Port J D, Hadcock J R, Malbon C C
Department of Pharmacology, School of Medicine, State University of New York, Stony Brook 11794-8651.
J Biol Chem. 1992 Apr 25;267(12):8468-72.
Cross-regulation from the stimulatory to the inhibitory adenylylcyclase pathways has been described (Hadcock, J. R., Ros, M., Watkins, D. C., and Malbon, C. C. (1990) J. Biol. Chem. 265, 14784-14790). More recently, persistent activation (48 h) of the inhibitory adenylylcyclase pathway has been shown to cross-regulate the stimulatory pathway (i) enhancing the maximal response of beta-adrenergic agonits, (ii) increasing the expression of beta-adrenergic receptor, and (iii) reducing the ED50 for the isoproterenol-stimulated response by 50-fold (Hadcock, J. R., Port, J. D., and Malbon, C. C. (1991) J. Biol. Chem. 266, 11915-11922). Here, we report that short term activation (60 min) of the inhibitory adenylylcyclase pathway of hamster smooth muscle DDT1MF-2 cells with the A1-adenosine receptor agonist N6-phenylisopropyladenosine (PIA) likewise enhances the stimulatory adenylylcyclase response to the beta-adrenergic agonist isoproterenol. The PIA effect was exerted at the level of the receptor, i.e., the beta-adrenergic receptor-mediated response was enhanced, whereas the guanosine 5'-O-(thiotriphosphate)- and forskolin-stimulated adenylylcyclase activities were largely unaffected. In contrast to longer term persistent activation of the inhibitory pathway, receptor number and affinity for 125I-labeled cyanopindolol were unaffected. Metabolic labeling of cells with [32P]orthophosphate and immuneprecipitation of beta-adrenergic receptors detected phosphorylation of the receptor in unstimulated cells and marked phosphorylation in cells challenged with epinephrine. When cells were challenged short term with PIA, the basal state of beta-adrenergic receptor phosphorylation was reduced by 75%. Treating cells with PIA in combination with the cAMP analog 8-(4-chlorophenylthio)adenosine cyclic AMP attenuated the enhanced receptor-mediated adenylylcyclase response observed in cells treated with PIA alone. These data suggest that short term cross-regulation from the inhibitory to stimulatory adenylylcyclase pathways results in the following: (i) decreased intracellular cAMP levels and protein kinase A activity, (ii) reduced phosphorylation of the beta 2-adrenergic receptor in the "basal" (i.e. unstimulated) state, and (iii) enhanced receptor-mediated activation of Gs.
从刺激性腺苷酸环化酶途径到抑制性腺苷酸环化酶途径的交叉调节已有报道（哈德科克，J.R.，罗斯，M.，沃特金斯，D.C.，和马尔邦，C.C.（1990）《生物化学杂志》265，14784 - 14790）。最近，已表明抑制性腺苷酸环化酶途径的持续激活（48小时）会对刺激性途径进行交叉调节：（i）增强β - 肾上腺素能激动剂的最大反应，（ii）增加β - 肾上腺素能受体的表达，以及（iii）将异丙肾上腺素刺激反应的半数有效剂量（ED50）降低50倍（哈德科克，J.R.，波特，J.D.，和马尔邦，C.C.（1991）《生物化学杂志》266，11915 - 11922）。在此，我们报道用A1 - 腺苷受体激动剂N6 - 苯基异丙基腺苷（PIA）对仓鼠平滑肌DDT1MF - 2细胞的抑制性腺苷酸环化酶途径进行短期激活（60分钟）同样会增强对β - 肾上腺素能激动剂异丙肾上腺素的刺激性腺苷酸环化酶反应。PIA的作用是在受体水平发挥的，即β - 肾上腺素能受体介导的反应增强，而鸟苷5'-O - （硫代三磷酸）和福斯可林刺激的腺苷酸环化酶活性基本未受影响。与抑制性途径的长期持续激活相反，受体数量以及对125I标记的氰胍心安的亲和力未受影响。用[32P]正磷酸盐对细胞进行代谢标记并对β - 肾上腺素能受体进行免疫沉淀，在未刺激的细胞中检测到受体的磷酸化，在肾上腺素刺激的细胞中检测到明显的磷酸化。当细胞用PIA进行短期刺激时，β - 肾上腺素能受体磷酸化的基础状态降低了75%。将细胞用PIA与环磷酸腺苷类似物8 - （4 - 氯苯硫基）腺苷酸环化一磷酸联合处理，可减弱在单独用PIA处理的细胞中观察到的增强的受体介导的腺苷酸环化酶反应。这些数据表明，从抑制性腺苷酸环化酶途径到刺激性途径的短期交叉调节会导致以下结果：（i）细胞内环磷酸腺苷水平和蛋白激酶A活性降低，（ii）“基础”（即未刺激）状态下β2 - 肾上腺素能受体的磷酸化减少，以及（iii）受体介导的Gs激活增强。
