Abstract
Previous research has indicated that the cyclic AMP (cAMP) signal transduction system plays an important role in the predisposition to and development of ethanol abuse in humans. Our laboratory has demonstrated that ethanol is capable of enhancing adenylyl cyclase (AC) activity. This effect is AC isoform-specific; type 7 AC (AC7) is most enhanced by ethanol. Therefore, we hypothesized that the expression of a specific AC isoform will play a role on the effect of ethanol on cAMP regulated gene expression. We employed NIH 3T3 cells transfected with AC7 or AC3 as a model system. To evaluate ethanol's effects on cAMP regulated gene expression, a luciferase reporter gene driven by a cAMP inducing artificial promoter was utilized. Stimulation of AC activity leads to an increase in the reporter gene activity. This increase was enhanced in the presence of ethanol in cells expressing AC7, while cells expressing AC3 did not respond to ethanol. cAMP reporter gene expression was increased in the presence of 8-bromo-cAMP; this expression was not enhanced by ethanol. These observations are consistent with our hypothesis. The basal level of CREB phosphorylation was high and did not change by cAMP stimulation or in the presence of ethanol. However, there were significant changes in the TORC3 amount in nuclei depending on stimulation conditions. The results suggest that nuclear translocation of TORC3 plays a more important role than CREB phosphorylation in the observed changes in the cAMP driven reporter gene activity.
Highlights
USA has implicated a link between cyclic AMP (cAMP) signaling and ethanol abuse [7,8,9,10,11]
As an indicator of changes in cAMP, normalized normalized FRET (nFRET) values were plotted over time (Fig. 1). nFRET quickly decreased after the addition of DA in cells expressing AC7 (Fig. 1A); this decrease indicates an increase in intracellular cAMP
After perfusion with DA, the value of nFRET reached its minimum within 20 s and remained constant during the remainder of the treatment; when DA þ ethanol were added, nFRET was further decreased, which indicated higher level of cAMP in cells compared to the cells treated with DA alone
Summary
Cyclic AMP signaling elements including specific isoforms of AC: AC1, AC5, and AC8 have been implicated to play a vital role in the behavioral and physiological responses to ethanol in animals [12,13,14]. Ethanol is capable of altering the activities of cAMP signaling pathways. This alteration is evident in animal models and model cell culture systems. ACs play an important role in cAMP-dependent activation of protein kinase A (PKA) as well as regulating cAMP response element binding protein (CREB) function. CREB mediates the activation of cAMP-responsive genes by binding as a dimer to CRE; phosphorylated CREB (pCREB) causes an increase in transcription.
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