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, 3–300μM), a prototypic P2X7 receptor agonist, concentration-dependently decreased [3H]GABA (14%) and [14C]glutamate (24%) uptake; BzATP decreased transport maximum velocity (Vmax) without affecting the Michaelis constant (Km) values. The selective P2X7 receptor antagonist, A-438079 (3μM), prevented inhibition of [3H]GABA and [14C]glutamate uptake by BzATP (100μM). 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 [3H]GABA and [14C]glutamate uptake and attenuated BzATP effects. Uptake inhibition by BzATP (100μM) 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.