Schaefer O P, Ethier M F, Madison J M
Department of Medicine, University of Massachusetts Medical Center, Worcester, USA.
Am J Respir Cell Mol Biol. 1995 Aug;13(2):217-26. doi: 10.1165/ajrcmb.13.2.7626289.
The goal of this study was to characterize the receptors and coupling mechanisms mediating muscarinic inhibition of adenylyl cyclase activity in bovine tracheal smooth muscle. In radioligand binding experiments, methoctramine and AF-DX 116 competed for approximately 85% of the 3H-quinuclidynyl benzilate (3H-QNB) binding sites on intact cells with high affinities (-log KI of 7.73 +/- 0.16 and 6.67 +/- 0.31, respectively) characteristic of binding to M2 receptors. The antagonist 4-diphenylacetoxy-N-methylpiperidine (4-DAMP) bound the receptors on intact cells with an affinity (-log KI = 7.76 +/- 0.21) characteristic of binding at M2 receptors. In experiments measuring 3',5'-cyclic adenosine monophosphate (cAMP) accumulation, methoctramine, AF-DX 116, and 4-DAMP antagonized the inhibitory effect of carbachol on isoproterenol-stimulated cAMP accumulation with potencies consistent with mediation by M2 muscarinic receptors (-log Kb of 8.01 +/- 0.22 to 7.58 +/- 0.25 for methoctramine; 7.43 +/- 0.36 to 7.02 +/- 0.30 for AF-DX 116; and 7.60 +/- 0.21 for 4-DAMP). In other experiments, 24 +/- 3% of the inhibitory effect of carbachol was not reversed by 60 min exposure to atropine. Moreover, pertussis toxin (10, 250, and 1,000 ng/ml) decreased only a portion of the inhibitory effect of carbachol (8 +/- 19%, 32 +/- 10%, and 33 +/- 8%, respectively) on cAMP accumulation. These findings indicated that M2 receptors were coupled to adenylyl cyclase in trachealis cells, but that coupling mechanisms in addition to those of pertussis toxin-sensitive guanine nucleotide binding proteins were involved. Since the inhibitory effect of carbachol (10(-8) M) on isoproterenol-stimulated cAMP accumulation was decreased from 20 +/- 4% to -1 +/- 5% (n = 6) by okadaic acid (1 microM), protein phosphatases may regulate the processes coupling muscarinic receptors to adenylyl cyclase.
本研究的目的是鉴定介导牛气管平滑肌中M型胆碱能受体对腺苷酸环化酶活性抑制作用的受体及偶联机制。在放射性配体结合实验中，甲氧基氨甲酰胆碱和AF-DX 116以高亲和力（-log KI分别为7.73±0.16和6.67±0.31）竞争完整细胞上约85%的3H-喹核醇基苯甲酸酯（3H-QNB）结合位点，这是与M2受体结合的特征。拮抗剂4-二苯基乙酰氧基-N-甲基哌啶（4-DAMP）以与M2受体结合的特征亲和力（-log KI = 7.76±0.21）结合完整细胞上的受体。在测量3',5'-环磷酸腺苷（cAMP）积累的实验中，甲氧基氨甲酰胆碱、AF-DX 116和4-DAMP拮抗了卡巴胆碱对异丙肾上腺素刺激的cAMP积累的抑制作用，其效能与M2型胆碱能受体介导作用一致（甲氧基氨甲酰胆碱的-log Kb为8.01±0.22至7.58±0.25；AF-DX 116的为7.43±0.36至7.02±0.30；4-DAMP的为7.60±0.21）。在其他实验中，60分钟的阿托品暴露并未逆转卡巴胆碱24±3%的抑制作用。此外，百日咳毒素（10、250和1000 ng/ml）仅降低了卡巴胆碱对cAMP积累抑制作用的一部分（分别为8±19%、32±10%和33±8%）。这些发现表明，M2受体与气管平滑肌细胞中的腺苷酸环化酶偶联，但除了百日咳毒素敏感的鸟嘌呤核苷酸结合蛋白外，还涉及其他偶联机制。由于冈田酸（1 microM）使卡巴胆碱（10^-8 M）对异丙肾上腺素刺激的cAMP积累的抑制作用从20±4%降至-1±5%（n = 6），蛋白磷酸酶可能调节M型胆碱能受体与腺苷酸环化酶的偶联过程。
