Drímal J
Institute of Experimental Pharmacology, Slovak Academy of Sciences, Bratislava.
Gen Physiol Biophys. 1992 Dec;11(6):555-65.
Two phenylalkylamine Ca2+ channel ligands, (+/-)-[3H]verapamil ((+/-)-[3H]V) (-)-[3H]desmethoxyverapamil ((-)-[3H]DV), were employed in whole cell binding assays to characterize the specific high affinity binding sites on Ca2+ channels, their cooperativity and modulations induced on cultured human embryonal vascular smooth muscle preparation (VSM) by: 1) Beta-adrenergic stimulation of the cell, 2) exposure to high K+ concentration, 3) exposure to high concentration of Mg2+ ions, 4) the presence of a benzothiazepine Ca2+ channel antagonist and modulator d-cis-diltiazem, and 5) guanylylimidodiphosphate. The total amounts of specific (+/-)-[3H]V and (-)-[3H]DV binding sites present on VSM cells increased significantly after beta-adrenergic receptor activation, following cell membrane depolarization induced by high concentrations of K+, in the presence of Ca2+ chelator Na3EDTA, and after incubation of VSM cells with a benzothiazine-type Ca2+ channel blocker d-cis-diltiazem. A marked reduction of (-)-[3H]DV binding was observed after permanent G-protein activation by a nonhydrolyzable analog of guanylylimidodiphosphate, after incubation of the cells with norepinephrine, and after incubation of VSM cells with millimolar concentration of Mg2+. The results suggest the existence of multiple modulations of specific (-)-[3H]DV binding sites on Ca2+ channel corresponding to the way of activation of the cell and also to the immediate "state" of the membrane bound Ca2+ channels present on VSM cells, the positive heterotropic interaction after beta-adrenergic stimulation, the homotropic positive allosteric interaction induced by d-cis-diltiazem and pure noncompetitive inhibition induced by guanylylimidodiphosphate. The presence of high concentrations of Mg2+ inhibited whereas the presence of Ca2+ chelator, of ethylenediamine-tetraacetic acid sodium salt, significantly increased the total number of specific high affinity (-)-[3H]DV binding sites on VSM cells.
两种苯烷基胺类钙离子通道配体，（±）-[3H]维拉帕米（（±）-[3H]V）和（-）-[3H]去甲氧基维拉帕米（（-）-[3H]DV），被用于全细胞结合试验，以表征钙离子通道上的特异性高亲和力结合位点、它们的协同性以及在培养的人胚胎血管平滑肌制剂（VSM）上由以下因素诱导的调节作用：1）细胞的β-肾上腺素能刺激；2）暴露于高钾浓度；3）暴露于高浓度镁离子；4）存在苯并硫氮䓬类钙离子通道拮抗剂和调节剂d-顺式地尔硫䓬；5）鸟苷酰亚胺二磷酸。在β-肾上腺素能受体激活后、在高浓度钾离子诱导细胞膜去极化后、在存在钙离子螯合剂Na3EDTA的情况下以及在VSM细胞与苯并噻嗪类钙离子通道阻滞剂d-顺式地尔硫䓬孵育后，VSM细胞上存在的特异性（±）-[3H]V和（-）-[3H]DV结合位点的总量显著增加。在用鸟苷酰亚胺二磷酸的非水解类似物永久性激活G蛋白后、在用去甲肾上腺素孵育细胞后以及在用毫摩尔浓度的镁孵育VSM细胞后，观察到（-）-[3H]DV结合显著减少。结果表明，钙离子通道上特异性（-）-[3H]DV结合位点存在多种调节，这与细胞的激活方式以及VSM细胞上存在的膜结合钙离子通道的即时“状态”相对应，β-肾上腺素能刺激后的正性异源性相互作用、d-顺式地尔硫䓬诱导的同型正性变构相互作用以及鸟苷酰亚胺二磷酸诱导的纯非竞争性抑制。高浓度镁的存在起抑制作用，而钙离子螯合剂乙二胺四乙酸钠盐的存在显著增加了VSM细胞上特异性高亲和力（-）-[3H]DV结合位点的总数。
