Fig. 2.

Pharmacology of Ca²⁺-dependent glutamate release from astrocytes. (A) ATP-induced glutamate release from astrocytic cultures detected by fluorescence enzymatic assay. BAPTA/AM (20 μM for 30 min) and the anion channel blocker NPPB (100 μM) attenuated the glutamate efflux. (B Upper) Comparison of ATP-induced and hypotonic-induced glutamate release. ATP-induced release was inhibited by BAPTA/AM (20 μM for 30 min) and thapsigargin (1 μM). Anion channel blockers, NPPB (100 μM), FFA (100 μM), and gossypol (10 μM) all eliminated ATP-induced glutamate release, whereas removal of extracellular Ca²⁺ had no effect. A glutamate transport blocker, dl-threo-β-benzyloxyaspartic acid (TBOA) (100 μM), had no effect. Similarly, the inhibition of vesicular release by bafilomycin A1 (1 μM for 1 h) or tetanus neurotoxin (TeNT; 2 μg/ml for 24 h) had no effect. A glutamine synthetase inhibitor, methionin sulfoximine (MSO; 1.5 mM for 2 h), increased ATP-induced glutamate releases. (n = 5; ^*, P < 0.01 compared with control, Tukey-Kramer test). The release from cultured astrocytes from Cx43 KO mice was not significantly different from the release from matched wild-type littermates (n = 4; P = 0.64, t test). (B Lower) Hypoosmotic stimulation (214 mOsM) induced glutamate release that was Ca²⁺-independent, but otherwise had the same pharmacological profile as ATP-induced release (n = 5, ^*, P < 0.01 compared with control, Tukey-Kramer test). (C) Glutamate release was mediated by P2YR activation. UTP (100 μM; a P2Y agonist) induced glutamate release with a potency comparable to that of ATP. By contrast, αβ-meATP (100 μM; a P2X agonist) and Bz-ATP (100 μM) elicited little glutamate release. Similarly, Ox-ATP (300 μM for 1 h) did not significantly attenuate the release. Reactive Blue 2 (RB2, 30 μM; a P2Y antagonist) blocked the release. A cycoloxygenase inhibitor indomethacin (10 μM) also failed to inhibit ATP-induced glutamate release. n = 4; ^*, P < 0.01 compared with ATP, Tukey-Kramer test. (D Left) Glutamate release by 10 μM ATP is smaller than the release by 100 μM ATP but retains sensitivity to NPPB (100 μM) and TeNT (10 μg/ml overnight) (n = 3-5). ^*, P < 0.01, Tukey-Kramer test, mean ± SEM. (Right) Dose-response curve of ATP-induced glutamate release (n = 3). (E) Cell swelling is required for ATP-induced glutamate release. ATP (100 μM) was added at the same time as the osmolarity change, which was accomplished by adding sucrose (for hypertonicity) or distilled water (for hypotonicity). Hyperosmolarity >15% completely inhibited glutamate release (n = 3-5).