓度（5微摩尔）的曲古抑菌素 -B-III不会增加细胞质标记酶乳酸脱氢酶从细胞中的释放。相反，高浓度（10微摩尔）的该肽会增加该酶的释放。曲古抑菌素 -B-III还会导致45Ca2+流入细胞并使细胞内游离Ca2+浓度增加。儿茶酚胺分泌和45Ca2+流入的增加与曲古抑菌素 -B-III浓度（3 - 10微摩尔）的关系相似。曲古抑菌素 -B-III诱导的分泌、45Ca2+流入和细胞内游离Ca2+浓度增加的时间进程也非常相似。曲古抑菌素 -B-III（5微摩尔）诱导的分泌不受孵育培养基中Na+去除的影响，也不受添加高选择性电压依赖性Na+通道阻滞剂河豚毒素或烟碱型乙酰胆碱受体阻滞剂六甲铵的影响。另一方面，电压依赖性Ca2+通道阻滞剂地尔硫卓（2 - 200微摩尔）和尼卡地平（1 - 200微摩尔）均以浓度依赖性方式抑制曲古抑菌素 -B-III（5微摩尔）诱导的分泌。向培养基中添加Mn2+（5毫摩尔）也会抑制曲古抑菌素 -B-III（5微摩尔）诱导的分泌。通过增加细胞外Ca2+浓度可逆转地尔硫卓（20微摩尔）对曲古抑菌素 -B-III（5微摩尔）诱导分泌的抑制作用。这些结果表明，曲古抑菌素 -B-III通过两种机制导致牛肾上腺嗜铬细胞分泌儿茶酚胺，即Ca2+依赖性机制和Ca2+非依赖性机制（仅在高浓度的曲古抑菌素 -B-III时）。此外，这些结果强烈表明，在Ca(2+)依赖性分泌中，曲古抑菌素 -B-III激活内源性电压依赖性Ca2+通道，或其自身在膜中形成通道，并诱导Ca2+流入细胞。
