title: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Laboratório de Farmacologia e Neurobiologia - Center for Drug Discovery and Innovative Medicines (MedInUP), Instituto de Ciências Biomédicas de Abel Salazar, Universidade do Porto, Porto, Portugal.
authors.forename: Aurora R
authors.initials: AR
authors.lastname: Barros-Barbosa
authors.affiliation: Laboratório de Farmacologia e Neurobiologia - Center for Drug Discovery and Innovative Medicines (MedInUP), Instituto de Ciências Biomédicas de Abel Salazar, Universidade do Porto, Porto, Portugal.
authors.forename: Ana L
authors.initials: AL
authors.lastname: Fonseca
authors.affiliation: Laboratório de Farmacologia e Neurobiologia - Center for Drug Discovery and Innovative Medicines (MedInUP), Instituto de Ciências Biomédicas de Abel Salazar, Universidade do Porto, Porto, Portugal.
authors.forename: Sónia
authors.initials: S
authors.lastname: Guerra-Gomes
authors.affiliation: Laboratório de Farmacologia e Neurobiologia - Center for Drug Discovery and Innovative Medicines (MedInUP), Instituto de Ciências Biomédicas de Abel Salazar, Universidade do Porto, Porto, Portugal.
authors.forename: Fátima
authors.initials: F
authors.lastname: Ferreirinha
authors.affiliation: Serviço de Patologia Forense, Instituto Nacional de Medicina Legal e Ciências Forenses - Delegação do Norte (INMLCF-DN), Porto, Portugal.
authors.forename: Agostinho
authors.initials: A
authors.lastname: Santos
authors.affiliation: Serviço de Neurocirurgia, Centro Hospitalar do Porto - Hospital Geral de Santo António (CHP - HGSA), Porto, Portugal.
authors.forename: Rui
authors.initials: R
authors.lastname: Rangel
authors.affiliation: Laboratório de Farmacologia e Neurobiologia - Center for Drug Discovery and Innovative Medicines (MedInUP), Instituto de Ciências Biomédicas de Abel Salazar, Universidade do Porto, Porto, Portugal.
authors.forename: M Graça
authors.initials: MG
authors.lastname: Lobo
authors.affiliation: Laboratório de Farmacologia e Neurobiologia - Center for Drug Discovery and Innovative Medicines (MedInUP), Instituto de Ciências Biomédicas de Abel Salazar, Universidade do Porto, Porto, Portugal.
authors.forename: Paulo
authors.identifier: http://orcid.org/0000-0002-6114-9189
authors.initials: P
authors.lastname: Correia-de-Sá
authors.affiliation: Laboratório de Farmacologia e Neurobiologia - Center for Drug Discovery and Innovative Medicines (MedInUP), Instituto de Ciências Biomédicas de Abel Salazar, Universidade do Porto, Porto, Portugal.
authors.forename: J Miguel
authors.initials: JM
authors.lastname: Cordeiro
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D001224:Aspartic Acid / Q000494:pharmacology; D002247:Carbon Isotopes / Q000378:metabolism; D002648:Child; D000076122:Disks Large Homolog 4 Protein; D004827:Epilepsy / Q000473:pathology*; D018683:Excitatory Amino Acid Agents / Q000494:pharmacology; D005260:Female; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism; D008875:Middle Aged; D019579:Neocortex / Q000187:drug effects / Q000378:metabolism / Q000473:pathology / Q000648:ultrastructure*; D058486:Receptors, Purinergic P2X7 / Q000378:metabolism*; D016708:Synaptophysin / Q000378:metabolism; D013574:Synaptosomes / Q000378:metabolism*; D015854:Up-Regulation / Q000187:drug effects / Q000502:physiology*; D050987:Vesicle-Associated Membrane Protein 1 / Q000378:metabolism; D055815:Young Adult; D005680:gamma-Aminobutyric Acid / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D002247:Carbon Isotopes; C501444:DLG4 protein, human; D000076122:Disks Large Homolog 4 Protein; D018683:Excitatory Amino Acid Agents; D005904:Glial Fibrillary Acidic Protein; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D058486:Receptors, Purinergic P2X7; D016708:Synaptophysin; D050987:Vesicle-Associated Membrane Protein 1; C120673:benzyloxyaspartate; D001224:Aspartic Acid; D005680:gamma-Aminobutyric Acid
keywords: Epilepsy; GABA; Glutamate; High-affinity transporters; MTLE; P2X7R