Garcia M L, King V F, Shevell J L, Slaughter R S, Suarez-Kurtz G, Winquist R J, Kaczorowski G J
Department of Membrane Biochemistry and Biophysics, Merck Institute for Therapeutic Research, Rahway, New Jersey 07065.
J Biol Chem. 1990 Mar 5;265(7):3763-71.
Three structural classes of commonly used amiloride analogs, molecules derivatized at the terminal guanidino-nitrogen, the five-position pyrazinoyl-nitrogen, or di-substituted at both of these positions, inhibit binding of the L-type Ca2+ channel modulators diltiazem, gallopamil, and nitrendipine to porcine cardiac sarcolemmal membrane vesicles. The rank order of inhibitory potencies among the various derivatives tested is well defined with amiloride being the least potent. Saturation binding studies indicate that inhibition of ligand binding results primarily from effects on Kd. Ligand dissociation measurements suggest that amiloride derivatives do not associate directly at any of the known sites in the Ca2+ entry blocker receptor complex. In addition, these compounds do not compete at the "Ca2+ coordination site" within the channel. However, studies with inorganic and substituted diphenylbutylpiperidine Ca2+ entry blockers reveal that amiloride analogs interact at a site on the channel where metal ions bind and occlude the pore. Photolysis experiments performed with amiloride photoaffinity reagents confirm that a specific interaction occurs between such probes and the channel protein. Upon photolysis, these agents produce concentration- and time-dependent irreversible inactivation of Ca2+ entry blocker binding activities, which can be protected against by either verapamil or diltiazem. 45Ca2+ flux and voltage-clamp experiments performed with GH3 anterior pituitary cells demonstrate that amiloride-like compounds inhibit L-type Ca2+ channels directly. Moreover, these compounds block contraction of isolated vascular tissue in pharmacological assays. Electrophysiological experiments indicate that they also inhibit T-type Ca2+ channels in GH3 cells. Taken together, these results demonstrate unequivocally that amiloride analogs display significant Ca2+ entry blocker activity in both ligand binding and functional assays. This property, therefore, can seriously complicate the interpretation of many in vitro and in vivo studies where amiloride analogs are used to elicit inhibition of other transport systems (e.g. Na-Ca and Na-H exchange).
常用的氨氯地平类似物有三类结构，分别是在末端胍基氮、5位吡嗪甲酰氮处衍生的分子，或在这两个位置都进行双取代的分子，它们能抑制L型钙通道调节剂地尔硫卓、加洛帕米和尼群地平与猪心肌肌膜囊泡的结合。在所测试的各种衍生物中，抑制效力的排序很明确，氨氯地平的效力最低。饱和结合研究表明，配体结合的抑制主要源于对解离常数（Kd）的影响。配体解离测量表明，氨氯地平衍生物不会在钙通道阻滞剂受体复合物的任何已知位点直接结合。此外，这些化合物不会在通道内的“钙配位位点”竞争。然而，对无机和取代二苯基丁基哌啶类钙通道阻滞剂的研究表明，氨氯地平类似物在通道上金属离子结合并堵塞孔道的位点相互作用。用氨氯地平光亲和试剂进行的光解实验证实，此类探针与通道蛋白之间发生了特异性相互作用。光解后，这些试剂会产生浓度和时间依赖性的不可逆钙通道阻滞剂结合活性失活，维拉帕米或地尔硫卓可对此起到保护作用。用GH3垂体前叶细胞进行的45Ca2+通量和电压钳实验表明，氨氯地平样化合物可直接抑制L型钙通道。此外，在药理学实验中，这些化合物可阻断离体血管组织的收缩。电生理实验表明，它们还能抑制GH3细胞中的T型钙通道。综上所述，这些结果明确表明，氨氯地平类似物在配体结合和功能测定中均表现出显著的钙通道阻滞剂活性。因此，在许多体外和体内研究中，当使用氨氯地平类似物来抑制其他转运系统（如钠钙和钠氢交换）时，这一特性会严重干扰对实验结果的解读。
