P2Y1 Receptor-evoked Glutamate Exocytosis from Astrocytes

Abstract

ATP, released by both neurons and glia, is an important mediator of brain intercellular communication. We find that selective activation of purinergic P2Y1 receptors (P2Y1R) in cultured astrocytes triggers glutamate release. By total internal fluores-cence reflection imaging of fluorescence-labeled glutamatergic vesicles, we document that such release occurs by regulated exo-cytosis. The stimulus-secretion coupling mechanism involves Ca 2 release from internal stores and is controlled by additional transductive events mediated by tumor necrosis factor-(TNF) and prostaglandins (PG). P2Y1R activation induces release of both TNF and PGE 2 and blocking either one significantly reduces glutamate release. Accordingly, astrocytes from TNF-deficient (TNF /) or TNF type 1 receptor-deficient (TNFR1 /) mice display altered P2Y1R-dependent Ca 2 sig-naling and deficient glutamate release. In mixed hippocampal cultures, the P2Y1R-evoked process occurs in astrocytes but not in neurons or microglia. P2Y1R stimulation induces Ca 2-dependent glutamate release also from acute hippocampal slices. The process in situ displays characteristics resembling those in cultured astrocytes and is distinctly different from synaptic glu-tamate release evoked by high K stimulation as follows: (a) it is sensitive to cyclooxygenase inhibitors; (b) it is deficient in preparations from TNF / and TNFR1 / mice; and (c) it is inhibited by the exocytosis blocker bafilomycin A1 with a different time course. No glutamate release is evoked by P2Y1R-dependent stimulation of hippocampal synaptosomes. Taken together, our data identify the coupling of purinergic P2Y1R to glutamate exocytosis and its peculiar TNF-and PG-dependent control, and we strongly suggest that this cascade operates selectively in astrocytes. The identified pathway may play physiological roles in glial-glial and glial-neuronal communication.