title: 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.
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.
authors.affiliation: Institute of Neuroscience, College of Life Science, National Yang Ming University, Taipei, Taiwan, ROC.
authors.forename: Chia-Mei
authors.initials: CM
authors.lastname: Wang
authors.forename: Yuan-Yi
authors.initials: YY
authors.lastname: Chang
authors.forename: Jon-Son
authors.initials: JS
authors.lastname: Kuo
authors.forename: Synthia H
authors.initials: SH
authors.lastname: Sun
mesh_terms: D000612:4-Aminobutyrate Transaminase / Q000037:antagonists & inhibitors / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D000838:Anions / Q000378:metabolism*; D001253:Astrocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D001639:Bicarbonates / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D002478:Cells, Cultured; D002490:Central Nervous System / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D002712:Chlorides / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005110:Extracellular Space / Q000187:drug effects / Q000378:metabolism; D050485:GABA Plasma Membrane Transport Proteins; D006801:Humans; D007424:Intracellular Fluid / Q000187:drug effects / Q000378:metabolism; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D026901:Membrane Transport Proteins; D027361:Organic Anion Transporters; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000378:metabolism*; D058910:Purinergic P2 Receptor Agonists; D058919:Purinergic P2 Receptor Antagonists; D018048:Receptors, Purinergic P2 / Q000378:metabolism*; D058486:Receptors, Purinergic P2X7; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D005680:gamma-Aminobutyric Acid / Q000378:metabolism*
chemical_list: D000838:Anions; D001639:Bicarbonates; D002352:Carrier Proteins; D002712:Chlorides; D004791:Enzyme Inhibitors; D050485:GABA Plasma Membrane Transport Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D027361:Organic Anion Transporters; C546468:P2RX7 protein, human; D047428:Protein Kinase Inhibitors; D058910:Purinergic P2 Receptor Agonists; D058919:Purinergic P2 Receptor Antagonists; D018048:Receptors, Purinergic P2; D058486:Receptors, Purinergic P2X7; D005680:gamma-Aminobutyric Acid; D000255:Adenosine Triphosphate; D000612:4-Aminobutyrate Transaminase; D011494:Protein Kinases; D002118:Calcium