title: Interaction between thiol-modifying agents and P1075, a K(ATP) channel opener, in rat isolated aorta.
abstract: 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.
authors.affiliation: Department of Pharmacology, Medical Faculty, University of Tübingen, Germany.
authors.forename: C
authors.initials: C
authors.lastname: Linde
authors.forename: C
authors.initials: C
authors.lastname: Löffler
authors.forename: C
authors.initials: C
authors.lastname: Kessler
authors.forename: U
authors.initials: U
authors.lastname: Quast
mesh_terms: D000818:Animals; D001011:Aorta / Q000187:drug effects; D004229:Dithiothreitol / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism; D006146:Guanidines / Q000378:metabolism / Q000494:pharmacology*; D006902:Hydroxymercuribenzoates / Q000494:pharmacology; D066298:In Vitro Techniques; D008297:Male; D009119:Muscle Contraction / Q000187:drug effects; D009126:Muscle Relaxation / Q000187:drug effects; D009131:Muscle, Smooth, Vascular / Q000187:drug effects* / Q000378:metabolism; D015221:Potassium Channels / Q000819:agonists* / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects; D011725:Pyridines / Q000378:metabolism / Q000494:pharmacology*; D011849:Radioactive Tracers; D051381:Rats; D017207:Rats, Sprague-Dawley; D012413:Rubidium; D013439:Sulfhydryl Reagents / Q000494:pharmacology*; D013849:Thimerosal / Q000494:pharmacology; D014665:Vasodilator Agents / Q000378:metabolism / Q000494:pharmacology*
chemical_list: D006146:Guanidines; D006902:Hydroxymercuribenzoates; D015221:Potassium Channels; D011725:Pyridines; D011849:Radioactive Tracers; D013439:Sulfhydryl Reagents; D014665:Vasodilator Agents; D013849:Thimerosal; C073896:N-cyano-N'-(1,1-dimethylpropyl)-N''-(3-pyridinyl)guanidine; D012413:Rubidium; D004229:Dithiothreitol