Activation of P2X7 receptors induced [3H]GABA release from the RBA-2 type-2 astrocyte cell line through a Cl−/HCO3−-dependent mechanism
✍ Scribed by Chia-Mei Wang; Yuan-Yi Chang; Jon-Son Kuo; Synthia H. Sun
- Publisher
- John Wiley and Sons
- Year
- 2001
- Tongue
- English
- Weight
- 313 KB
- Volume
- 37
- Category
- Article
- ISSN
- 0894-1491
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✦ Synopsis
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 2ϩ 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 3Ј-O-(4-benoylbenoyl)-adenosine-5Ј-triphosphate (BzATP) both stimulated [ 3 H]GABA release in a concentration dependent manner, while the nonselective P2 receptor antagonist pyridoxalphosphate-6-azophenyl-2Ј,4Ј-disulfonic acid (PPADS), the P2X 7 -sensitive antagonist oxidized ATP (oATP), and high extracellular Mg 2ϩ all inhibited the ATP-stimulated [ 3 H]GABA release. The ATP-stimulated [ 3 H]GABA release was not affected neither by removing extracellular Na ϩ nor by changes in the intracellular or extracellular Ca 2ϩ concentration. The GABA transporter inhibitors nipecotic acid and -alanine also had no effect. The ATP-stimulated [ 3 H]GABA release was blocked, however, when media Cl Ϫ was replaced with gluconate and when extracellular HCO 3 Ϫ was removed. The Cl Ϫ channel/ exchanger blockers 4,4Ј-diisothiocyanatostilbene-2Ј,2Ј-disulfonic acid (DIDS) and 4-acetamido-4Ј-isothiocyanatostilbene-2Ј,2Ј-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 Ϫ Ͼ Cl Ϫ Ͼ Br Ϫ which is the same as that reported for volume-sensitive Cl Ϫ conductance. Treating cells with phorbol-12-myristate 13-acetate (PMA), forskolin, dibutyryl-cAMP, PD98059, neomycin, and D609 all inhibited the ATPstimulated [ 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 Ϫ /HCO 3 Ϫ -dependent mechanism that is regulated by PKC,