Agonist-induced desensitization of D1-dopamine receptors linked to adenylyl cyclase activity in cultured NS20Y neuroblastoma cells.
NS20Y neuroblastoma cells expressing a homogeneous population of D1-dopamine receptors were used in the present study as a model system to investigate the mechanisms of agonist-induced stimulation and desensitization of D1 receptor-coupled adenylyl cyclase activity. Membrane prepared from NS20Y cells showed a pharmacologically specific, dose-dependent increase in cAMP production in response to various dopaminergic agonists. Dopamine exhibited an EC50 of 5 microM, and at 100 microM a maximal stimulation of 3-4-fold over basal enzyme activity was observed, which could be selectively antagonized by the active stereoisomers of SCH-23390 and butaclamol. Preincubation of NS20Y cells with dopamine induced homologous desensitization of D1 receptor-coupled adenylyl cyclase activity, decreasing dopamine- but not prostaglandin-, adenosine-, or forskolin-stimulated cAMP production. Desensitization did not affect the EC50 for dopamine but resulted in an 85-90% reduction in the maximal response. Dopamine-induced desensitization of adenylyl cyclase activity was found to be both dose and time dependent. As early as 5 min after preincubation with dopamine, cAMP production was decreased by 45-50%, with maximal desensitization occurring by 90 min. Preincubation of NS20Y cells with dopamine also induced a decrease in D1 receptor ligand binding activity, as assessed with the radiolabeled antagonist [3H]SCH-23390. This decrease in binding activity occurred more slowly than the loss of enzyme activity, not achieving maximal levels until after 3 hr. [3H]SCH-23390 saturation binding isotherms in control and maximally desensitized NS20Y cell membranes revealed no change in affinity (KD); however, a 65-70% decrease in receptor number (Bmax) was observed. Because the maximal and temporal decrease in D1 receptors does not correlated with the decrease in dopamine-stimulated enzyme activity, the desensitization may involve a functional uncoupling of the D1 receptor in addition to receptor down-regulation. This is further suggested by a loss in high affinity agonist binding observed in agonist/[3H]SCH-23390 competition experiments after desensitization. Removal of dopamine after maximal desensitization/down-regulation results in recovery to control values by 24 hr. This recovery is mostly, but not completely, blocked by protein synthesis inhibitors, suggesting an involvement of receptor degradation in the desensitization process.
Barton
A C
AC
Experimental Therapeutics Branch, National Institute for Neurological Disorders and Stroke, National Institute of Health, Bethesda, Maryland.
Sibley
D R
DR
D000262:Adenylyl Cyclases / Q000032:analysis*; D001552:Benzazepines / Q000378:metabolism; D004298:Dopamine / Q000494:pharmacology; D015259:Dopamine Agents / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D006801:Humans; D009447:Neuroblastoma / Q000378:metabolism; D010766:Phosphorylation; D011954:Receptors, Dopamine / Q000187:drug effects*; D017447:Receptors, Dopamine D1; D014407:Tumor Cells, Cultured
D001552:Benzazepines; D015259:Dopamine Agents; D011954:Receptors, Dopamine; D017447:Receptors, Dopamine D1; D006165:Guanylyl Imidodiphosphate; D000262:Adenylyl Cyclases; D004298:Dopamine