Erlenkamp S, Gretzer B, Zillikens S, Glitsch H G, Pusch H, Staroske T, Welzel P
Arbeitsgruppe Muskelphysiologie, Ruhr-Universität, Bochum, Germany.
Naunyn Schmiedebergs Arch Pharmacol. 1998 Jan;357(1):54-62. doi: 10.1007/pl00005138.
Affinity labeling might be used to localize the binding site(s) of the lactone ring of cardioactive steroids on the Na+/K+-ATPase. The aim of the experiments described below was to identify C-22-substituted derivatives of digitoxigenin suitable for this purpose. The positive inotropic effect of digitoxigenin, 22-benzoyloxy-digitoxigenin, 22-acetoxy-digitoxigenin, 22-allyl-digitoxigenin, and 22-hydroxy-digitoxigenin was studied in sheep cardiac Purkinje fibres. In addition, the inhibition of the Na+/K+ pump by these drugs was investigated by means of simultaneous measurements of membrane current and intracellular Na+ concentration in voltage-clamped Purkinje fibres and by means of whole-cell recording in isolated sheep Purkinje cells. The experiments were performed at 5.4 mM K+ and 30 to 33 degrees C. All compounds exerted a reversible positive inotropic effect. The concentrations required for the half maximal effect (EC50 value) amounted to approximately 5 x 10(-7) M digitoxigenin, 22-acetoxy-digitoxigenin or 22-hydroxy-digitoxigenin. The EC50 values for 22-benzoyloxy-digitoxigenin and 22-allyl-digitoxigenin were estimated to be 1.3 x 10(-6) M and 1.1 x 10(-5) M, respectively. From measurements on voltage-clamped Purkinje fibres the concentrations required for half maximal Na+/K+ pump inhibition (K'D value) were calculated to be approximately 10(-6) M for digitoxigenin, 22-acetoxy-digitoxigenin or 22-hydroxy-digitoxigenin. The K'D value for 22-benzoyloxy-digitoxigenin was 10 times larger. The K'D value for 22-allyl-digitoxigenin was even larger and amounted to approximately 4 x 10(-5) M. The K'D values of the drugs derived from whole-cell recording on single Purkinje cells tended to be smaller by a factor 2 to 8. Measurements of drug binding and unbinding revealed that the apparent association rate constant of 22-benzoyloxy-digitoxigenin (approximately 9 x 10(2) s(-1) M[-1]) was smaller than the association rate constant of digitoxigenin (approximately 2 x 10(4) s(-1) M[-1]), whereas the apparent dissociation rate constants of both compounds were similar (approximately 4 x 10(-3) s[-1]). Compared to digitoxigenin 22-allyl-digitoxigenin displayed a lower association rate constant (approximately 3 x 10(3) s(-1) M[-1]) and a larger dissociation rate constant (approximately 8 x 10(-2) M[-1]). The structure-activity relationships of the drugs are discussed. We conclude that esters derived from 22-hydroxy-digitoxigenin might be suitable to localize the binding site(s) of the lactone moiety on the Na+/K+ pump by affinity labeling.
亲和标记可用于定位强心甾体内酯环在钠钾ATP酶上的结合位点。以下所述实验的目的是鉴定适用于此目的的洋地黄毒苷元C-22取代衍生物。研究了洋地黄毒苷元、22-苯甲酰氧基洋地黄毒苷元、22-乙酰氧基洋地黄毒苷元、22-烯丙基洋地黄毒苷元和22-羟基洋地黄毒苷元对绵羊心脏浦肯野纤维的正性肌力作用。此外，通过在电压钳制的浦肯野纤维中同时测量膜电流和细胞内钠浓度以及通过在分离的绵羊浦肯野细胞中进行全细胞记录，研究了这些药物对钠钾泵的抑制作用。实验在5.4 mM钾离子浓度和30至33摄氏度下进行。所有化合物均产生可逆的正性肌力作用。产生半数最大效应所需的浓度（EC50值）约为5×10⁻⁷ M洋地黄毒苷元、22-乙酰氧基洋地黄毒苷元或22-羟基洋地黄毒苷元。22-苯甲酰氧基洋地黄毒苷元和22-烯丙基洋地黄毒苷元的EC50值估计分别为1.3×10⁻⁶ M和1.1×10⁻⁵ M。根据对电压钳制的浦肯野纤维的测量，洋地黄毒苷元、22-乙酰氧基洋地黄毒苷元或22-羟基洋地黄毒苷元产生半数最大钠钾泵抑制所需的浓度（K'D值）计算约为10⁻⁶ M。22-苯甲酰氧基洋地黄毒苷元的K'D值大10倍。22-烯丙基洋地黄毒苷元的K'D值甚至更大，约为4×10⁻⁵ M。从单个浦肯野细胞的全细胞记录得出的药物K'D值往往小2至8倍。药物结合和解离的测量表明，22-苯甲酰氧基洋地黄毒苷元的表观缔合速率常数（约9×10² s⁻¹ M⁻¹）小于洋地黄毒苷元的缔合速率常数（约2×10⁴ s⁻¹ M⁻¹），而两种化合物的表观解离速率常数相似（约4×10⁻³ s⁻¹）。与洋地黄毒苷元相比，22-烯丙基洋地黄毒苷元表现出较低的缔合速率常数（约3×10³ s⁻¹ M⁻¹）和较大的解离速率常数（约8×10⁻² s⁻¹）。讨论了这些药物的构效关系。我们得出结论，22-羟基洋地黄毒苷元衍生的酯可能适用于通过亲和标记定位内酯部分在钠钾泵上的结合位点。
