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Activation of P2X 7 receptors induced [ 3 H]GABA release from the RBA‐2 type‐2 astrocyte cell line through a Cl − /HCO 3 − ‐dependent mechanism
Author(s) -
Wang ChiaMei,
Chang YuanYi,
Kuo JonSon,
Sun Synthia H.
Publication year - 2002
Publication title -
glia
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.954
H-Index - 164
eISSN - 1098-1136
pISSN - 0894-1491
DOI - 10.1002/glia.10004
Subject(s) - dids , ppads , extracellular , ionotropic effect , p2 receptor , biophysics , metabotropic receptor , receptor , biology , biochemistry , glutamate receptor , purinergic receptor , membrane
ATP is an important signaling molecule in the nervous system and it's signaling is mediated through the metabotropic P2Y and ionotropic P2X receptors. ATP is known to stimulate Ca 2+ influx and phospholipase D (PLD) activity in the type‐2 astrocyte cell line, RBA‐2; in this study, we show that the release of preloaded [ 3 H]GABA from RBA‐2 cells is mediated through the P2X 7 receptors. ATP and the ATP analogue 3′‐O‐(4‐benoylbenoyl)‐adenosine‐5′‐triphosphate (BzATP) both stimulated [ 3 H]GABA release in a concentration dependent manner, while the nonselective P2 receptor antagonist pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulfonic acid (PPADS), the P2X 7 ‐sensitive antagonist oxidized ATP (oATP), and high extracellular Mg 2+ all inhibited the ATP‐stimulated [ 3 H]GABA release. The ATP‐stimulated [ 3 H]GABA release was not affected neither by removing extracellular Na + nor by changes in the intracellular or extracellular Ca 2+ concentration. The GABA transporter inhibitors nipecotic acid and β‐alanine also had no effect. The ATP‐stimulated [ 3 H]GABA release was blocked, however, when media Cl − was replaced with gluconate and when extracellular HCO 3 − was removed. The Cl − channel/exchanger blockers 4,4′‐diisothiocyanatostilbene‐2′,2′‐disulfonic acid (DIDS) and 4‐acetamido‐4′‐ isothiocyanatostilbene‐2′,2′‐disulfonic acids (SITS), but not diphenylamine‐2‐carboxylic acid (DPC) and furosemide, blocked the ATP‐stimulated [ 3 H]GABA release. The anionic selectivity of the process was F − > Cl − > Br − which is the same as that reported for volume‐sensitive Cl − conductance. Treating cells with phorbol‐12‐myristate 13‐acetate (PMA), forskolin, dibutyryl‐cAMP, PD98059, neomycin, and D609 all inhibited the ATP‐stimulated [ 3 H]GABA release. We concluded that in RBA‐2 cells, ATP stimulates [ 3 H]GABA release through the P2X 7 receptors via a Cl − /HCO 3 − ‐dependent mechanism that is regulated by PKC, PKA, MEK/ERK, and PLD. GLIA 37:8–18, 2002. © 2002 Wiley‐Liss, Inc.