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.