Regulation and functional characterization of a rat recombinant dopamine D3 receptor.
We stably expressed a rat D3 receptor cDNA in C6 glioma cells (C6-D3 cells), quantifying receptor expression with the radioligands [125I]epidepride (KD = 0.1 nM) and [3H]spiperone (KD = 0.7 nM). As reported previously for D2 receptors, quinpirole induced a 9-16% increase in the rate of extracellular acidification by C6-D3 cells. The acidification was inhibited by epidepride and by the Na+/H+ antiporter inhibitors, amiloride and methylisobutylamiloride, but pertussis toxin treatment had no effect on quinpirole-induced extracellular acidification. These data suggest that D3 receptor stimulation of Na+/H+ exchange in C6 glioma cells is not mediated by the pertussis toxin-sensitive G proteins, Gi or G(o). Overnight treatment of C6-D3 cells with N-propylnorapomorphine, dopamine, or quinpirole resulted in large concentration-dependent increases (up to 500%) in the density of D3 receptors on membranes prepared from the cells. Antagonists had smaller, variable effects on the density of D3 receptors in C6-D3 cells, except for domperidone, which significantly increased the density of D3 receptors. Treatment with pertussis toxin had no effect on the agonist-induced receptor up-regulation, indicating that an interaction with pertussis toxin-sensitive G proteins was not required. Densitometry analysis of Northern blots of RNA prepared from C6-D3 cells showed no significant N-propylnorapomorphine-induced increase in D3 receptor message. Treatment with cycloheximide, however, completely prevented receptor up-regulation by N-propylnorapomorphine. Pretreatment of C6-D2 cells with 10 microM DA resulted in a substantial heterologous sensitization, in which isoproterenol-stimulated adenylyl cyclase activity was enhanced more than twofold.(ABSTRACT TRUNCATED AT 250 WORDS)
Cox
B A
BA
Department of Psychiatry, Oregon Health Sciences University, Portland 97201, USA.
Rosser
M P
MP
Kozlowski
M R
MR
Duwe
K M
KM
Neve
R L
RL
Neve
K A
KA
D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D018076:DNA, Complementary; D004305:Dose-Response Relationship, Drug; D015870:Gene Expression; D005910:Glioma / Q000235:genetics*; D011869:Radioligand Assay; D051381:Rats; D011919:Rats, Inbred Strains; D011954:Receptors, Dopamine / Q000187:drug effects* / Q000502:physiology*; D011995:Recombination, Genetic; D015398:Signal Transduction / Q000502:physiology*; D013997:Time Factors
D018076:DNA, Complementary; D011954:Receptors, Dopamine; D000584:Amiloride