REGULATION AND FUNCTIONAL-CHARACTERIZATION OF A RAT RECOMBINANT DOPAMINE D3 RECEPTOR

We stably expressed a rat D3 receptor cDNA in C-6 glioma cells (C6-D3 cells), quantifying receptor expression with the radioligands [I-125]e pidepride (K-D = 0.1 nM) and [H-3]spiperone (K-D = 0.7 nM). As reporte d previously for D2 receptors, quinpirole induced a 9-16% increase in the rate of extracellular acidification by C6-D3 cells. The acidificat ion was inhibited by epidepride and by the Na+/H+ antiporter inhibitor s, amiloride and methylisobutylamiloride, but pertussis toxin treatmen t had no effect on quinpirole-induced extracellular acidification. The se data suggest that D3 receptor stimulation of Na+/H+ exchange in C-6 glioma cells is not mediated by the pertussis toxin-sensitive G prote ins, G(i) or G(o). Overnight treatment of C6-D3 cells with N-propylnor apomorphine, dopamine, or quinpirole resulted in large concentration-d ependent increases (up to 500%) in the density of D3 receptors on memb ranes prepared from the cells. Antagonists had smaller, variable effec ts on the density of D3 receptors in C6-D3 cells, except for domperido ne, which significantly increased the density of D3 receptors. Treatme nt with pertussis toxin had no effect on the agonist-induced receptor up-regulation, indicating that an interaction with pertussis toxin-sen sitive G proteins was not required. Densitometry analysis of Northern blots of RNA prepared from C6-D3 cells showed no significant N-propyln orapomorphine-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 mu M DA r esulted in a substantial heterologous sensitization, in which isoprote renol-stimulated adenylyl cyclase activity was enhanced more than twof old. In contrast, isoproterenol-stimulated enzyme activity was inhibit ed by greater than 50% in C6-D3 cells pretreated with dopamine. These results confirm one functional response to activation of D3 receptors and demonstrate that the density of D3 receptors, like D2 receptors, i s increased after incubation of intact cells with agonists. (C) 1995 W iley-Liss, Inc.()