Zhu Q L, He H M, Xiao W B, Wang H
Institute of Pharmacology and Toxicology, Academy of Military Medical Sciences, Beijing, China.
J Cardiovasc Pharmacol. 2001 May;37(5):522-31. doi: 10.1097/00005344-200105000-00004.
Radioligand binding techniques were employed to determine the modulation by nucleotides of the specific [3H]glibenclamide (Gli) binding to rat aortic and cardiac ventricular preparations. Saturation analysis revealed a single binding site with K(D) value of 31.3 nM and Bmax of 180 fmol/mg wet weight in aortic preparations. We also observed that [3H]Gli bound reversibly and specifically to cardiac membranes. Unlabeled glibenclamide displaced [3H]Gli-specific binding of cardiac membranes completely with K(I) of 54.4 nM. In cardiac membranes, adenosine triphosphate (ATP), adenosine diphosphate (ADP), and uridine diphosphate (UDP) (from 0.01-5 mM) concentration dependently inhibited [3H]Gli binding independent of Mg2+. The values of K(I) were 0.47, 0.22, and 0.58 mM, respectively. However, in aortic preparations, [3H]Gli-specific binding was increased by ATP of 5 and 10 mM and showed a biphasic response to ADP. At concentrations to 1 mM, ADP inhibited binding; above 5 mM, the specific [3H]Gli binding was increased. UDP did not alter the binding up to 5 mM. In the presence of Mg2+ (20 mM), the inhibitory effects of ATP (0.01-1 mM) or ADP (0.01-5 mM) on the binding in cardiac membranes were abolished, whereas the facilitatory effects of ATP or ADP in aortic preparations were strengthened. Analysis of kinetics showed that the time of [3H]Gli association and dissociation in cardiac and aortic preparations was monophasic. The association was delayed with dissociation unchanged by ATP, ADP, and UDP of 1 mM, respectively, in cardiac membranes. In aorta, however, at the same concentration ATP accelerated association and retarded dissociation and vice versa for ADP. Association and dissociation were not changed by UDP of 5 mM. We conclude that ATP, ADP, and UDP are all major allosteric modulators of K(ATP) channels and they affect the antagonist binding to heart (sulfonylurea receptor 2A) and aorta (sulfonylurea receptor 2B) differently.
采用放射性配体结合技术来确定核苷酸对特异性[3H]格列本脲（Gli）与大鼠主动脉和心室肌制剂结合的调节作用。饱和分析显示，在主动脉制剂中存在一个单一结合位点，其解离常数（K(D)）值为31.3 nM，最大结合容量（Bmax）为180 fmol/mg湿重。我们还观察到[3H]Gli与心肌膜可逆且特异性地结合。未标记的格列本脲能完全取代心肌膜上[3H]Gli的特异性结合，其抑制常数（K(I)）为54.4 nM。在心肌膜中，三磷酸腺苷（ATP）、二磷酸腺苷（ADP）和二磷酸尿苷（UDP）（浓度范围为0.01 - 5 mM）浓度依赖性地抑制[3H]Gli结合，且与镁离子（Mg2+）无关。其K(I)值分别为0.47、0.22和0.58 mM。然而，在主动脉制剂中，5 mM和10 mM的ATP可增加[3H]Gli的特异性结合，且ADP呈现双相反应。在浓度达1 mM时，ADP抑制结合；高于5 mM时，特异性[3H]Gli结合增加。UDP在浓度达5 mM时不改变结合。在存在镁离子（20 mM）的情况下，ATP（0.01 - 1 mM）或ADP（0.01 - 5 mM）对心肌膜结合的抑制作用被消除，而ATP或ADP在主动脉制剂中的促进作用增强。动力学分析表明，[3H]Gli在心肌和主动脉制剂中的结合和解离时间均为单相。在心肌膜中，1 mM的ATP、ADP和UDP分别使结合延迟，解离不变。然而，在主动脉中，相同浓度下ATP加速结合并延迟解离，而ADP则相反。5 mM的UDP不改变结合和解离。我们得出结论，ATP、ADP和UDP均为钾离子通道（K(ATP)）的主要变构调节剂，它们对拮抗剂与心脏（磺脲类受体2A）和主动脉（磺脲类受体2B）的结合影响不同。
