Neuronal dopamine internalization and intracellular stimulation of D₅-receptor-dependent CDP-diacylglycerol biosynthesis for phosphoinositide signaling

Abstract ID 100107 Poster Board 529 Dopamine receptor stimulation facilitates phosphatidylinositol resynthesis, thus amplifying subsequent responses to activation of phospholipase C-coupled receptors. Phosphatidylinositol synthesis critically depends on the nucleolipid CDP-diacylglycerol. Dopamine robustly increases microsomal CDP-diacylglycerol biosynthesis through stimulation of D₁-like receptors, particularly the D₅ subtype the majority of which are intracellularly localized. Here, we explored the mechanism by which extracellular dopamine acts to modulate intracellular CDP-diacylglycerol biosynthesis. Dopamine concentration-dependently stimulated CDP-diacylglycerol synthesis in organotypic and primary neuronal cultures devoid of the presynaptic dopamine transporter. Dopamine was saturably transported into cortical primary neurons or B35 neuroblastoma cells expressing wild-type plasmalemmal monoamine transporter (PMAT), which is known to be the principal component of classical Uptake₂ transporters in the forebrain. Dopamine uptake and CDP-diacylglycerol biosynthesis in brain slices or cultured cells were inhibited by microtubule disrupters which block cytoskeletal transport, and by decynium-22 which blocks Uptake₂-like transporters. Dopamine effects were selectively mimicked by D₁-like agonists SKF38393 and SKF83959, competitively inhibited by D₁-like antagonist SCH23390, and unaffected by D₂-like agonist or antagonist. These observations indicate that dopamine is actively internalized by Uptake₂ into postsynaptic-type cells where the monoamine can stimulate its intracellular D₅-type receptors to increase CDP-diacylglycerol production. This finding counters the conventional notion that postsynaptic-type cells internalize supra-threshold levels of synaptic dopamine only to inactivate the transmitter. Given the critical involvement of CDP-diacylglycerol in phospholipase C and phosphatidylinositol-3-kinase signaling systems, our findings imply that intracellular dopamine could play an important role in cellular responses and adaptation to high levels of extracellular dopamine. Supported in part by NIH/NIDA Grant R01DA017614 to ASU; NIH/NINDS Grant R16NS129675 to ASU; the New York State Spinal Cord Injury Research Board Grants C32247GG; C37715GG to ASU.