Pharmacological and functional characterization of D₂, D₃ and D₄ dopamine receptors in fibroblast and dopaminergic cell lines

In order to study the properties of the D₂-like dopamine receptors, D₂, D₃ and D₄ clones were transfected into mouse Ltk^- fibroblasts, CCL1.3, and a neuronal mesencephalic cell line, MN9D. Most of the derived antagonist and agonist inhibition constants were the same for a given receptor in either cell line as determined by saturation and competition binding experiments. The rank order potencies for antagonists are: eticlopride, D₂ > D₃ > D₄; YM-09151-2, D₂ = D₄ > D₃; spiperone, D₂ = D₃ > D₄; (+)-butaclamol, D₂ > D₃ > D₄; clozapine, D₄ > D₂ > D₃; and for agonists, quinpirole, D₃ = D₄ > D₂; 7-hydroxy-2-(di-n-propyl)-aminotetralin, D₃ > D₂ = D₄. Functionally, D₂ stimulation increases inositol phosphate levels in CCL1.3 cells but not in MN9D, whereas D₂ activation inhibits forskolin-stimulated cyclic AMP levels in both cell lines. D₄ stimulation has no effect on inositol phosphate metabolism in either cell type, but inhibits adenylate cyclase in MN9D cells. Both the D₂ and D₄ mediated decreases in cyclic AMP can be blocked by preincubation with pertussis toxin. D₃ does not couple to these pathways in either cell line. Reverse transcription/polymerase chain reaction techniques were used to determine the availability of cellular signalling systems. Both CCL 1.3 and MN9D cells have high levels of Gα(i2) expression, whereas neither cell expresses Gα(i1) or Gα(i3). These data imply that the D₂ receptor couples to the Gα(i2) subtype in both cell lines, whereas D₄ does not. The differential coupling of D₂ and D₄ receptors demonstrates the utility of model central nervous system cell lines in addressing receptor-effector mechanisms.