Tanaka M, Muramatsu M, Aihara H, Otomo S
Research Center, Taisho Pharmaceutical Co., Ltd., Saitama, Japan.
Biochem Pharmacol. 1990 Sep 1;40(5):991-6. doi: 10.1016/0006-2952(90)90484-3.
The characteristics of the binding of the 1,4-dihydropyridine Ca2+ antagonist, 2-nitratopropyl 3-nitratopropyl 2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine 3,5-dicarboxylate (CD-349), to calmodulin (CaM) and the effect of CD-349 on the Ca2+/CaM-dependent enzyme, cyclic GMP (cGMP) phosphodiesterase (PDE), were investigated. CD-349 showed a Ca2(+)-dependent binding to CaM, in equilibrium column binding studies. CD-349 inhibited the [3H]CD-349 binding to CaM, at a concentration producing a 50% inhibition (IC50) of 2.4 microM, whereas the CaM antagonist, trifluoperazine hydrochloride (TFP), stimulated the [3H]CD-349 binding to CaM. Scatchard plot analysis of the binding of CD-349 to CaM revealed that the apparent dissociation constant (Kapp) of CD-349 was 2.1 microM and the maximal number of binding sites (Bmax) of CD-349 was 1.0 nmol/nmol CaM. In the presence of TFP, the Kapp and Bmax values of CD-349 binding to CaM were changed to 1.1 microM and 1.5 nmol/nmol CaM respectively. Although the CaM antagonists, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7) and TFP, decreased and increased, respectively, the fluorescence intensity caused by 2-p-toluidinylnaphthalene-6-sulfonic acid (TNS)-CaM binding, CD-349 only slightly decreased the fluorescence of TNS bound CaM. CD-349 inhibited both basal and Ca2+/CaM-activated cGMP PDE activity. However, CaM did not competitively antagonize the CD-349-induced inhibition of the Ca2+/CaM-activated PDE activity. In addition, the kinetic study showed that CD-349 inhibited both basal and Ca2+/CaM-activated cGMP PDE activity, competitively with cGMP, with almost the same inhibition constant (Ki). These results suggest that CD-349 binds to CaM, with Ca2+ dependency, at sites differing from those which bind to the CaM antagonist. The inhibitory activity of CD-349 on Ca2+/CaM-dependent PDE does not seem to be due to a CaM antagonistic action.
研究了1,4 - 二氢吡啶类钙离子拮抗剂2 - 硝酸丙酯 - 3 - 硝酸丙酯 - 2,6 - 二甲基 - 4 - (3 - 硝基苯基)-1,4 - 二氢吡啶 - 3,5 - 二羧酸酯(CD - 349)与钙调蛋白(CaM)的结合特性以及CD - 349对Ca2+/CaM依赖性酶环磷酸鸟苷(cGMP)磷酸二酯酶(PDE)的影响。在平衡柱结合研究中，CD - 349表现出与CaM的Ca2+依赖性结合。CD - 349在产生50%抑制作用(IC50)的浓度为2.4 μM时抑制[3H]CD - 349与CaM的结合，而钙调蛋白拮抗剂盐酸三氟拉嗪(TFP)则刺激[3H]CD - 349与CaM的结合。对CD - 349与CaM结合的Scatchard图分析表明，CD - 349的表观解离常数(Kapp)为2.1 μM，CD - 349的最大结合位点数(Bmax)为1.0 nmol/nmol CaM。在TFP存在下，CD - 349与CaM结合的Kapp和Bmax值分别变为1.1 μM和1.5 nmol/nmol CaM。尽管钙调蛋白拮抗剂N - (6 - 氨基己基)-5 - 氯 - 1 - 萘磺酰胺盐酸盐(W - 7)和TFP分别降低和增加了由2 - (对甲苯氨基)萘 - 6 - 磺酸(TNS)-CaM结合引起的荧光强度，但CD - 349仅轻微降低了与TNS结合的CaM的荧光。CD - 349抑制基础和Ca2+/CaM激活的cGMP PDE活性。然而，CaM并未竞争性拮抗CD - 349对Ca2+/CaM激活的PDE活性的抑制作用。此外，动力学研究表明，CD - 349抑制基础和Ca2+/CaM激活的cGMP PDE活性，与cGMP竞争性结合，抑制常数(Ki)几乎相同。这些结果表明，CD - 349以Ca2+依赖性方式与CaM结合，其结合位点不同于与钙调蛋白拮抗剂结合的位点。CD - 349对Ca2+/CaM依赖性PDE的抑制活性似乎不是由于钙调蛋白拮抗作用。
