Cyclic AMP-mediated desensitization of D1 dopamine receptor-coupled adenylyl cyclase in NS20Y neuroblastoma cells
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L E Black1, E M Smyk-Randall, D R Sibley
1Molecular Neuropharmacology Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892.
Abstract:
Cyclic AMP-mediated desensitization of D1 dopamine receptor-coupled adenylyl cyclase was investigated using NS20Y neuroblastoma cells. Pretreatment of the cells for 24 h with 8-(4-chlorophenylthio)-adenosine-3':5'-cyclic monophosphate (CPT-cAMP), a membrane-permeable analog of cAMP, resulted in an approximately 90% reduction of the maximum dopamine-stimulated adenylyl cyclase activity. In addition, there was a twofold reduction in the potency of dopamine for stimulating cAMP production that was not dependent on the concentration of Mg2+ in the assay. These effects of CPT-cAMP pretreatment were time dependent, showing a t1/2 of about 3 h and a maximum reduction after about 8 h. Receptor-binding activity, as measured using the D1-selective antagonist [3H]SCH-23390, also declined following CPT-cAMP pretreatment with a t1/2 of about 5 h and a maximum reduction of about 70% after 20 h. Saturation analysis indicated that the loss in radioligand binding was due to a reduction in maximum binding capacity (Bmax) with no alteration in receptor affinity (KD). The EC50 of CPT-cAMP for producing enzyme desensitization and D1 receptor downregulation was determined to be about 30 microM with a maximal response occurring at 1 mM. These regulatory effects of CPT-cAMP were pharmacologically specific as other analogs of cAMP, such as dibutryl-cAMP, 8-bromo-cAMP, and Sp-cAMPS, were capable of inducing D1 receptor desensitization and downregulation, whereas treatment of the cells with the cAMP antagonist Rp-cAMPS had no effect. Conversely, Rp-cAMPS was capable of blocking the regulatory effects of CPT-cAMP but was apparently without effect in blocking dopamine-induced desensitization.(ABSTRACT TRUNCATED AT 250 WORDS)
Insights
Cyclic adenosine monophosphate (cAMP) desensitizes D1 dopamine receptors and adenylyl cyclase in neuroblastoma cells. This desensitization involves reduced enzyme activity and receptor downregulation, mediated by specific cAMP analogs.
Area of Science:
- Neuroscience
- Molecular Pharmacology
- Cell Signaling
Background:
- Dopamine D1 receptors are coupled to adenylyl cyclase, regulating cyclic adenosine monophosphate (cAMP) production.
- Receptor desensitization is a crucial cellular mechanism for adapting to prolonged agonist stimulation.
- Understanding the molecular basis of D1 receptor desensitization is vital for neurological drug development.
Purpose of the Study:
- To investigate the role of cyclic adenosine monophosphate (cAMP) in the desensitization of D1 dopamine receptor-coupled adenylyl cyclase.
- To characterize the effects of a membrane-permeable cAMP analog (CPT-cAMP) on D1 receptor and adenylyl cyclase function.
- To determine the time course and pharmacological specificity of cAMP-mediated receptor and enzyme desensitization.
Main Methods:
- NS20Y neuroblastoma cells were pretreated with 8-(4-chlorophenylthio)-adenosine-3':5'-cyclic monophosphate (CPT-cAMP).
- Adenylyl cyclase activity was measured by dopamine stimulation.
- D1 receptor expression was assessed using radioligand binding assays with [3H]SCH-23390.
Main Results:
- CPT-cAMP pretreatment significantly reduced maximum dopamine-stimulated adenylyl cyclase activity (approx. 90%) and dopamine potency.
- D1 receptor binding capacity (Bmax) decreased by about 70% with no change in affinity (KD) following CPT-cAMP treatment.
- The desensitization and downregulation effects were time-dependent and pharmacologically specific to cAMP analogs, with Rp-cAMPS blocking CPT-cAMP effects.
Conclusions:
- Elevated intracellular cAMP levels induce desensitization and downregulation of D1 dopamine receptors and their coupled adenylyl cyclase.
- This cAMP-mediated process involves a reduction in receptor number rather than affinity.
- The findings highlight a critical intracellular feedback mechanism in dopamine receptor signaling.