Activation of P2X7 receptors induced [3H]GABA release from the RBA‐2 type‐2 astrocyte cell line through a Cl−/HCO3−‐dependent mechanism
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Abstract
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 Ca2+ 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 [3H]GABA from RBA‐2 cells is mediated through the P2X7 receptors. ATP and the ATP analogue 3′‐O‐(4‐benoylbenoyl)‐adenosine‐5′‐triphosphate (BzATP) both stimulated [3H]GABA release in a concentration dependent manner, while the nonselective P2 receptor antagonist pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulfonic acid (PPADS), the P2X7‐sensitive antagonist oxidized ATP (oATP), and high extracellular Mg2+ all inhibited the ATP‐stimulated [3H]GABA release. The ATP‐stimulated [3H]GABA release was not affected neither by removing extracellular Na+ nor by changes in the intracellular or extracellular Ca2+ concentration. The GABA transporter inhibitors nipecotic acid and β‐alanine also had no effect. The ATP‐stimulated [3H]GABA release was blocked, however, when media Cl− was replaced with gluconate and when extracellular HCO3− 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 [3H]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 [3H]GABA release. We concluded that in RBA‐2 cells, ATP stimulates [3H]GABA release through the P2X7 receptors via a Cl−/HCO3−‐dependent mechanism that is regulated by PKC, PKA, MEK/ERK, and PLD. GLIA 37:8–18, 2002.Keywords
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