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 a denylyl cyclase was investigated using NS20Y neuroblastoma cells. Pret reatment of the cells for 24 h with 8-(4-chlorophenylthio)-adenosine-3 ':5'cyclic monophosphate (CPT-cAMP), a membrane-permeable analog of cA MP, resulted in an similar to 90% reduction of the maximum dopamine-st imulated adenylyl cyclase activity. In addition, there was a twofold r eduction in the potency of dopamine for stimulating cAMP production th at 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-bindin g activity, as measured using the D-1-selective antagonist [H-3]SCH-23 390, also declined following CPT-cAMP pretreatment with a t(1/2) of ab out 5 h and a maximum reduction of about 70% after 20 h. Saturation an alysis indicated that the loss in radioligand binding was due to a red uction in maximum binding capacity (B-max) with no alteration in recep tor affinity (K-D). The EC(50) of CPT-cAMP for producing enzyme desens itization and D-1 receptor downregulation was determined to be about 3 0 mu M with a maximal response occurring at 1 mM. These regulatory eff ects of CPT-cAMP were pharmacologically specific as other analogs of c AMP, such as dibutryl-cAMP, 8-bromo-cAMP, and Sp-cAMPS, were capable o f inducing D-1 receptor desensitization and downregulation, whereas tr eatment 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-induc ed desensitization. In addition to desensitization and receptor downre gulation, CPT-cAMP treatment was found to promote functional uncouplin g of the receptor as suggested by a loss of high-affinity agonist bind ing observed in dopamine/[H-3]SCH-23390 competition assays following d esensitization. Removal of CPT-cAMP resulted in recovery to control ac tivities within 24 h. This recovery could be completely blocked throug h inhibition of cellular protein synthesis using cycloheximide. These data indicate that increasing the intracellular levels of cAMP can pro mote desensitization of the D-1 receptor/adenylyl cyclase system and c an induce a downregulation of D-1 receptor-binding activity, presumabl y involving degradation of receptor protein. (C) 1994 Academic Press, Inc.