[3H]Verapamil binding sites in brain and skeletal muscle: regulation by calcium.
[3H]Verapamil binding sites in brain and skeletal muscle: regulation by calcium.
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[3H]维拉帕米在大脑和骨骼肌中的结合位点：钙的调节。
DOI：
10.1016/0014-2999(83)90655-6
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发表时间：
1983
影响因子：
5
通讯作者：
Snyder,SH
中科院分区：
文献类型：
--
作者：
Reynolds,IJ;Gould,RJ;Snyder,SH
几种不同种类的钙通道拮抗剂药物。二氢吡啶类钙通道拮抗剂的结合位点可用[3 H]尼群地平标记。虽然维拉帕米和地尔硫卓类药物不作用于二氢吡啶位点，但这些药物影响[3 H]尼群地平在与尼群地平位点不同的共同位点的结合（Murphy等人，1983年）。为了直接研究维拉帕米样药物作用的部位，我们探索了[3 H]维拉帕米与组织膜的结合。将大鼠大脑皮质或骨骼肌以10-20体积均质化。50 mM HEPES（N-2-羟乙基哌嗪-N '-2-乙磺酸），pH 7.4，含有1-10 mM EDTA（乙二胺四乙酸）。以50000× g离心10 min后，将沉淀物重悬于EDTA缓冲液中。重复该离心两次后，在不存在EDTA的情况下将膜重悬至5-10 mg/ml湿重的浓度。在室温下孵育45分钟，体积为0.5-1 ml，含有3-5 mg组织和2 nM [3 H]维拉帕米。在3~ M甲氧基维拉帕米（D-600）存在下评估非特异性结合。通过预先浸泡在0.1%聚乙烯亚胺中的Schleicher和Schuell No.32玻璃纤维过滤器快速过滤终止孵育。用5-10 ml冰冷的50 mM HEPES（pH 7.4）洗涤膜，并测量过滤器保留的放射性。药物来源为：获得[3 H]维拉帕米（75 C1/mmol
Several different classes of calcium channel antagonist drugs ernst. Binding sites for the dihydropyridine calcium channel antagonists can be labeled with [3H] nitrendipine. While drugs of the verapamil and diltiazem classes do not act at the dlhydropyridine sites, these drugs influence [3H] nltrendipine binding at a common site, distinct from the nitrendipine site (Murphy et al., 1983). In an effort to study directly the sites where verapamil-like drugs act, we have explored the binding of [3H] verapamil to tissue membranes. Rat cerebral cortices or skeletal muscle were homogenized in 10-20 vols. of 50 mM HEPES (N-2-hydroxyethyl piperazine-N'-2-ethanesulfonxc acid), pH 7.4, containing 1-10 mM EDTA (ethylenedlamine-tetraacetlc acid). After centrifugtng at 50000× g for 10 nun the pellet was resuspended in EDTA buffer. After repeating this centrifugation two times, the membranes were resuspended in the absence of EDTA to a concentration of 5-10 mg/ml wet weight. Incubations were carried out at room temperature for 45 min in a volume of 0.5-1 ml, containing 3-5 mg tissue and 2 nM [3H] verapamil. Nonspecific binding was assessed in the presence of 3~ M methoxyverapamil (D-600). Incubations were stopped by rapid filtration over Schleicher and Schuell No. 32 glass fiber filters previously soaked in 0.1% polyethyleneimine. Membranes were washed with 5-10 ml ice-cold 50 mM HEPES (pH 7.4) and radioactivity retained by the filters was measured. Drug sources were:[3H] verapamil (75 C1/mmol) was obtained