Yang C M, Chen F F, Sung T C, Hsu H F, Wu D
Department of Pharmacology, Chang Gung Medical College, Tao-Yuan, Taiwan, Republic of China.
Am J Physiol. 1993 Sep;265(3 Pt 1):C666-73. doi: 10.1152/ajpcell.1993.265.3.C666.
[N-methyl-3H]scopolamine methylchloride ([3H]NMS) was used to characterize the muscarinic receptors (mAChRs) in the intact cardiomyocytes. The specific binding of [3H]NMS was proportional to cell concentration, saturable with respect to [3H]NMS concentration, and time dependent. Scatchard analysis of binding isotherms showed that [3H]NMS bound to the freshly isolated and cultured cardiomyocytes with dissociation constants of 275 +/- 64 and 207 +/- 20 pM as well as maximum receptor densities of 0.13 +/- 0.09 and 5.36 +/- 0.20 fmol/10(5) cells, respectively. Heterogeneity of mAChRs was demonstrated by competitive binding experiments against [3H]NMS with M2 and M3 antagonists. These receptors (80%) exhibited high affinities for 11-([2-[(diethylamino)methyl]-1-piperidinyl]-acetyl)-5,11-dihydro- 6H-pyrido[2,3-b][1,4]benzodiazepine-6-one (AF-DX-116) and methoctramine similar to those of M2 subtype. The low-affinity M2 antagonist binding constants were close to those reported for M3 receptors and possessed high affinity for 4-diphenylacetoxyl-N-methylpiperidine (4-DAMP) and hexahydrosiladifenidol. On the basis of biochemical studies, AF-DX-116 blocked adenosine 3',5'-cyclic monophosphate (cAMP) inhibition with high affinity (pKB 7.4), while it antagonized inositol phosphate formation with low affinity (pKB 6.5). 4-DAMP possessed high affinity in blocking inositol phosphate formation (pKB 9.0) and low affinity for antagonism of cAMP inhibition (pKB 7.7). Although no other muscarinic receptor mRNA has been detected in these cells, these data suggest the presence of a second population of mAChRs, which may not be identical to the classical cardiac "M2" receptors.
[甲基 - 3H]东莨菪碱甲基氯（[3H]NMS）用于表征完整心肌细胞中的毒蕈碱受体（mAChRs）。[3H]NMS的特异性结合与细胞浓度成正比，相对于[3H]NMS浓度可饱和，且具有时间依赖性。结合等温线的Scatchard分析表明，[3H]NMS与新鲜分离和培养的心肌细胞结合的解离常数分别为275±64和207±20 pM，最大受体密度分别为0.13±0.09和5.36±0.20 fmol/10(5)个细胞。通过用M2和M3拮抗剂对[3H]NMS进行竞争性结合实验证明了mAChRs的异质性。这些受体（80%）对11 - （[2 - [（二乙氨基）甲基] - 1 - 哌啶基] - 乙酰基）-5,11 - 二氢 - 6H - 吡啶并[2,3 - b][1,4]苯并二氮杂卓 - 6 - 酮（AF - DX - 116）和甲奥克胺表现出与M2亚型相似的高亲和力。低亲和力的M2拮抗剂结合常数与报道的M3受体的常数接近，并且对4 - 二苯基乙酰氧基 - N - 甲基哌啶（4 - DAMP）和六氢硅二苯并环辛烯具有高亲和力。基于生化研究，AF - DX - 116以高亲和力（pKB 7.4）阻断腺苷3',5'-环磷酸（cAMP）的抑制，而以低亲和力（pKB 6.5）拮抗肌醇磷酸的形成。4 - DAMP在阻断肌醇磷酸形成方面具有高亲和力（pKB 9.0），而在拮抗cAMP抑制方面具有低亲和力（pKB 7.7）。尽管在这些细胞中未检测到其他毒蕈碱受体mRNA，但这些数据表明存在第二类mAChRs，其可能与经典的心脏“M2”受体不同。
