Wei X Y, Rutledge A, Triggle D J
Department of Biochemical Pharmacology, School of Pharmacy, State University of New York, Buffalo 14260.
Mol Pharmacol. 1989 Apr;35(4):541-52.
Binding of 1,4-dihydropyridine Ca2+ channel ligands was characterized as a function of membrane potential using saturation, competition, and kinetic measurements in cultured neonatal rat ventricular myocytes. The 1,4-dihydropyridine antagonist [3H]PN 200-110 bound to polarized cells (5.8 mM K+) with a KD value of 3.53 X 10(-9) M and a Bmax value of 50.1 fmol/mg of protein. In depolarized cells (50 mM K+), a KD value of 6.33 X 10(-11) M was found, reflecting a 55-fold increase in affinity; Bmax did not change upon depolarization. Dissociation rates (k-1) of [3H]PN 200-110 binding were faster in polarized cells (0.53 min-1) than in depolarized cells (0.018 min-1), but association rates (k1 of 2.17 X 10(8) and 2.27 X 10(8) min-1M-1 were not different in polarized and depolarized cells. The KD values calculated from the ratio of k-1/k1 accorded well with those determined from equilibrium binding assays. The enantiomers of Bay K 8644 and 202-791 and a series of nifedipine analogs inhibited specific binding of [3H]PN 200-110 in depolarized cells. In polarized cells, the affinities of the S-enantiomers (activators) were close to those in depolarized cells; however, the affinities of R-enantiomers (antagonists) were 50- to 65-fold lower. The effects of both (S)- and (R)-Bay K 8644 on [3H]PN 200-110 binding were mediated through increased apparent KD values, without changes in Bmax and nH. In depolarized cells, l-D600 and d-D600 partially inhibited [3H]PN 200-110 binding to a maximum of 71% and 56%, respectively; in polarized cells, l-D600 (d-D600 not measured) was ineffective on [3H]PN 200-110 binding. d-(cis)-Diltiazem, but not l-(cis)-diltiazem, partially inhibited (maximum 30%) specific binding of [3H]PN 200-110 in depolarized cells, but potentiated (maximum 79%) binding in polarized cells. The potentiating effect of d-(cis)-diltiazem was mediated through an increase in affinity without change in Bmax of [3H]PN 200-110 binding. (S)-Bay K 8644 potentiated 45Ca2+ uptake into the cells, with an EC50 value of 4.26 X 10(-10) M; concentrations higher than 10(-7) M were inhibitory, producing a biphasic concentration-response relationship. (R)-Bay K 8644 inhibited 80 mM K+-stimulated 45Ca2+ uptake with an IC50 value of 2.11 X 10(-9) M. These pharmacologic values correlate well with the binding affinities.(ABSTRACT TRUNCATED AT 400 WORDS)
利用培养的新生大鼠心室肌细胞的饱和、竞争和动力学测量，将1,4 - 二氢吡啶钙通道配体的结合特性表征为膜电位的函数。1,4 - 二氢吡啶拮抗剂[3H]PN 200 - 110与极化细胞（5.8 mM K+）结合，KD值为3.53×10(-9) M，Bmax值为50.1 fmol/mg蛋白质。在去极化细胞（50 mM K+）中，发现KD值为6.33×10(-11) M，反映亲和力增加了55倍；去极化后Bmax不变。[3H]PN 200 - 110结合的解离速率（k-1）在极化细胞（0.53 min-1）中比在去极化细胞（0.018 min-1）中更快，但结合速率（k1分别为2.17×10(8)和2.27×10(8) min-1M-1）在极化和去极化细胞中没有差异。由k-1/k1比值计算出的KD值与平衡结合测定确定的值非常吻合。Bay K 8644和202 - 791的对映体以及一系列硝苯地平类似物在去极化细胞中抑制[3H]PN 200 - 110的特异性结合。在极化细胞中，S - 对映体（激活剂）的亲和力与去极化细胞中的接近；然而，R - 对映体（拮抗剂）的亲和力低50至65倍。（S） - 和（R） - Bay K 8644对[3H]PN 200 - 110结合的影响是通过表观KD值增加介导的，Bmax和nH没有变化。在去极化细胞中，l - D600和d - D600分别部分抑制[3H]PN 200 - 110结合，最大抑制率分别为71%和56%；在极化细胞中，l - D600（未测量d - D600）对[3H]PN 200 - 110结合无效。d -（顺式） - 地尔硫卓而非l -（顺式） - 地尔硫卓在去极化细胞中部分抑制（最大30%）[3H]PN 200 - 110的特异性结合，但在极化细胞中增强（最大79%）结合。d -（顺式） - 地尔硫卓的增强作用是通过增加[3H]PN 200 - 110结合的亲和力介导的，Bmax不变。（S） - Bay K 8644增强45Ca2+摄取进入细胞，EC50值为4.26×10(-10) M；高于10(-7) M的浓度具有抑制作用，产生双相浓度 - 反应关系。（R） - Bay K 8644抑制80 mM K+刺激的45Ca2+摄取，IC50值为2.11×10(-9) M。这些药理学值与结合亲和力密切相关。（摘要截断于400字）
