Relationship between dopamine agonist stimulation of inositol phosphate formation and cytidine diphosphate-diacylglycerol accumulation in brain slices.
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-[3H]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-[3H]inositol-prelabeled slices, the CDP-DG responses were proportionately greater, while the agonist EC50 values were similar between the two assays. The D1-receptor antagonist SCH23390 inhibited SKF38393-mediated responses at 0.1-10 microM 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 D1-receptor antagonist SCH23390 is a partial agonist at the D1-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.
Undie
A S
AS
Neuroscience and Pharmacology Groups, Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, 20 North Pine Street, Room 450, Baltimore, MD 21201-1180, USA. asundie@umaryland.edu
D015647:2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine / Q000494:pharmacology; D000818:Animals; D001921:Brain / Q000187:drug effects* / Q000378:metabolism; D003565:Cytidine Diphosphate / Q000378:metabolism*; D004075:Diglycerides / Q000378:metabolism*; D018491:Dopamine Agonists / Q000494:pharmacology*; D006868:Hydrolysis; D066298:In Vitro Techniques; D007295:Inositol Phosphates / Q000096:biosynthesis*; D008297:Male; D009355:Neomycin / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D013268:Stimulation, Chemical
D004075:Diglycerides; D018491:Dopamine Agonists; D007295:Inositol Phosphates; D003565:Cytidine Diphosphate; D015647:2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine; D009355:Neomycin