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 fluorescence reflection imaging of fluorescence-labeled glutamatergic vesicles, we document that such release occurs by regulated exocytosis. 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؉ signaling 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 glutamate 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.

Astrocyte signaling and neurodegeneration

“…55,56 Among these molecules, there are several cytokines and enzymes involved in the metabolic pathways of arachidonic acid, and thus in the generation of eicosanoids. 55,56 This finding is particularly interesting considering that some of these molecules, such as TNFα and prostaglandins, control the Ca 2+ -dependent release of glutamate from astrocytes, 32,38,51,52 which has been implicated in the excitotoxic death of neuronal cells in vitro, in neuron-glia co-culture systems. 38 Additional alterations of reactive astrocyte physiology have been described in a pioneer study by Aguado and colleagues.…”

“…Among the GPCRs competent to initiate the astrocytic release of gliotransmitters, there are group I metabotropic glutamate receptors (mGluRs), CXCR4 chemokine receptors and P2Y 1 purinergic receptors (P2Y 1 Rs). 38,[51][52][53][54] Notably, Ca 2+ -dependent release of glutamate from cultured astrocytes, in response to stimulation of the CXCR4 chemokine receptors or the purinergic P2Y 1 receptors, was reported to be controlled by pro-inflammatory mediators, such as prostaglandins and cytokines, particularly the tumor necrosis factor α (TNFα). 32,38,51,52 Since the levels of these mediators are subjected to dramatic increases in several neurodegenerative diseases, it is reasonable to postulate that the molecular pathway controlling the glial release of glutamate can become over stimulated in pathological conditions, and this may perturb the astrocyte-to-neuron signaling and, possibly, trigger neurodamaging events.…”

Suppression of SNARE‐dependent exocytosis in retinal glial cells and its effect on ischemia‐induced neurodegeneration

“…Since photolysis of NP‐EGTA may induce nonphysiological levels of intracellular calcium, we tested next, whether extracellular ATP triggers purinergic receptor‐dependent glutamate release from Müller glia as observed previously in astrocytes (Domercq et al, 2006; Jourdain et al, 2007). Photolysis of caged ATP caused a P2Y 1 receptor‐dependent release of glutamate from Müller cells: it was attenuated by the P2Y 1 antagonist MRS2179 (30 µM) and it was reduced in Müller cells isolated from P2Y 1 KO mice (Figure 2e,f).…”

Fine-tuning the central nervous system: microglial modelling of cells and synapses

“…R. Soc. B 369: 20130593 triggers glutamate release [98,99], which yields an increased frequency of EPSCs, through the activation of mGluR5 in astrocytes [21]. By means such as this, microglial activation may modulate both the threshold and intensity of synaptic activity in local networks.…”