Cyclic AMP-Mediated Desensitization of D1 Dopamine Receptor-Coupled Adenylyl Cyclase in NS20Y Neuroblastoma Cells

• Published in: Molecular and cellular neuroscience Vol. 5; no. 6; pp. 567 - 575
• Main Authors: Black, Lauren E., Smyk-Randall, Elzbieta M., Sibley, David R.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.12.1994
• Subjects: Adenylyl Cyclases - metabolism; Benzazepines - metabolism; Binding, Competitive; Cyclic AMP - analogs & derivatives; Cyclic AMP - pharmacology; Cyclic AMP - physiology; Dopamine - metabolism; Down-Regulation; Neuroblastoma - metabolism; Neuroblastoma - pathology; Receptors, Dopamine D1 - metabolism; Thionucleotides - pharmacology; Tumor Cells, Cultured
• ISSN: 1044-7431; 1095-9327
• DOI: 10.1006/mcne.1994.1069

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 ∼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 D2-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 μM 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. In addition to desensitization and receptor downregulation, CPT-cAMP treatment was found to promote functional uncoupling of the receptor as suggested by a loss of high-affinity agonist binding observed in dopamine/[3H]SCH-23390 competition assays following desensitization. Removal of CPT-cAMP resulted in recovery to control activities within 24 h. This recovery could be completely blocked through inhibition of cellular protein synthesis using cycloheximide. These data indicate that increasing the intracellular levels of cAMP can promote desensitization of the D1 receptor/adenylyl cyclase system and can induce a downregulatlon of D1 receptor-binding activity, presumably involving degradation of receptor protein.