Linde C, Löffler C, Kessler C, Quast U
Department of Pharmacology, Medical Faculty, University of Tübingen, Germany.
Naunyn Schmiedebergs Arch Pharmacol. 1997 Oct;356(4):467-74. doi: 10.1007/pl00005078.
In vascular smooth muscle, openers of ATP-dependent potassium channels (K(ATP) channels), such as P1075 (N-cyano-N'-(1,1-dimethylpropyl)-N"-3-pyridylguanidine), produce relaxation. In this study we have investigated the effects of thiol-modifying agents on the binding of P1075 and on the 86Rb+ efflux stimulating and vasorelaxant effects of the opener in rat aortic rings. The increase in 86Rb+ efflux induced by P1075 was taken as a qualitative measure of K+ channel opening. The hydrophilic SH-group-oxidizing substance, thimerosal (1 to 100 microM), abolished specific binding of [3H]-P1075 with an IC50 value of 7.6+/-1.2 microM; at 30 microM, the half time for inhibition was 38 min. Two other thioloxidizing agents, PMB (4-hydroxy-mercuribenzoic acid) and DTBNP (2,2'-dithio-bis(5-nitropyridine)), inhibited binding up to 86% and 44%, respectively. The disulphide bond reducing substance, DTT (1,4-dithiothreitol, 0.1 to 1 mM), reduced [3H]-P1075 binding by up to 20% and partially reversed the inhibitory effect of thimerosal. In 86Rb+ efflux experiments, thimerosal (3 to 100 microM) concentration-dependently increased basal efflux but inhibited P1075-stimulated tracer efflux with an IC50 value of 7+/-1 microM. The inhibitory effect occurred with a half-time of approximately 8 min and was essentially reversed by DTT. In rings precontracted with noradrenaline, thimerosal inhibited the vasorelaxant effect in a noncompetitive manner, shifting the concentration-relaxation curves to the right and reducing maximum relaxation. The data show that oxidation of thiol groups interferes with the binding of the K(ATP) channel opener, P1075; concomitantly, the 86Rb+ efflux stimulating and the vasorelaxant effects are inhibited. Reduction of disulphide bonds by DTT has only minor effects on the action of P1075. Collectively, the results suggest that intact thiol groups are essential for the functioning of the K(ATP) channel in rat aorta. The different kinetics governing the inhibition of opener binding and of opener-stimulated 86Rb+ efflux suggest that the SH-groups involved in the two processes differ in their accessibility to thimerosal and/or in their reactivity.
在血管平滑肌中，ATP依赖性钾通道（K(ATP)通道）的开放剂，如P1075（N-氰基-N'-(1,1-二甲基丙基)-N"-3-吡啶基胍），可引起舒张。在本研究中，我们研究了硫醇修饰剂对P1075结合以及对大鼠主动脉环中开放剂刺激86Rb+外流和血管舒张作用的影响。P1075诱导的86Rb+外流增加被用作钾通道开放的定性指标。亲水性巯基氧化物质硫柳汞（1至100 microM）可消除[3H]-P1075的特异性结合，IC50值为7.6±1.2 microM；在30 microM时，抑制的半衰期为38分钟。另外两种巯基氧化剂，对羟基汞苯甲酸（PMB）和2,2'-二硫代双（5-硝基吡啶）（DTBNP），分别抑制结合达86%和44%。二硫键还原物质二硫苏糖醇（DTT，1,4-二硫代苏糖醇，0.1至1 mM）可使[3H]-P1075结合减少达20%，并部分逆转硫柳汞的抑制作用。在86Rb+外流实验中，硫柳汞（3至100 microM）浓度依赖性地增加基础外流，但抑制P1075刺激的示踪剂外流，IC50值为7±1 microM。抑制作用的半衰期约为8分钟，基本上可被DTT逆转。在去甲肾上腺素预收缩的血管环中，硫柳汞以非竞争性方式抑制血管舒张作用，使浓度-舒张曲线右移并降低最大舒张程度。数据表明，巯基的氧化会干扰K(ATP)通道开放剂P1075的结合；同时，86Rb+外流刺激和血管舒张作用也会受到抑制。DTT对二硫键的还原对P1075的作用影响较小。总体而言，结果表明完整的巯基对于大鼠主动脉中K(ATP)通道的功能至关重要。开放剂结合抑制和开放剂刺激的86Rb+外流抑制的不同动力学表明，参与这两个过程的巯基对硫柳汞的可及性和/或反应性不同。
