In Vivo Stimulation of D1 Receptors Increases the Phosphorylation of Proteins in the Striatum

Abstract

In vivo changes in levels of DARPP-32 [dopamine (DA)- and cyclic AMP-regulated phosphoprotein, Mr = 32,000] protein phosphorylation in response to DA agonists in the rat striatum were measured using a novel assay that combines the benefits of rapid quenching of enzyme activity by focused microwave irradiation with a back-phosphorylation assay. The basal level of phospho-DARPP-32 was 5.6% of total DARPP-32. Injections of L-3,4-dihydroxyphenylalanine (100 mg/kg) increased this level to 44.4%. This effect was not as great if focused microwave irradiation was not used. The D1-specific agonist SKF 38393 (10 mg/kg) increased the level of phospho-DARPP-32 to 36.4%. A further modification of the back-phosphorylation assay was used to detect other phosphoproteins that appear to be regulated by DA. These results establish an assay for in vivo studies of postsynaptic responses involving second messengers in the DA system and provide direct in vivo evidence for the hypothesis that stimulation of D1 receptors increases the phosphorylation of DARPP-32, as well as several other proteins.