Lara B, Zapater P, Montiel C, de la Fuente M T, Martínez-Sierra R, Ballesta J J, Gandía L, García A G
Departamento de Farmacología, Facultad de Medicina, Universidad Autónoma de Madrid, Spain.
Biochem Pharmacol. 1995 May 17;49(10):1459-68. doi: 10.1016/0006-2952(94)00524-p.
Three objectives were defined when planning this study: (i) to identify binding sites for [125I]-apamin in intact bovine adrenal medulla chromaffin cells and to estimate their density and selectivity; (ii) to determine whether apamin modified the release of catecholamines evoked by brief pulses of dimethylphenylpiperazinium (DMPP, 1 or 5 microM for 10 sec), histamine (10 microM for 10 sec) or high K+ (20, 35 or 70 mM for 10 sec) applied to superfused cells; and (iii) to test whether apamin affected the profiles of the changes in cytosolic Ca2+ concentrations [Ca2+]i obtained in suspensions of cells loaded with fura-2 and stimulated with DMPP or histamine. At equilibrium, increasing concentrations of [125I]-apamin gave a saturation curve whose Scatchard transformation produced a Kd of 132 pM and a Bmax of 0.72 fmol/10(6) cells. Quinine, tetraethylammonium, charybdotoxin or glibenclamide (blockers of various subtypes of K+ channels) did not inhibit [125I]apamin binding. Binding was blocked by apamin and by d-tubocurarine, two blockers of small-conductance Ca(2+)-activated K+ channels (SK channels). The number of binding sites for [125I]apamin amounted to approx. 900 per single chromaffin cell, 0.72 sites per micron 2 surface area. Apamin (1 microM) enhanced the secretory response to histamine (10 microM), DMPP (1 or 5 microM) and high K+ (20 or 35 mM) by 2-3-fold. The response to 70 mM K+, however, was unaffected. Apamin also enhanced the peak [Ca2+]i increase produced by DMPP or histamine by approx. 30%. Overall, these results strongly support the hypothesis that under physiological conditions, SK channels control some of the electrical activity of chromaffin cells and indirectly, the opening of voltage-dependent Ca2+ channels, the access of Ca2+ to the secretory machinery and the rate of catecholamine release to the circulation from the intact adrenal gland.
(i) 确定[125I] - 蜂毒明肽在完整牛肾上腺髓质嗜铬细胞中的结合位点，并估计其密度和选择性；(ii) 确定蜂毒明肽是否改变由短暂脉冲施加于灌流细胞的二甲基苯基哌嗪鎓（DMPP，1或5 microM，持续10秒）、组胺（10 microM，持续10秒）或高钾（20、35或70 mM，持续10秒）所诱发的儿茶酚胺释放；以及(iii) 测试蜂毒明肽是否影响在用fura - 2加载并由DMPP或组胺刺激的细胞悬液中获得的胞质Ca2 +浓度[Ca2 +]i变化曲线。在平衡状态下，[125I] - 蜂毒明肽浓度增加产生一条饱和曲线，其Scatchard转换得出Kd为132 pM，Bmax为0.72 fmol / 10(6)个细胞。奎宁、四乙铵、蝎毒素或格列本脲（各种亚型K +通道的阻滞剂）不抑制[1(25I]蜂毒明肽结合。结合被蜂毒明肽和d - 筒箭毒碱阻断，这两种是小电导Ca(2 +)激活K +通道（SK通道）的阻滞剂。[125I]蜂毒明肽的结合位点数约为每个嗜铬细胞900个，每平方微米表面积0.72个位点。蜂毒明肽（1 microM）使对组胺（10 microM）、DMPP（1或5 microM）和高钾（20或35 mM）的分泌反应增强2 - 3倍。然而，对70 mM K +的反应未受影响。蜂毒明肽还使由DMPP或组胺产生 的[Ca2 +]i峰值增加约30%。总体而言，这些结果有力地支持了以下假设：在生理条件下，SK通道控制嗜铬细胞的一些电活动，并间接控制电压依赖性Ca2 +通道的开放、Ca2 +进入分泌机制以及儿茶酚胺从完整肾上腺释放到循环中的速率。
