Desensitization of alpha 2-adrenergic receptors in NG 108 15 cells by (-)-adrenaline and phorbol 12-myristate 13-acetate.
alpha 2-Adrenergic receptors on NG 108 15 cell membranes were identified by [3H]rauwolscine binding: Bmax. = 661 +/- 81 fmol/mg of protein, Kd = 6.9 +/- 2.5 nM (mean +/- S.E.M., n = 6). On intact cells, stimulation of these receptors by (-)-adrenaline inhibited the prostaglandin-E1-stimulated adenylate cyclase activity by about 60%. The effect of (-)-adrenaline was pertussis-toxin-sensitive, indicating the involvement of an inhibitory G protein. (-)-Adrenaline/[3H]rauwolscine competition-binding experiments revealed that only 50% of the alpha 2 receptors were coupled to G proteins (i.e. displayed high agonist affinity). Pre-treatment of the cells with 20 microM-(-)-adrenaline provoked homologous desensitization of the alpha 2 receptors. The alpha 2-adrenergic response decreased after a time lag of about 2 h, to reach a minimum after 12 h. The bradykinin and muscarinic responses were not affected. The alpha 2-receptor concentration decreased without time lag. The high-agonist-affinity sites disappeared more rapidly (t1/2 = 42 min) than did the low-affinity uncoupled sites (t1/2 approx. 20 h). In contrast, pertussis-toxin-mediated [32P]ADP-ribosylation of inhibitory G proteins was unaffected by the pre-treatment. Pretreatment of intact NG 108 15 cells with 1 microM-phorbol 12-myristate 13-acetate (PMA) provoked a rapid decrease of the alpha 2-adrenergic response. The effect was nearly complete after 40 min. PMA also decreased the bradykinin response, suggesting a heterologous desensitization process. The alpha 2-receptor concentration, the (-)-adrenaline competition-binding curves and the pertussis- and cholera-toxin-mediated [32P]ADP-ribosylation of their respective G proteins were not affected.
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Department of Protein Chemistry, Vrije Universiteit Brussel, Belgium.
De Backer
J P
JP
André
C
C
Vauquelin
G
G
D000262:Adenylyl Cyclases / Q000378:metabolism; D000527:Alprostadil / Q000378:metabolism; D000818:Animals; D001667:Binding, Competitive; D001920:Bradykinin / Q000494:pharmacology; D002217:Carbachol / Q000494:pharmacology; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism; D004837:Epinephrine / Q000494:pharmacology*; D019204:GTP-Binding Proteins / Q000378:metabolism; D009474:Neurons; D011942:Receptors, Adrenergic, alpha / Q000187:drug effects*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D015016:Yohimbine / Q000378:metabolism
D011942:Receptors, Adrenergic, alpha; D015016:Yohimbine; D002217:Carbachol; D000242:Cyclic AMP; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases; D000527:Alprostadil; D013755:Tetradecanoylphorbol Acetate; D001920:Bradykinin; D004837:Epinephrine