Characterization of calcium antagonist receptors in coronary artery

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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.