Regulation and functional characterization of a rat recombinant dopamine D3 receptor

Abstract

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)

Increases in Dopamine D3 Receptor Binding in Rats Receiving a Cocaine Challenge at Various Time Points after Cocaine Self-Administration: Implications for Cocaine-Seeking Behavior

“…It was somewhat surprising that repeated 7-OH-DPAT administration attenuated the time-dependent increase in D 3 receptor binding given that previous research suggests D 3 receptors are upregulated by dopamine and dopamine agonists (Cox et al, 1995;Fauchey et al, 2000;Stanwood et al, 2000b). However, repeated administration of dopamine agonists does not always follow this general rule.…”

“…Thus, the region-specific effects observed in the present study suggest that the changes in D 3 receptor binding may be functionally related to plasticity in the neural systems thought to underlie the rewarding and incentive motivational effects of cocaine. Dopamine D 3 receptors typically exhibit a paradoxical downregulation in response to dopamine depletion (Guillin et al, 2001;Lévesque et al, 1995;Morissette et al, 1998;Stanwood et al, 2000b) and upregulation in response to dopamine and dopamine agonist administration (Cox et al, 1995;Fauchey et al, 2000;Stanwood et al, 2000b). Therefore, we suggest that the increase in D 3 receptor binding during the course of abstinence from cocaine self-administration may involve enhancement in synaptic concentrations of dopamine.…”

“…In these studies, phasic dopamine levels were likely abnormally high, and we speculate that the increase in phasic dopamine may invoke D 3 receptor regulatory responses. Furthermore, it is reasonable to suggest that an episode leading to cocaine overdose would be sufficient to upregulate D 3 receptors given that agonist-induced upregulation has been shown to occur rapidly in cell lines, reaching a maximal level within 6-8 h during acute exposure (Cox et al, 1995). In contrast, another postmortem analysis of brains from drug abusers failed to observe a change in D 3 receptor mRNA (Meador-Woodruff et al, 1995).…”

“…In contrast, increased dopamine associated with knockout of the dopamine transporter gene upregulates D 3 receptor mRNA in the CPu and NAc (Fauchey et al, 2000). Furthermore, exposure to D 3 -preferring agonists upregulates D 3 receptors in C6 glioma cells in vitro (Cox et al, 1995) and upregulates D 3 receptors in the ventral pallidum and substantia nigra when administered in vivo (Cox et al, 1995;Stanwood et al, 2000b).…”

Regulation of Dopamine D1Receptor Trafficking by Protein Kinase A-Dependent Phosphorylation

“…Cells were maintained at 37°C in a humidified atmosphere with 10% CO 2 in Dulbecco's modified Eagle's medium (DMEM; Sigma Chemical, St. Louis, MO) supplemented with 5% fetal bovine serum and 5% calf bovine serum, with 600 g/ml G418 sulfate (Calbiochem, San Diego, CA) . Clonal C 6 cell lines expressing untagged wild-type and mutant D 1 receptors were created by cotransfection with pBabe-Puro (Morgenstern and Land, 1990) and selection with puromycin (wild-type D 1 and T268A, 2 g/ml) or by transfection (in pcDNA3) and selection with G418 (S380A, 600 g/ml), exactly as described previously (Cox et al, 1995). C 6 cells expressing wild-type and mutant D 1 receptors were maintained in DMEM supplemented with 2% fetal bovine serum, 3% iron-supplemented calf bovine serum, 50 g/ml penicillin, 50 g/ml streptomycin, and either puromycin or G418.…”