Murphy M G, Byczko Z
Department of Pharmacology, Dalhousie University, Halifax, Nova Scotia, Canada.
Biochem Pharmacol. 1989 Oct 1;38(19):3289-95. doi: 10.1016/0006-2952(89)90627-8.
We have examined the effects of R-phenylisopropyladenosine (R-PIA) and other adenosine analogues on basal, prostaglandin E1 (PGE1)- and forskolin-stimulated cyclic AMP (cAMP) formation in intact N1E-115 neuroblastoma cells, to determine whether the cells contain A1 adenosine receptors that are negatively coupled with adenylate cyclase. Basal levels of cAMP (68 +/- 7 pmol/mg protein; mean +/- SE, N = 15) were not altered by low concentrations of R-PIA. The apparent lack of inhibition was not due to increases in cAMP due to activation of a stimulatory A2 receptor by endogenously-synthesized adenosine. By comparison, low levels of R-PIA did reduce significantly (P less than 0.05) PGE1-dependent increases in cAMP formation (maximum response to PGE1, 972 +/- 77 pmol cAMP/mg protein; EC50 for PGE1, 0.2 microM). Inhibition was dose dependent, and resulted in a 30-50% maximum reduction in production stimulated by PGE1. Nanomolar concentrations of R-PIA elicited half-maximal inhibition; the inhibitory response was blocked by 8-phenyltheophylline (8-PT). The order of potencies of several adenosine analogues in eliciting this response suggested that inhibition was mediated by an A1 adenosine receptor. Examination of the effects of R-PIA on forskolin-stimulated cAMP formation yielded several interesting findings. First, stimulation by the diterpene by itself was blocked by both adenosine deaminase (ADA) and 8-PT (40 and 25% inhibition respectively). Low concentrations of R-PIA (less than 10(-6) M) had no effect on forskolin-stimulated cAMP production. At higher levels (greater than or equal to 10(-6) M) the analogues acted synergistically with the diterpene, to yield cAMP levels that were up to 3-fold higher than the additive effect of the two agents. Potentiation was stereospecific, Ca2+ dependent, and was blocked by 8-PT. The results of this study suggest that, in N1E-115 neuroblastoma cells, inhibitory A1 receptors are not stimulated in response to non-specific elevations in cAMP, but are associated with specific stimulatory receptors such as those activated by PGE1.
我们研究了R-苯异丙基腺苷（R-PIA）和其他腺苷类似物对完整的N1E-115神经母细胞瘤细胞中基础的、前列腺素E1（PGE1）和福斯高林刺激的环磷酸腺苷（cAMP）生成的影响，以确定这些细胞中是否含有与腺苷酸环化酶负偶联的A1腺苷受体。低浓度的R-PIA不会改变cAMP的基础水平（68±7 pmol/mg蛋白质；平均值±标准误，N = 15）。这种明显缺乏抑制作用并非是由于内源性合成的腺苷激活刺激性A2受体导致cAMP增加所致。相比之下，低水平的R-PIA确实显著降低了（P < 0.05）PGE1依赖性的cAMP生成增加（对PGE1的最大反应，972±77 pmol cAMP/mg蛋白质；PGE1的EC50，0.2 μM）。抑制作用呈剂量依赖性，导致PGE1刺激产生的cAMP最大减少30 - 50%。纳摩尔浓度的R-PIA引起半数最大抑制；该抑制反应被8-苯基茶碱（8-PT）阻断。几种腺苷类似物引发此反应的效力顺序表明抑制作用是由A1腺苷受体介导的。研究R-PIA对福斯高林刺激的cAMP生成的影响产生了几个有趣的发现。首先，二萜单独刺激被腺苷脱氨酶（ADA）和8-PT阻断（分别抑制40%和25%）。低浓度的R-PIA（< 10^(-6) M）对福斯高林刺激的cAMP产生没有影响。在较高水平（≥10^(-6) M）时，类似物与二萜协同作用，使cAMP水平比两种药物的加和效应高出多达3倍。增强作用具有立体特异性、Ca2+依赖性，并被8-PT阻断。本研究结果表明，在N1E-115神经母细胞瘤细胞中，抑制性A1受体不会因cAMP的非特异性升高而被激活，但与特定的刺激性受体相关，如那些被PGE1激活的受体。
