Selective coupling of α2-adrenergic receptor subtypes to cyclic AMP-dependent reporter gene expression in transiently transfected JEG-3 cells

David J. Pepperl, John W Regan

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40 Citations (Scopus)

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 α2-adrenergic receptor subtypes. The reporter gene consists of a cAMP response element linked to the gene for chloramphenicol acetyltransferase (CAT). Plasmids encoding the α2-C10 (α2A), α2-C2 (α2B), or α2-C4 (α2C) receptor subtypes were co-transfected with a plasmid containing the reporter gene, and the ability of α2 receptor agonists to influence forskolin-stimulated CAT expression was examined. For α2-C10, agonists had a biphasic effect on forskolin-stimulated CAT expression. Thus, low (nanomolar) concentrations of agonist inhibited CAT expression by ∼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 α2 receptor subtypes. For α2-C4, agonists only inhibited forskolin-stimulated CAT expression, whereas for α2-C2 only potentiation of expression was seen. Each of these responses was specifically blocked by α2- but not α1- or β-adrenergic receptor antagonists. For α2-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 α2-C10. The potentiation of CAT expression, however, was not prevented by pertussis toxin pretreatment in cells transfected with either α2-C2 or α2-C10. In this transient expression system, each α2-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.

Original languageEnglish (US)
Pages (from-to)802-809
Number of pages8
JournalMolecular Pharmacology
Volume44
Issue number4
StatePublished - Oct 1993

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Chloramphenicol O-Acetyltransferase
Reporter Genes
Cyclic AMP
Adrenergic Receptors
Gene Expression
Colforsin
Pertussis Toxin
Second Messenger Systems
Plasmids
Choriocarcinoma
Adrenergic Antagonists
Response Elements
GTP-Binding Proteins
Adenylyl Cyclases
Complement System Proteins

ASJC Scopus subject areas

  • Pharmacology

Cite this

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title = "Selective coupling of α2-adrenergic receptor subtypes to cyclic AMP-dependent reporter gene expression in transiently transfected JEG-3 cells",
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 α2-adrenergic receptor subtypes. The reporter gene consists of a cAMP response element linked to the gene for chloramphenicol acetyltransferase (CAT). Plasmids encoding the α2-C10 (α2A), α2-C2 (α2B), or α2-C4 (α2C) receptor subtypes were co-transfected with a plasmid containing the reporter gene, and the ability of α2 receptor agonists to influence forskolin-stimulated CAT expression was examined. For α2-C10, agonists had a biphasic effect on forskolin-stimulated CAT expression. Thus, low (nanomolar) concentrations of agonist inhibited CAT expression by ∼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 α2 receptor subtypes. For α2-C4, agonists only inhibited forskolin-stimulated CAT expression, whereas for α2-C2 only potentiation of expression was seen. Each of these responses was specifically blocked by α2- but not α1- or β-adrenergic receptor antagonists. For α2-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 α2-C10. The potentiation of CAT expression, however, was not prevented by pertussis toxin pretreatment in cells transfected with either α2-C2 or α2-C10. In this transient expression system, each α2-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.",
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N2 - 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 α2-adrenergic receptor subtypes. The reporter gene consists of a cAMP response element linked to the gene for chloramphenicol acetyltransferase (CAT). Plasmids encoding the α2-C10 (α2A), α2-C2 (α2B), or α2-C4 (α2C) receptor subtypes were co-transfected with a plasmid containing the reporter gene, and the ability of α2 receptor agonists to influence forskolin-stimulated CAT expression was examined. For α2-C10, agonists had a biphasic effect on forskolin-stimulated CAT expression. Thus, low (nanomolar) concentrations of agonist inhibited CAT expression by ∼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 α2 receptor subtypes. For α2-C4, agonists only inhibited forskolin-stimulated CAT expression, whereas for α2-C2 only potentiation of expression was seen. Each of these responses was specifically blocked by α2- but not α1- or β-adrenergic receptor antagonists. For α2-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 α2-C10. The potentiation of CAT expression, however, was not prevented by pertussis toxin pretreatment in cells transfected with either α2-C2 or α2-C10. In this transient expression system, each α2-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.

AB - 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 α2-adrenergic receptor subtypes. The reporter gene consists of a cAMP response element linked to the gene for chloramphenicol acetyltransferase (CAT). Plasmids encoding the α2-C10 (α2A), α2-C2 (α2B), or α2-C4 (α2C) receptor subtypes were co-transfected with a plasmid containing the reporter gene, and the ability of α2 receptor agonists to influence forskolin-stimulated CAT expression was examined. For α2-C10, agonists had a biphasic effect on forskolin-stimulated CAT expression. Thus, low (nanomolar) concentrations of agonist inhibited CAT expression by ∼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 α2 receptor subtypes. For α2-C4, agonists only inhibited forskolin-stimulated CAT expression, whereas for α2-C2 only potentiation of expression was seen. Each of these responses was specifically blocked by α2- but not α1- or β-adrenergic receptor antagonists. For α2-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 α2-C10. The potentiation of CAT expression, however, was not prevented by pertussis toxin pretreatment in cells transfected with either α2-C2 or α2-C10. In this transient expression system, each α2-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.

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