Zhang L B, Buxton I L
Department of Pharmacology, University of Nevada School of Medicine, Reno 89557.
Mol Pharmacol. 1991 Dec;40(6):952-9.
We have, in the accompanying work, demonstrated the coexistence of M2 and M3 muscarinic receptors in the circular smooth muscle of canine colon. In the present study, the effects of muscarinic receptor stimulation on phosphoinositide turnover and adenylate cyclase activity were examined. In myo-[3H]inositol-labeled circular smooth muscle strips, carbachol caused a concentration-dependent (EC50 = 5 microM) increase in [3H]inositol phosphate production. The more M3 receptor-selective muscarinic antagonist pirenzepine (KB = 53 nM) was approximately 60 times more potent than the more M2-selective agent AF-DX 116 (KB = 3 microM) in blocking carbachol-elicited accumulation of [3H]inositol phosphates. The carbachol-stimulated increase in [3H]inositol phosphate accumulation was not affected by pretreatment of the tissue with pertussis toxin (200 ng/ml, 3 hr). Within the first minute, carbachol (100 microM) caused a rapid and transient increase of [3H]inositol 1,4,5-trisphosphate production that oscillated continuously in the presence of agonist (120 min). The accumulation of [3H]inositol 1,3,4-trisphosphate was also extremely rapid, reaching a peak at 15 sec. The accumulation of [3H]inositol monophosphate was delayed and progressively increased over 30 min. [3H]inositol 1,3,4,5-tetrakisphosphate, although not detectable in the first minute, accumulated to significant levels over 30 min in the presence of agonist. Addition of carbachol in the adenylate cyclase assay caused inhibition of forskolin-stimulated [32P]cAMP production and blocked forskolin-stimulated cAMP accumulation in the intact tissue. The inhibitory effects of carbachol on adenylate cyclase were blocked by atropine, AF-DX 116, and 4-diphenylacetoxy-N-methylpiperidine methobromide but were unaffected by the more M3-selective agent pirenzepine (1 microM). Pretreatment of tissues with pertussis toxin completely eliminated M2 receptor-mediated inhibition of adenylate cyclase activity, without altering inositol 1,4,5-trisphosphate accumulation. We conclude that muscarinic receptor stimulation of inositol trisphosphate production is mediated by the M3 receptor coupled to a pertussis toxin-insensitive GTP-binding protein and results in the rapid formation of inositol tetrakisphosphate, whereas inhibition of adenylate cyclase activity is mediated by the M2 subtype of muscarinic receptor coupled to the pertussis toxin-sensitive GTP-binding protein Gi.
在随附的研究中，我们已证明M2和M3毒蕈碱受体共存于犬结肠的环形平滑肌中。在本研究中，检测了毒蕈碱受体刺激对磷酸肌醇代谢和腺苷酸环化酶活性的影响。在肌醇-[3H]标记的环形平滑肌条中，卡巴胆碱引起[3H]肌醇磷酸生成呈浓度依赖性增加（EC50 = 5 microM）。在阻断卡巴胆碱引起的[3H]肌醇磷酸积累方面，M3受体选择性更强的毒蕈碱拮抗剂哌仑西平（KB = 53 nM）的效力比M2选择性更强的药物AF-DX 116（KB = 3 microM）高约60倍。用百日咳毒素（200 ng/ml，3小时）预处理组织后，卡巴胆碱刺激引起的[3H]肌醇磷酸积累不受影响。在第一分钟内，卡巴胆碱（100 microM）引起[3H]肌醇1,4,5-三磷酸生成迅速且短暂增加，在激动剂存在下持续振荡（120分钟）。[3H]肌醇1,3,4-三磷酸的积累也极其迅速，在15秒时达到峰值。[3H]肌醇单磷酸的积累延迟，并在30分钟内逐渐增加。[3H]肌醇1,3,4,5-四磷酸虽然在第一分钟内无法检测到，但在激动剂存在下30分钟内积累到显著水平。在腺苷酸环化酶测定中加入卡巴胆碱导致福斯高林刺激的[32P]cAMP生成受到抑制，并阻断了福斯高林刺激的完整组织中cAMP的积累。卡巴胆碱对腺苷酸环化酶的抑制作用被阿托品、AF-DX 116和4-二苯基乙酰氧基-N-甲基哌啶甲溴化物阻断，但不受M3受体选择性更强的药物哌仑西平（1 microM）影响。用百日咳毒素预处理组织完全消除了M2受体介导的腺苷酸环化酶活性抑制，而不改变肌醇1,4,5-三磷酸的积累。我们得出结论，毒蕈碱受体刺激肌醇三磷酸生成是由与百日咳毒素不敏感的GTP结合蛋白偶联的M3受体介导的，并导致肌醇四磷酸的快速形成，而腺苷酸环化酶活性的抑制是由与百日咳毒素敏感的GTP结合蛋白Gi偶联的M2毒蕈碱受体亚型介导的。
