Up-regulation of P2X7 receptor-mediated inhibition of GABA uptake by nerve terminals of the human epileptic neocortex

Abstract

Objective: Thirty percent of patients with epilepsy are refractory to medication. The majority of these patients have mesial temporal lobe epilepsy (MTLE). This prompts for new pharmacologic targets, like ATP-mediated signaling pathways, since the extracellular levels of the nucleotide dramatically increase during in vitro epileptic seizures. In this study, we investigated whether sodium-dependent high-affinity γ-aminobutyric acid (GABA) and glutamate uptake by isolated nerve terminals of the human neocortex could be modulated by ATP acting via slow-desensitizing P2X7 receptor (P2X7R).

Methods: Modulation of [(3) H]GABA and [(14) C]glutamate uptake by ATP, through activation of P2X7R, was investigated in isolated nerve terminals of the neocortex of cadaveric controls and patients with drug-resistant epilepsy (non-MTLE or MTLE) submitted to surgery. Tissue density and distribution of P2X7R in the human neocortex was assessed by Western blot analysis and immunofluorescence confocal microscopy.

Results: The P2X7R agonist, 2'(3')-O-(4-benzoylbenzoyl)ATP (BzATP, 3-100 μm) decreased [(3) H]GABA and [(14) C]glutamate uptake by nerve terminals of the neocortex of controls and patients with epilepsy. The inhibitory effect of BzATP (100 μm) was prevented by the selective P2X7R antagonist, A-438079 (3 μm). Down-modulation of [(14) C]glutamate uptake by BzATP (100 μm) was roughly similar in controls and patients with epilepsy, but the P2X7R agonist inhibited more effectively [(3) H]GABA uptake in the epileptic tissue. Neocortical nerve terminals of patients with epilepsy express higher amounts of the P2X7R protein than control samples.

Significance: High-frequency cortical activity during epileptic seizures releases huge amounts of ATP, which by acting on low-affinity slowly desensitizing ionotropic P2X7R, leads to down-modulation of neuronal GABA and glutamate uptake. Increased P2X7R expression in neocortical nerve terminals of patients with epilepsy may, under high-frequency firing, endure GABA signaling and increase GABAergic rundown, thereby unbalancing glutamatergic neuroexcitation. This study highlights the relevance of the ATP-sensitive P2X7R as an important negative modulator of GABA and glutamate transport and prompts for novel antiepileptic therapeutic targets.

Keywords: Epilepsy; GABA; Glutamate; High-affinity transporters; MTLE; P2X7R.