Characterization of calcium antagonist receptors in coronary artery

为了鉴定和表征1,4-二氢吡啶（DEP） Ca^<++>通道拮抗剂在冠状动脉中的受体位点，我们用放射线受体法测定了[^3H]尼群地平（NTD）和(+)-[^3H]PN200-110（PN）在猪冠状动脉（PCA）粗膜中的特异性结合。[^3H]NTD和[^3H]PN在PCA中的特异性结合是饱和、可逆和高亲和力的，具有药理特异性和立体选择性，表征了DHP Ca^<++>拮抗剂的受体位点。DHP拮抗剂（0.1+nM-1 muM）竞争两种配体的结合顺序为：（+）PN>美吡咯地平>尼索地平>尼卡地平>尼替地平>尼莫地平>硝苯地平>(-)PN。（+）PN和美吡咯地平对[^3H]PN结合位点的亲和力是硝苯地平的10 ~ 20倍。（+）PN的效力大约是（-）同分异构体的140倍。8种DHP Ca^<++>拮抗剂在PCA中竞争配体结合位点的效价（pki）与其药理效价（pA_2）具有良好的相关性。d-顺式地尔硫卓可增强PCA中特异性[^3H]PN结合，维拉帕米和D-600可不完全抑制其结合。EDTA能有效抑制[^3H]NTD和[^3H]PN的特异性结合，其Ki值约为60 muM。edta预处理PCA后，特异[^3H]PN结合的最大结合位点（Bmax）明显减少（80%），添加Ca^<++>和Mg^<++>后恢复到未处理的水平。我们得出结论：1)[^3H]NTD和[^3H]PN选择性标记PCA中与药理相关的DHP Ca^<++>拮抗剂受体；2) d-顺式地尔硫卓和维拉帕米是血管组织DHP受体位点的变构调节剂；DHP拮抗剂与血管受体的结合是一个极度依赖于二价阳离子存在的过程。

To identify and characterize the receptor sites for 1,4-dihydropyridine(DEP) Ca^<++> channel antagonists in coronary artery, we have measured specific binding of [^3H]nitrendipine(NTD) and (+)-[^3H]PN200-110(PN) in crude membranes of porcine coronary artery(PCA) by radioreceptor assay. Specific binding of [^3H]NTD and [^3H]PN in PCA was saturable, reversible and of high affinity, it showed a pharmacological specificity as well as stereoselectivity which characterized the receptor sites for DHP Ca^<++> antagonists. DHP antagonists(0.1+nM-1 muM) competed for the binding of both ligand in order:(+)PN> mepirodipine > nisoldipine > nicardipine > nitrendipine > nimodipine > nifedipine >(-)PN. (+)PN and mepirodipine exhibited 10 to 20 times greater affinity for [^3H]PN binding sites than nifedipine. (+)PN was approximately 140 times as potent as the (-)isomer. The potencies(pki) of these eight DHP Ca^<++> antagonists in competing for the ligand binding sites in PCA correlated well with their pharmacological potencies(pA_2)- Specific [^3H]PN binding in PCA was enhanced by d-cis-diltiazem and was inhibited incompletely by verapamil and D-600. Specific binding of [^3H]NTD and [^3H]PN was effectively inhibited by EDTA, and their Ki values were approximately 60 muM. In EDTA-pretreated PCA, the maximal number of binding sites(Bmax) for specific [^3H]PN binding was reduced(80 %) markedly, and it was restored to the untreated level by the addition of Ca^<++> and Mg^<++>. We conclude: 1) [^3H]NTD and [^3H]PN selectively label the pharmacological relevant DHP Ca^<++> antagonist receptors in PCA; 2) d-cis-diltiazem and verapamil are allosteric modulators of DHP receptor sites in the vascular tissues; 3) the binding of DHP antagonists to the vascular receptors is an extremely dependent process of the presence of divalent cations.