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Selective coupling of alpha 2-adrenergic receptor subtypes to cyclic AMP-dependent reporter gene expression in
1Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson 85721.
Abstract:
A cAMP-dependent reporter gene has been used in transiently transfected human choriocarcinoma (JEG-3) cells to examine the second messenger coupling of the human alpha 2-adrenergic receptor subtypes. The reporter gene consists of a cAMP response element linked to the gene for chloramphenicol acetyltransferase (CAT). Plasmids encoding the alpha 2-C10 (alpha 2A), alpha 2-C2 (alpha 2B), or alpha 2-C4 (alpha 2C) receptor subtypes were co-transfected with a plasmid containing the reporter gene, and the ability of alpha 2 receptor agonists to influence forskolin-stimulated CAT expression was examined. For alpha 2-C10, agonists had a biphasic effect on forskolin-stimulated CAT expression. Thus, low (nanomolar) concentrations of agonist inhibited CAT expression by approximately 60%, whereas high (micromolar) concentrations reversed this inhibition and could even potentiate CAT expression by as much as 140%. A significantly different pattern of coupling was observed for the other alpha 2 receptor subtypes. For alpha 2-C4, agonists only inhibited forskolin-stimulated CAT expression, whereas for alpha 2-C2 only potentiation of expression was seen. Each of these responses was specifically blocked by alpha 2- but not alpha 1- or beta-adrenergic receptor antagonists. For alpha 2-C4, the inhibition of forskolin-stimulated CAT expression was prevented by pretreatment of the cells with pertussis toxin. This was also true for the inhibition obtained with alpha 2-C10. The potentiation of CAT expression, however, was not prevented by pertussis toxin pretreatment in cells transfected with either alpha 2-C2 or alpha 2-C10. In this transient expression system, each alpha 2-adrenergic receptor subtype had access to the same complement of G proteins, adenylyl cyclase, and other second messengers. It would appear, therefore, that the potential for the activation of unique intracellular responses exists even among closely related receptor subtypes.