Le. Black et al., CYCLIC AMP-MEDIATED DESENSITIZATION OF D-1 DOPAMINE RECEPTOR-COUPLED ADENYLYL-CYCLASE IN NS20Y NEUROBLASTOMA-CELLS, Molecular and cellular neurosciences, 5(6), 1994, pp. 567-575
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.