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.
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.
Wang
Chia-Mei
CM
Institute of Neuroscience, College of Life Science, National Yang Ming University, Taipei, Taiwan, ROC.
Chang
Yuan-Yi
YY
Kuo
Jon-Son
JS
Sun
Synthia H
SH
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*
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