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P2 receptors have been implicated in the release of neurotransmitter and proinflammatory cytokines by the response to neuroexcitatory substances in astrocytes. In the present study, we examined the mechanisms of ADP and adenosine 5′‐O‐2‐thiodiphosphate (ADPbetaS, ADP analogue) on glutamate release from cultured dorsal spinal cord astrocytes by using confocal laser scanning microscopy and HPLC. Immunofluorescence activity showed that P2Y1 receptor protein is expressed in cultured astrocytes. ADP and ADPbetaS‐induced [Ca2+]i increase and glutamate release are mediated by P2Y1 receptor. Ca2+ release from IP3‐sensitive calcium stores and protein kinase C (PKC) activation is important for glutamate release from astrocytes. Furthermore, P2Y1 receptor‐evoked glutamate release is regulated by volume‐sensitive Cl− channels and anion co‐transporter, which open up the possibility that P2Y1 receptor activation causes the increase of cell volume. Release of glutamate by ADPbetaS was abolished by 5‐nitro‐2 (3‐phenyl propy lamino)–benzoate plus furosemide but was unaffected by botulinum toxin A. These observations indicate that P2Y1 receptor‐evoked glutamate may be mediated via volume‐sensitive Cl− channel but not via exocytosis of glutamate containing vesicles. We speculate that P2Y1 receptors‐evoked glutamate efflux, occurring under pathological condition, may modulate the activity of synapses in spinal cord.

It was reported that ATP, an excitatory chemical mediator, exerts its effects by activation of the P2X (ligand-gated cationic channels) and P2Y (G protein-coupled receptors) purinoceptors in the nervous system. In the present work, we used confocal laser scanning microscopy and high-performance liquid chromatography to assess the role of the P2Y₁ receptor in ATP-evoked Ca²⁺ mobilization and glutamate release from cultured dorsal spinal cord astrocytes. ATP (0.01–100 µmol/l) produces a dose-dependent rise in the Ca²⁺ relative fluorescence intensity in cultured astrocytes. N6methyl-2′-deoxyadenosine-3′,5′-bisphosphate (MRS2179, 0.01–100 µmol/l), a P2Y₁-specific antagonist, could dose-dependently inhibit ATP-evoked Ca²⁺ mobilization. In addition, 100 µmol/l ATP caused glutamate efflux from cultured dorsal spinal cord astrocytes in a time-dependent manner. 100 µmol/l MRS2179 significantly inhibited the glutamate efflux induced by ATP, which suggests that P2Y₁ receptor activation is responsible for the ATP-induced glutamate efflux from astrocytes. Taken together, our results demonstrate that P2Y₁ receptor plays an important role in modulating the function of astrocytes, which raises the possibility that MRS2179, a potent P2Y₁-specific antagonist, may become a potential drug in treating many chronic neurological diseases characterized by astrocytic activation in the nervous system.

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