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Keywords:

  • Adenylyl Cyclase;
  • Ethanol;
  • cAMP;
  • Fluorescence Resonance Energy Transfer;
  • Real-Time Measurement

Background

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.

Methods

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.

Results

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.

Conclusions

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.