ATP stimulates calcium-dependent glutamate release from cultured astrocytes

ATP caused a dose-dependent, receptor-mediated increase in the release of glutamate and aspartate from cultured astrocytes. Using calcium imaging in combination HPLC we found that the increase in intracellular calcium coincided with an increase in glutamate and aspartate release. competitive antagonists of P, receptors blocked the response to ATP. The increase in intracellular calcium and release of glutamate evoked by ATP were not abolished in low Ca2+-EGTA saline, suggesting the involvement of intracellular calcium stores. Pre-treatment of glial cultures with an intracellular Ca2+ chelator abolished the stimulatory effects of ATP. Thapsigargin (1 muM), an inhibitor of Ca2+-ATPase from the Ca2+ pump of internal stores, significantly reduced the calcium transients and the release of aspartate and glutamate evoked by ATP. U73122 (10 muM), a phospholipase C inhibitor, attenuated the ATP-stimulatory effect on calcium transients and blocked ATP-evoked glutamate release in astrocytes. Replacement of extracellular sodium with choline failed to influence ATP-induced glutamate release. Furthermore, inhibition of the glutamate transporters p-chloromercuri-phenyl-sulfonic acid and L-transpyrolidine-2,4-dicarboxylate failed to impair the ability of ATP to stimulate glutamate release from astrocytes. However, an anion transport inhibitor, furosemide, and a potent Cl channel blocker, 5-nitro-2(3-phenylpropylamino)-benzoate, reduced ATP-induced glutamate release. These results suggest that ATP stimulates excitatory amino acid release from astrocytes via a calcium-dependent anion-transport sensitive mechanism.