Williams L T, Tremble P
J Clin Invest. 1982 Jul;70(1):209-12. doi: 10.1172/jci110596.
[(3)H]Nitrendipine, a potent calcium channel antagonist [3-ethyl-5-methyl-1-1,4-dihydro-2,6 - dimethyl - 4 - (3 - nitrophenyl) - 3,5 - pyridine carboxylate], was used to label high affinity binding sites on membranes prepared from bovine aortic smooth muscle. The binding of [(3)H]nitrendipine is rapid (t(1/2) < 5 min) and reversible at 37 degrees C. The binding sites have a high affinity for [(3)H]nitrendipine with an equilibrium dissociation constant of 2.1 nM. The density of sites is 40-60 fmol/mg of membrane protein. Analogues of nitrendipine compete for the binding sites with affinities consistent with their known biological effects as calcium antagonists. Nisoldipine, [isobutyl methyl 1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)-3,5-pyridine carboxylate], a calcium antagonist more potent than nifedipine [2,6-dimethyl-3,5-dicarbomethoxy-4-(2-nitrophenyl)-1,4-dihydropyridine] in relaxing vascular smooth muscle, has an affinity three-fold higher than that of nifedipine in competing for the binding sites. A biologically inactive derivative of nifedipine does not compete for [(3)H]nitrendipine binding. Verapamil (alpha-isopropyl-alpha[(N-methyl - N-homoveratryl) -alpha-aminopropyl]-3,4-dimethyoxyphenyl acetonitrile), a structurally different calcium antagonist, only partially (25%) inhibits binding at high concentrations (1 muM). Prazosin, an alpha adrenergic antagonist does not compete for [(3)H]nitrendipine binding sites. The binding of [(3)H]nitrendipine is not affected by 1.5 mM calcium. Canine cardiac membranes also have high affinity [(3)H]nitrendipine binding sites, (K(D) = 6 nM) but bovine erythrocytes do not. The relative affinities of nisoldipine and nifedipine for the cardiac membrane binding sites reflect the relative activities of these compounds as calcium channel antagonists. These results suggest that the [(3)H]nitrendipine binding sites are the sites through which dihydropyridines act as calcium channel antagonists.
[³H]尼群地平，一种强效钙通道拮抗剂[3 - 乙基 - 5 - 甲基 - 1,4 - 二氢 - 2,6 - 二甲基 - 4 - (3 - 硝基苯基) - 3,5 - 吡啶羧酸酯]，用于标记从牛主动脉平滑肌制备的膜上的高亲和力结合位点。[³H]尼群地平的结合迅速（半衰期<5分钟），在37℃时可逆。结合位点对[³H]尼群地平具有高亲和力，平衡解离常数为2.1 nM。位点密度为40 - 60 fmol/mg膜蛋白。尼群地平类似物以与其作为钙拮抗剂的已知生物学效应一致的亲和力竞争结合位点。尼索地平，[异丁基甲基1,4 - 二氢 - 2,6 - 二甲基 - 4 - (2 - 硝基苯基) - 3,5 - 吡啶羧酸酯]，一种在舒张血管平滑肌方面比硝苯地平[2,6 - 二甲基 - 3,5 - 二羧酸甲氧基 - 4 - (2 - 硝基苯基) - 1,4 - 二氢吡啶]更有效的钙拮抗剂，在竞争结合位点时的亲和力比硝苯地平高3倍。硝苯地平的一种无生物学活性的衍生物不竞争[³H]尼群地平的结合。维拉帕米（α - 异丙基 - α[(N - 甲基 - N - 高藜芦基) - α - 氨基丙基] - 3,4 - 二甲氧基苯基乙腈），一种结构不同的钙拮抗剂，在高浓度（1 μM）时仅部分（25%）抑制结合。哌唑嗪，一种α肾上腺素能拮抗剂，不竞争[³H]尼群地平结合位点。[³H]尼群地平的结合不受1.5 mM钙的影响。犬心肌膜也有高亲和力的[³H]尼群地平结合位点，（解离常数K(D)=6 nM），但牛红细胞没有。尼索地平和硝苯地平对心肌膜结合位点的相对亲和力反映了这些化合物作为钙通道拮抗剂的相对活性。这些结果表明，[³H]尼群地平结合位点是二氢吡啶类作为钙通道拮抗剂发挥作用的位点。
