title: P2X7 receptor activation downmodulates Na(+)-dependent high-affinity GABA and glutamate transport into rat brain cortex synaptosomes.
abstract: Sodium-dependent high-affinity amino-acid transporters play crucial roles in terminating synaptic transmission in the central nervous system (CNS). However, there is lack of information about the mechanisms underlying the regulation of amino-acid transport by fast-acting neuromodulators, like ATP. Here, we investigated whether activation of the ATP-sensitive P2X7 receptor modulates Na(+)-dependent high-affinity γ-aminobutyric acid (GABA) and glutamate uptake into nerve terminals (synaptosomes) of the rat cerebral cortex. Radiolabeled neurotransmitter accumulation was evaluated by liquid scintillation spectrometry. The cell-permeant sodium-selective fluorescent indicator, SBFI-AM, was used to estimate Na(+) influx across plasma membrane. 2'(3')-O-(4-benzoylbenzoyl)ATP (BzATP, 3-300 μM), a prototypic P2X7 receptor agonist, concentration-dependently decreased [(3)H]GABA (14%) and [(14)C]glutamate (24%) uptake; BzATP decreased transport maximum velocity (Vmax) without affecting the Michaelis constant (Km) values. The selective P2X7 receptor antagonist, A-438079 (3 μM), prevented inhibition of [(3)H]GABA and [(14)C]glutamate uptake by BzATP (100 μM). The inhibitory effect of BzATP coincided with its ability to increase intracellular Na(+) and was mimicked by Na(+) ionophores, like gramicidin and monensin. Increases in intracellular Na(+) (with veratridine or ouabain) or substitution of extracellular Na(+) by N-methyl-D-glucamine (NMDG)(+) all decreased [(3)H]GABA and [(14)C]glutamate uptake and attenuated BzATP effects. Uptake inhibition by BzATP (100 μM) was also attenuated by calmidazolium, which selectively inhibits Na(+) currents through the P2X7 receptor pore. In conclusion, disruption of the Na(+) gradient by P2X7 receptor activation downmodulates high-affinity GABA and glutamate uptake into rat cortical synaptosomes. Interference with amino-acid transport efficacy may constitute a novel target for therapeutic management of cortical excitability.
authors: Laboratório de Farmacologia e Neurobiologia, Instituto de Ciências Biomédicas de Abel Salazar (ICBAS), Universidade do Porto (UP), Porto, Portugal; Center for Drug Discovery and Innovative Medicines (MedInUP), Instituto de Ciências Biomédicas de Abel Salazar (ICBAS), Universidade do Porto (UP), Porto, Portugal.
authors: A R
authors: AR
authors: Barros-Barbosa
authors: Laboratório de Farmacologia e Neurobiologia, Instituto de Ciências Biomédicas de Abel Salazar (ICBAS), Universidade do Porto (UP), Porto, Portugal; Center for Drug Discovery and Innovative Medicines (MedInUP), Instituto de Ciências Biomédicas de Abel Salazar (ICBAS), Universidade do Porto (UP), Porto, Portugal.
authors: M G
authors: MG
authors: Lobo
authors: Laboratório de Farmacologia e Neurobiologia, Instituto de Ciências Biomédicas de Abel Salazar (ICBAS), Universidade do Porto (UP), Porto, Portugal; Center for Drug Discovery and Innovative Medicines (MedInUP), Instituto de Ciências Biomédicas de Abel Salazar (ICBAS), Universidade do Porto (UP), Porto, Portugal.
authors: F
authors: F
authors: Ferreirinha
authors: Laboratório de Farmacologia e Neurobiologia, Instituto de Ciências Biomédicas de Abel Salazar (ICBAS), Universidade do Porto (UP), Porto, Portugal; Center for Drug Discovery and Innovative Medicines (MedInUP), Instituto de Ciências Biomédicas de Abel Salazar (ICBAS), Universidade do Porto (UP), Porto, Portugal. Electronic address: farmacol@icbas.up.pt.
authors: P
authors: P
authors: Correia-de-Sá
authors: Laboratório de Farmacologia e Neurobiologia, Instituto de Ciências Biomédicas de Abel Salazar (ICBAS), Universidade do Porto (UP), Porto, Portugal; Center for Drug Discovery and Innovative Medicines (MedInUP), Instituto de Ciências Biomédicas de Abel Salazar (ICBAS), Universidade do Porto (UP), Porto, Portugal. Electronic address: mcordeiro73@gmail.com.
authors: J M
authors: JM
authors: Cordeiro
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D026922:Amino Acid Transport Systems, Acidic / Q000187:drug effects / Q000493:pharmacokinetics*; D000818:Animals; D001572:Benzofurans / Q000493:pharmacokinetics; D002250:Carbon Radioisotopes; D002540:Cerebral Cortex / Q000000981:diagnostic imaging / Q000187:drug effects / Q000378:metabolism*; D005260:Female; D018698:Glutamic Acid / Q000493:pharmacokinetics*; D008297:Male; D010795:Phthalic Acids / Q000493:pharmacokinetics; D058911:Purinergic P2X Receptor Agonists / Q000494:pharmacology; D058920:Purinergic P2X Receptor Antagonists / Q000494:pharmacology; D011725:Pyridines / Q000494:pharmacology; D011877:Radionuclide Imaging; D051381:Rats; D017208:Rats, Wistar; D058486:Receptors, Purinergic P2X7 / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism; D013574:Synaptosomes / Q000000981:diagnostic imaging / Q000187:drug effects / Q000378:metabolism*; D013777:Tetrazoles / Q000494:pharmacology; D014316:Tritium; D005680:gamma-Aminobutyric Acid / Q000493:pharmacokinetics*
chemical_list: C523668:3-(5-(2,3-dichlorophenyl)-1H-tetrazol-1-yl)methylpyridine; D026922:Amino Acid Transport Systems, Acidic; D001572:Benzofurans; D002250:Carbon Radioisotopes; D010795:Phthalic Acids; D058911:Purinergic P2X Receptor Agonists; D058920:Purinergic P2X Receptor Antagonists; D011725:Pyridines; D058486:Receptors, Purinergic P2X7; D013777:Tetrazoles; C103805:sodium binding benzofuran isophthalate acetoxymethyl ester; D014316:Tritium; D018698:Glutamic Acid; C033901:3'-O-(4-benzoyl)benzoyladenosine 5'-triphosphate; D005680:gamma-Aminobutyric Acid; D000255:Adenosine Triphosphate; D012964:Sodium
keywords: GABA; P2X7 receptor; glutamate; high-affinity transporters; sodium influx