Kanda S, Kurokawa J, Adachi-Akahane S, Nagao T
Department of Toxicology and Pharmacology, Faculty of Pharmaceuticals Sciences, University of Tokyo, Japan.
Eur J Pharmacol. 1997 Jan 14;319(1):101-7. doi: 10.1016/s0014-2999(96)00826-6.
To examine whether the modulation of the 1,4-dihydropyridine-binding by diltiazem derivatives, which has been shown in cardiac and skeletal muscle membranes, takes place in intact cardiac myocytes, effects of diltiazem on the specific binding of 3H-PN200-110 to freshly isolated adult rat ventricular myocytes were investigated in normal Tyrode solution at 37 degrees C. Diltiazem consistently potentiated the 3H-PN200-110-binding in a concentration-dependent manner, while DTZ323 (3-(acetyloxy)-5-[2-[[2- (3,4-dimethoxyphenyl)ethyl]-methylamino]ethyl]-2,3-dihydro-2(-4 methoxyphenyl)-1,5-benzothiazepin-4-(5H)-one), a potent diltiazem derivative, inhibited it in a non-competitive manner. In saturation studies, 100 microM decreased the Kd value of the 3H-PN200-110-binding (control, 0.102 +/- 0.008 vs. diltiazem, 0.074 +/- 0.004 (nM, n = 6), P < 0.05) without significant effect on Bmax (control, 65.7 +/- 6.4 vs. diltiazem, 76.7 +/- 4.4 (fmol/mg protein, n = 6). Moreover, membrane-impermeant quaternary diltiazem also potentiated the 3H-PN200-110-binding in intact myocytes. These results suggest that diltiazem modulates the 1,4-dihydro-pyridine-binding even in intact cardiac myocytes, and the binding site of diltiazem is accessible from the extracellular side of the L-type Ca2+ channels.
为了研究在心肌和骨骼肌细胞膜中已得到证实的地尔硫䓬衍生物对1,4-二氢吡啶结合的调节作用是否也发生在完整的心肌细胞中，我们在37℃的正常台氏液中研究了地尔硫䓬对新鲜分离的成年大鼠心室肌细胞中3H-PN200-110特异性结合的影响。地尔硫䓬始终以浓度依赖的方式增强3H-PN200-110的结合，而强效地尔硫䓬衍生物DTZ323（3-(乙酰氧基)-5-[2-[[2-(3,4-二甲氧基苯基)乙基]-甲基氨基]乙基]-2,3-二氢-2(-4-甲氧基苯基)-1,5-苯并硫氮杂䓬-4-(5H)-酮）则以非竞争性方式抑制其结合。在饱和研究中，100μM降低了3H-PN200-110结合的Kd值（对照组，0.102±0.008对用地尔硫䓬后，0.074±0.004（nM，n = 6），P < 0.05），而对Bmax无显著影响（对照组，65.7±6.4对用地尔硫䓬后，76.7±4.4（fmol/mg蛋白，n = 6）。此外，膜不透性的季铵化地尔硫䓬也增强了完整心肌细胞中3H-PN200-110的结合。这些结果表明，地尔硫䓬即使在完整的心肌细胞中也能调节1,4-二氢吡啶的结合，并且地尔硫䓬的结合位点可从L型钙通道的细胞外侧进入。
