CYCLIC AMP-MEDIATED DESENSITIZATION OF D-1 DOPAMINE RECEPTOR-COUPLED ADENYLYL-CYCLASE IN NS20Y NEUROBLASTOMA-CELLS

Cyclic AMP-mediated desensitization of D-1 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 similar to 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 t(1/2) of about 3 h and a maximum reduction after about 8 h. Receptor-binding activity, as measured using the D-1-selective antagonist [H-3]SCH-23390, also declined following CPT-cAMP pretreatment with a t(1/2) 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 (B-max) with no alteration in receptor affinity (K-D). The EC(50) of CPT-cAMP for producing enzyme desensitization and D-1 receptor downregulation was determined to be about 30 mu M 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 D-1 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/[H-3]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 D-1 receptor/adenylyl cyclase system and can induce a downregulation of D-1 receptor-binding activity, presumably involving degradation of receptor protein. (C) 1994 Academic Press, Inc.