Sankary R M, Jones C A, Madison J M, Brown J K
Medical Service, Veterans Administration Medical Center, San Francisco, CA 94121.
Am Rev Respir Dis. 1988 Jul;138(1):145-50. doi: 10.1164/ajrccm/138.1.145.
Muscarinic agonists are potent constrictors of airway smooth muscle. In many tissues, muscarinic agonists also reduce intracellular cyclic AMP by inhibiting its synthesis. In airway smooth muscle, the role muscarinic agonists have in the regulation of cyclic AMP content is not established. The hypothesis of our study was that muscarinic agonists reduce cyclic AMP accumulation in dog tracheal smooth muscle, and that this reduction involves a pertussis toxin-sensitive regulatory protein (Gi) that couples occupancy of the muscarinic receptor by the agonist to inhibition of adenylate cyclase. We measured cyclic AMP accumulation in tracheal smooth muscle from 4 dogs, and found that acetylcholine (10(-4) M) diminished basal and isoproterenol-stimulated cyclic AMP accumulation by 37.6 +/- 12.1% and 39.4 +/- 1.9%, respectively (mean +/- SEM, p less than 0.05). This reduction of cyclic AMP was dose-dependent and inhibited by atropine (10(-5) M). Incubation of dog tracheal smooth muscle with pertussis toxin (12.5 micrograms/ml) for 21 h catalyzed covalent modification of a membrane protein with an approximate Mr of 40,000. In control strips, acetylcholine decreased isoproterenol-stimulated cyclic AMP content by 33.7 +/- 5.6% (p less than 0.05). However, in strips treated with pertussis toxin (10 micrograms/ml), acetylcholine decreased cyclic AMP by only 7.9 +/- 4.8%; this change was not significant. Thus, pertussis toxin (10 micrograms/ml) attenuated muscarinic cholinergic regulation of cyclic AMP. These findings are consistent with muscarinic cholinergic regulation of adenylate cyclase via Gi in dog tracheal smooth muscle. In addition, the techniques we employed should permit the evaluation of other functions of pertussis toxin-sensitive G proteins in airway smooth muscle.
毒蕈碱激动剂是气道平滑肌的强效收缩剂。在许多组织中，毒蕈碱激动剂还通过抑制细胞内环状AMP的合成来降低其含量。在气道平滑肌中，毒蕈碱激动剂在调节环状AMP含量方面的作用尚未明确。我们研究的假设是，毒蕈碱激动剂会减少犬气管平滑肌中环状AMP的积累，且这种减少涉及一种对百日咳毒素敏感的调节蛋白（Gi），该蛋白将激动剂对毒蕈碱受体的占据与腺苷酸环化酶的抑制联系起来。我们测量了4只犬的气管平滑肌中环状AMP的积累情况，发现乙酰胆碱（10⁻⁴ M）分别使基础状态和异丙肾上腺素刺激后的环状AMP积累减少了37.6±12.1%和39.4±1.9%（平均值±标准误，p<0.05）。环状AMP的这种减少呈剂量依赖性，并被阿托品（10⁻⁵ M）抑制。将犬气管平滑肌与百日咳毒素（12.5微克/毫升）孵育21小时，可催化一种分子量约为40000的膜蛋白的共价修饰。在对照条带中，乙酰胆碱使异丙肾上腺素刺激后的环状AMP含量降低了33.7±5.6%（p<0.05）。然而，在用百日咳毒素（10微克/毫升）处理的条带中，乙酰胆碱仅使环状AMP降低了7.9±4.8%；这种变化不显著。因此，百日咳毒素（10微克/毫升）减弱了毒蕈碱胆碱能对环状AMP的调节作用。这些发现与毒蕈碱胆碱能通过Gi对犬气管平滑肌中腺苷酸环化酶的调节作用一致。此外，我们采用的技术应该能够评估百日咳毒素敏感的G蛋白在气道平滑肌中的其他功能。
