Rossier J R, Cox J A, Niesor E J, Bentzen C L
Research Laboratory of Symphar S.A., Geneva, Switzerland.
J Biol Chem. 1989 Oct 5;264(28):16598-607.
Tetrabutyl-2(2-phenoxyethyl)-1,3-propylidene diphosphonate (SR-7037) completely displaced dihydropyridine [( 3H]PN200-110), phenylalkylamine [( 3H]D888), and benzothiazepine [( 3H]diltiazem) ligands from brain L-type calcium channels. Half-maximal inhibition of [3H]PN200-110 binding occurred at 19 nM with a Hill coefficient of 0.96. SR-7037 primarily decreased the affinity for [3H]PN200-110 with a small, but significantly, effect on the maximal binding capacity. Kinetic studies showed that this was due to an increased radioligand dissociation rate from 0.04 min-1 to 0.43 min-1 in the presence of the diphosphonate. Displacement of [3H]D888 by SR-7037 was biphasic with respective IC50 of 44 and 8400 nM. Likewise, unlabeled (-)-D888 identified two sites with IC50 values of 0.9 and 27 nM. Both SR-7037 (1000 nM) and D888 (200 nM) accelerated radioligand dissociation about 2-fold. [3H]Diltiazem binding was inhibited by SR-7037 with an IC50 value of 29 nM. The inhibition of dihydropyridine binding by SR-7037 is enhanced by most divalent cations at millimolar concentrations with the following potency: Mn2+ greater than Mg2+ greater than Ca2+ greater than Co2+. Barium has the opposite effect. The half-maximal effect of calcium occurred at 6 microM free ion. Specific binding of [3H]D888 was antagonized in the presence of 1 mM CaCl2. It is concluded that SR-7037 has allosteric interactions with the dihydropyridine receptor of the L-type calcium channel. The differential effect of Ca2+ on the potency of D888 and diltiazem relative to that of SR-7037 indicates that the three drugs may bind to nonequivalent sites. These results support specific calcium channel inhibition, possibly at a novel site, as the primary mechanism of the diphosphonate's pharmacological actions.
四丁基-2(2-苯氧基乙基)-1,3-亚丙基二膦酸酯（SR-7037）完全取代了脑L型钙通道上的二氢吡啶类（[3H]PN200-110）、苯烷基胺类（[3H]D888）和苯并硫氮杂䓬类（[3H]地尔硫䓬）配体。[3H]PN200-110结合的半数抑制浓度为19 nM，希尔系数为0.96。SR-7037主要降低了对[3H]PN200-110的亲和力，对最大结合容量有较小但显著的影响。动力学研究表明，这是由于在二膦酸酯存在下，放射性配体的解离速率从0.04 min-1增加到0.43 min-1。SR-7037对[3H]D888的取代呈双相性，IC50分别为44和8400 nM。同样，未标记的（-）-D888确定了两个位点，IC50值分别为0.9和27 nM。SR-7037（1000 nM）和D888（200 nM）均使放射性配体解离加速约2倍。SR-7037抑制[3H]地尔硫䓬结合，IC50值为29 nM。大多数毫摩尔浓度的二价阳离子增强了SR-7037对二氢吡啶结合的抑制作用，其效力顺序为：Mn2+＞Mg2+＞Ca2+＞Co2+。钡则有相反的作用。钙的半数最大效应出现在游离离子浓度为6 μM时。在1 mM CaCl2存在下，[3H]D888的特异性结合受到拮抗。结论是，SR-7037与L型钙通道的二氢吡啶受体存在变构相互作用。Ca2+对D888和地尔硫䓬相对于SR-7037效力的不同影响表明，这三种药物可能结合于不等价位点。这些结果支持特异性钙通道抑制，可能是在一个新位点，作为二膦酸酯药理作用的主要机制。
