Relationship between dopamine agonist stimulation of inositol phosphate formation and cytidine diphosphate–diacylglycerol accumulation in brain slices

Abstract

Dopamine receptor-coupled stimulation of inositol phosphate formation has been characterized extensively, but little is known about the diacylglycerol arm of this dual-signaling pathway. This study examined several parameters of cytidine diphosphate–diacylglycerol (CDP–DG) accumulation as an index of agonist-stimulated DG formation. Rat brain slices pre-labeled with 5-[ 3 H ]cytidine were incubated with various test agents in the presence of LiCl and accumulated CDP–DG analyzed. Dopamine and SKF38393 significantly and dose-dependently stimulated CDP–DG accumulation. SKF38393 responses were inhibited by neomycin and reversed by myo-inositol or by exclusion of LiCl. Compared to inositol phosphate formation in 2-[ 3 H ]inositol-prelabeled slices, the CDP–DG responses were proportionately greater, while the agonist EC 50 values were similar between the two assays. The D 1-receptor antagonist SCH23390 inhibited SKF38393-mediated responses at 0.1–10 μM concentrations, whereas greater concentrations reversed the inhibition. SKF38393 effects were completely blocked by the DG kinase inhibitor R59022, thus precluding any role for phospholipase-D or de novo phosphatidate synthesis in the dopaminergic response. D609 which inhibits phosphatidylcholine-specific phospholipase-C (PLC), potently inhibited both CDP–DG accumulation and inositol phosphate formation. These findings demonstrate that the selective D 1-receptor antagonist SCH23390 is a partial agonist at the D 1-like dopamine receptor that couples to phosphoinositide signaling, that dopaminergic facilitation of phosphoinositide signaling is independent of de novo phosphatidate synthesis, and that the widely used enzyme inhibitor, D-609, is probably not selective for phosphatidylcholine-specific PLC in brain slice preparations. The greater sensitivity of the CDP–DG measurement presents this assay as a reliable and possibly superior index of dopamine receptor-coupled PLC activation in intact tissues.