Entzeroth M, Mayer N
Department of Biochemical Research, Dr. Karl Thomae GmbH Biberach, F.R.G.
J Recept Res. 1991;11(1-4):141-52. doi: 10.3109/10799899109066395.
The tritiated cardioselective muscarinic antagonist AF-DX 384 (5,11-dihydro-11-[2-(-[8-dipropylamino)methyl]-1-piperidinyl]-ethyl] amino]-carbonyl]-6H-pyrido [2,3-b] [1,4]benzodiazepin-6-one) was used to label muscarinic receptors in the rat ileum. Saturation binding to membrane suspensions revealed a high affinity binding site with a Kd of 9.2 nM. The maximal number of binding sites labeled in this tissue (Bmax) is 237 fmol/mg protein. The association and dissociation kinetics were well represented by single exponential reactions, and the dissociation constant obtained from the ratio of rate constants was in agreement with that derived from saturation experiments. Specific binding was inhibited by muscarinic antagonists with a rank order of potencies of atropine (pKi: 8.80) greater than 4-DAMP (pKi: 8.23) = AF-DX 384 (pKi: 8.20) greater than AF-DX 116 (pKi: 7.09) = hexahydro-sila-difenidol (pKi: 6.97) greater than pirenzepine (pKi: 6.49) and is consistent with the interaction of [3H]AF-DX 384 with muscarinic receptors of the M2 subtype. It can be concluded that [3H]AF-DX 384 can be used to selectively label M2 muscarinic receptors in heterogeneous receptor populations.
用氚标记的心脏选择性毒蕈碱拮抗剂AF-DX 384（5,11-二氢-11-[2-(-[8-二丙基氨基)甲基]-1-哌啶基]-乙基]氨基]-羰基]-6H-吡啶并[2,3-b][1,4]苯并二氮杂卓-6-酮）标记大鼠回肠中的毒蕈碱受体。与膜悬浮液的饱和结合显示出一个高亲和力结合位点，其解离常数（Kd）为9.2 nM。该组织中标记的结合位点最大数量（Bmax）为237 fmol/mg蛋白质。结合和解离动力学由单指数反应很好地描述，从速率常数比值获得的解离常数与从饱和实验得出的结果一致。毒蕈碱拮抗剂抑制特异性结合，其效力顺序为阿托品（pKi：8.80）大于4-DAMP（pKi：8.23）=AF-DX 384（pKi：8.20）大于AF-DX 116（pKi：7.09）=六氢硅二苯醇（pKi：6.97）大于哌仑西平（pKi：6.49），这与[3H]AF-DX 384与M2亚型毒蕈碱受体的相互作用一致。可以得出结论，[3H]AF-DX 384可用于在异质受体群体中选择性标记M2毒蕈碱受体。
