Activation of P2X₇ receptors induced [³H]GABA release from the RBA‐2 type‐2 astrocyte cell line through a Cl⁻/HCO₃⁻‐dependent mechanism

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 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 beta-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.

Involvement of Chloride in Apoptotic Cell Death Induced by Activation of ATP-sensitive P2X7 Purinoceptor

“…Future studies will elucidate the mechanism of the apoptotic cell death mediated by increase in intracellular Cl Ϫ . To our knowledge, Wang et al (39) have only reported the presence of a Cl Ϫ /HCO 3 Ϫ -dependent mechanism in the P2X 7 receptor-induced ␥-aminobutyric acid release from astrocytes, but their system is different from our system in its sensitivity for HCO 3 Ϫ . The role of extracellular Cl Ϫ in the signal transduction pathway of apoptotic cell death by the activation of P2X 7 receptor has not been addressed before.…”

Pathophysiology and Therapeutic Potential of Purinergic Signaling

“…When cortical astrocytes were subjected to mechanical strain, ATP was released, leading to Akt activation; PPADS attenuated the Akt activation . ATP can activate P2X 7 receptors in astrocytes to release glutamate, GABA, and ATP, which regulate the excitability of neurons (Wang et al, 2002). ATP release during neuronal excitation or injury can enhance the inflammatory effects of cytokines and prostaglandin E 2 in astrocytes and may contribute to the chronic inflammation seen in Alzheimer's disease (Xu et al, 2003).…”

P2X7Receptors Mediate ATP Release and Amplification of Astrocytic Intercellular Ca2+Signaling

“…Although GABA and glutamate release through P2X 7 Rs was reported to occur in astrocytes (Wang et al, 2002;Duan et al, 2003), the only evidence favoring the participation of P2X 7 Rs in ATP release was provided by Ballerini et al (1996). These authors showed that stimulation of rat astrocytes with a P2X 7 R agonist ([2Ј-3Ј-O-(4-Benzoyl-benzoyl) adenosine 5Ј-triphosphate (BzATP)) induced Lucifer yellow (LY) uptake and purine release, whereas the antagonist oxidized ATP (oATP) prevented these responses.…”