Ethanol uses cAMP-independent signal transduction mechanisms to activate proenkephalin promoter activity in rat C6 glioma cells.
BACKGROUND
Previous in vivo studies show that acute ethanol exposure sequentially increases protein kinase A (PKA) activity, the phosphorylation of the adenosine 3':5'-cyclic monophosphate (cAMP)-dependent transcription factor, CREB, and finally proenkephalin gene expression. The present study was conducted to determine if ethanol could activate directly the adenylyl cyclase pathway and thus enhance proenkephalin promoter activity.


METHODS
Cultured rat C6 glioma cells stably transfected with a segment of the five prime flanking region of rat proenkephalin promoter (nucleotide -2700+/-53) ligated to the chloramphenicol acetyltransferase (CAT) reporter gene were employed to study the effects of ethanol on proenkephalin promoter activity. This region of proenkephalin promoter contains two cAMP response elements (CRE-1 and CRE-2) and one AP2 site located in the region upstream of the TATA box. Cultures were exposed to ethanol, isoproterenol, and phorbol-12, myristate 13-acetate (PMA) alone and in combination, in the presence and absence of PKA and protein kinase C (PKC) inhibitors.


RESULTS
Ethanol and isoproterenol increased proenkephalin promoter activity in a dose-dependent manner. Ethanol had an additive effect on maximal isoproterenol-stimulated proenkephalin promoter activity, which suggested that ethanol used a cAMP-independent signal transduction pathway to increase proenkephalin promoter activation. In contrast with isoproterenol, ethanol exposure did not increase cAMP accumulation, PKA activity, or the phosphorylated form of CREB. However, ethanol exposure modestly increased PKC activity. The PKA-specific inhibitor, Rp-cAMP, dampened isoproterenol-induced activation of CAT activity but did not alter ethanol's ability to increase CAT activity. However, the PKC inhibitors, chelerthyrine and G07874, abrogated ethanol's effect of CAT activity but did not alter isoproterenol's effects.


CONCLUSIONS
Ethanol enhanced proenkephalin promoter activity and potentiated isoproterenol-stimulated promoter activity through a cAMP-independent pathway.
Yang
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X
Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
Wand
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D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D000818:Animals; D002492:Central Nervous System Depressants / Q000494:pharmacology*; D002795:Choline O-Acetyltransferase / Q000187:drug effects* / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D004745:Enkephalins / Q000187:drug effects* / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D000431:Ethanol / Q000494:pharmacology*; D005910:Glioma / Q000378:metabolism; D007545:Isoproterenol / Q000494:pharmacology; D011401:Promoter Regions, Genetic / Q000187:drug effects / Q000502:physiology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D011498:Protein Precursors / Q000187:drug effects* / Q000378:metabolism; D051381:Rats; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D014407:Tumor Cells, Cultured
D000318:Adrenergic beta-Agonists; D002492:Central Nervous System Depressants; D004745:Enkephalins; D004791:Enzyme Inhibitors; D011498:Protein Precursors; C029992:proenkephalin; D000431:Ethanol; D000242:Cyclic AMP; D002795:Choline O-Acetyltransferase; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D007545:Isoproterenol