Bruni P, Burns D L, Hewlett E L, Moss J
Mol Pharmacol. 1985 Aug;28(2):229-34.
As noted previously, in N1E-115 neuroblastoma cells, carbamylcholine, a muscarinic cholinergic agonist, increased cGMP over 15-fold and decreased basal and prostaglandin E1 (PGE1)-stimulated cAMP content. In contrast to the stimulatory effects of PGE1 on cAMP, which were immediate, the carbamylcholine-induced decrease in basal and PGE1-stimulated cAMP exhibited a delay. The delay in carbamylcholine inhibition was independent of the extent of adenylate cyclase activation. Although basal cAMP content was suppressed within 30 sec after addition of carbamylcholine, inhibition was not maximal for at least 2 min following agonist addition; the delay was similar in cells exposed to PGE1 for 10 min prior to carbamylcholine but could be eliminated by incubation of the cells with muscarinic cholinergic agonist for 5 min prior to addition of prostaglandin. N1E-115 neuroblastoma cells possess a 41,000-Da membrane protein believed to be a component of the inhibitory GTP-binding protein of adenylate cyclase that is ADP ribosylated by pertussis toxin. Incubation of the cells with pertussis toxin prior to the addition of carbamylcholine reduced the maximal extent of inhibition of cAMP content and prevented the [32P]ADP-ribosylation of a 41,000-Da protein by toxin and [32P]NAD in membrane preparations from these cells. Incubation of cells with pertussis toxin, however, did not significantly alter the dose-response curve for carbamylcholine effects on cGMP. Even high concentrations of carbamylcholine, effective in stimulating cGMP, had minimal effects on cAMP content in toxin-treated cells; thus, ADP-ribosylation of Gi converts the adenylate cyclase but not the guanylate cyclase system to an agonist-insensitive state.
如前所述，在N1E - 115神经母细胞瘤细胞中，毒蕈碱型胆碱能激动剂氨甲酰胆碱使环鸟苷酸（cGMP）增加超过15倍，并降低基础和前列腺素E1（PGE1）刺激的环磷酸腺苷（cAMP）含量。与PGE1对cAMP的即时刺激作用相反，氨甲酰胆碱诱导的基础和PGE1刺激的cAMP降低存在延迟。氨甲酰胆碱抑制作用的延迟与腺苷酸环化酶激活程度无关。尽管在加入氨甲酰胆碱后30秒内基础cAMP含量就受到抑制，但在加入激动剂后至少2分钟抑制作用才达到最大；在加入氨甲酰胆碱前先用PGE1处理10分钟的细胞中延迟情况类似，但在加入前列腺素前先用毒蕈碱型胆碱能激动剂孵育细胞5分钟可消除这种延迟。N1E - 115神经母细胞瘤细胞具有一种41000道尔顿的膜蛋白，据信它是腺苷酸环化酶抑制性GTP结合蛋白的一个组分，该蛋白可被百日咳毒素进行ADP核糖基化。在加入氨甲酰胆碱前先用百日咳毒素孵育细胞，可降低cAMP含量的最大抑制程度，并阻止毒素和[32P]NAD对这些细胞膜制剂中41000道尔顿蛋白的[32P]ADP核糖基化。然而，用百日咳毒素孵育细胞并未显著改变氨甲酰胆碱对cGMP作用的剂量反应曲线。即使是高浓度的氨甲酰胆碱，虽能有效刺激cGMP，但对毒素处理过的细胞中的cAMP含量影响极小；因此，Gi的ADP核糖基化使腺苷酸环化酶系统而非鸟苷酸环化酶系统转变为对激动剂不敏感的状态。
