Department of Comparative Biomedical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, Louisiana.
Alcohol Clin Exp Res. 2013 Sep;37(9):1456-65. doi: 10.1111/acer.12133. Epub 2013 Jun 3.
In previous studies, we have shown that ethanol (EtOH) enhances the activity of stimulatory G protein (Gs)-stimulated membrane-bound adenylyl cyclase (AC). The effect is AC isoform specific, and the type 7 AC (AC7) is most responsive to EtOH. In this study, we employed a fluorescence resonance energy transfer (FRET)-based cyclic AMP (cAMP) sensor, Epac1-camps, to examine real-time temporal dynamics of EtOH effects on cAMP concentrations. To our knowledge, this is the first report on real-time detection of the EtOH effect on intracellular cAMP.
Hela cells were transfected with Epac1-camps, dopamine (DA) receptor D1a , and 1 isoform of AC (AC7 or AC3). Fluorescent images were captured using a specific filter set for cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and FRET, respectively, and FRET intensity was calculated on a pixel-by-pixel basis to examine changes in cAMP.
During 2-minute stimulation with DA, the cytoplasmic cAMP level quickly increased and then decreased to a plateau, where the cAMP level was higher than the level prior to stimulation with DA. EtOH concentration dependently increased cytoplasmic cAMP in cells transfected with AC7, while EtOH did not have effect on cells transfected with AC3. Similar trends were observed for cAMP at the plasma membrane and in the nucleus during 2-minute stimulation with DA. Unexpectedly, when cells expressing AC7 were stimulated with DA or other Gs-coupled receptor's ligand plus EtOH for 5 seconds, EtOH reduced cAMP concentration.
These results suggest that EtOH has 2 opposing effects on the cAMP-generating system in an AC isoform-specific manner, the enhancing effect on AC activity and the short-lived inhibitory effect. Thus, EtOH may have a different effect on cAMP depending on not only AC isoform but also the duration of exposure.
在之前的研究中，我们已经表明乙醇（EtOH）增强了刺激性 G 蛋白（Gs）刺激的膜结合腺苷酸环化酶（AC）的活性。这种作用是 AC 同工型特异性的，并且 7 型 AC（AC7）对 EtOH 的反应最敏感。在这项研究中，我们使用了基于荧光共振能量转移（FRET）的环 AMP（cAMP）传感器 Epac1-camps，来检测 EtOH 对 cAMP 浓度的实时时间动力学的影响。据我们所知，这是第一个关于实时检测 EtOH 对细胞内 cAMP 影响的报告。
Hela 细胞转染 Epac1-camps、多巴胺（DA）受体 D1a 和 1 型 AC（AC7 或 AC3）。使用特定的青色荧光蛋白（CFP）、黄色荧光蛋白（YFP）和 FRET 的滤光片组分别捕获荧光图像，并逐像素计算 FRET 强度，以检查 cAMP 的变化。
在 2 分钟的 DA 刺激期间，细胞质 cAMP 水平迅速增加，然后下降到一个平台，该平台上的 cAMP 水平高于刺激前的水平。EtOH 浓度依赖性地增加了转染有 AC7 的细胞中的细胞质 cAMP，而 EtOH 对转染有 AC3 的细胞没有影响。在 2 分钟的 DA 刺激期间，在质膜和核内也观察到类似的 cAMP 趋势。出乎意料的是，当表达 AC7 的细胞用 DA 或其他 Gs 偶联受体配体加 EtOH 刺激 5 秒时，EtOH 降低了 cAMP 浓度。
这些结果表明，EtOH 以 AC 同工型特异性的方式对 cAMP 生成系统具有 2 种相反的作用，即对 AC 活性的增强作用和短暂的抑制作用。因此，EtOH 对 cAMP 的影响不仅取决于 AC 同工型，还取决于暴露时间。
