Elsevier

Neurochemistry International

Volume 112, January 2018, Pages 81-95
Neurochemistry International

Under stressful conditions activation of the ionotropic P2X7 receptor differentially regulates GABA and glutamate release from nerve terminals of the rat cerebral cortex

https://doi.org/10.1016/j.neuint.2017.11.005Get rights and content

Highlights

  • P2X7-receptors activation favors GABA and Glu release from cortical neurons.

  • GABA and Glu release is differentially modulated by P2X7-receptors activation.

  • GABA outflow triggered by P2X7 receptors depends on extracellular Ca2+ ([Ca2+]o).

  • In low [Ca2+]o, P2X7-induced GABA release switches from exocytosis to GAT1 reversal.

  • Connexin hemichannels participate on P2X7-induced Glu release independently of [Ca2+]o.

Abstract

γ-Aminobutyric acid (GABA) and glutamate (Glu) are the main inhibitory and excitatory neurotransmitters in the central nervous system (CNS), respectively. Fine tuning regulation of extracellular levels of these amino acids is essential for normal brain activity. Recently, we showed that neocortical nerve terminals from patients with epilepsy express higher amounts of the non-desensitizing ionotropic P2X7 receptor. Once activated by ATP released from neuronal cells, the P2X7 receptor unbalances GABAergic vs. glutamatergic neurotransmission by differentially interfering with GABA and Glu uptake. Here, we investigated if activation of the P2X7 receptor also affects [3H]GABA and [14C]Glu release measured synchronously from isolated nerve terminals (synaptosomes) of the rat cerebral cortex. Data show that activation of the P2X7 receptor consistently increases [14C]Glu over [3H]GABA release from cortical nerve terminals, but the GABA/Glu ratio depends on extracellular Ca2+ concentrations. While the P2X7-induced [3H]GABA release is operated by a Ca2+-dependent pathway when external Ca2+ is available, this mechanism shifts towards the reversal of the GAT1 transporter in low Ca2+ conditions. A different scenario is verified regarding [14C]Glu outflow triggered by the P2X7 receptor, since the amino acid seems to be consistently released through the recruitment of connexin-containing hemichannels upon P2X7 activation, both in the absence and in the presence of external Ca2+. Data from this study add valuable information suggesting that ATP, via P2X7 activation, not only interferes with the high-affinity uptake of GABA and Glu but actually favors the release of these amino acids through distinct molecular mechanisms amenable to differential therapeutic control.

Introduction

GABA and Glu are key neurotransmitters in the CNS. The fine-tuning regulation of the extracellular amounts of these two amino acid neurotransmitters is crucial for normal synaptic transmission in the brain. Both neurotransmitters can be released from neuronal cells by untraditional molecular mechanisms different from the most studied vesicular exocytosis triggered by Ca2+ influx secondary to neuronal depolarization. Examples of non-vesicular and non-exocytotic mechanisms identified for Glu release include those involving the Glu-cystine antiporter (Zhou and Danbolt, 2014), anion channels (Zhou and Danbolt, 2014), the reversal of high-affinity Glu transporters (Allen et al., 2004), and hemichannels containing connexins (Cx) and pannexins (Di Cesare Mannelli et al., 2015, Jiang et al., 2011, Orellana et al., 2011). Regarding GABA, it can be released through the bestrophin 1 anion channel (Lee et al., 2010) or through a more widespread mechanism involving reversal of Na+-coupled high-affinity GABA transporters (Allen et al., 2004, Richerson and Wu, 2003, Wu et al., 2007). Notwithstanding this, the balance between all these release pathways (and uptake systems) is not fully understood. There is also a gap in our knowledge concerning the most relevant mechanisms operating during paroxysmal neuronal activity, such as during and after epileptic seizures.

Mounting evidence indicate that activation of the non-desensitizing ionotropic P2X7 receptor by ATP released during neuronal cell damage or excessive neuronal firing (for instance during status epilepticus) favors the release of both Glu (Cervetto et al., 2013, Di Cesare Mannelli et al., 2015, Marcoli et al., 2008) and GABA (Papp et al., 2004, Wang et al., 2002). There is, however, some controversy regarding the mechanisms responsible for the P2X7-induced release of Glu and GABA and whether the synchronous release of both amino acids is amenable to differential modulation by ATP, via the P2X7 receptor. Recently, our group showed that activation of the P2X7 receptor releases five-fold more [14C]Glu than [3H]GABA from isolated nerve terminals of the rat cerebral cortex when both neurotransmitters were measured simultaneously in Ca2+-free conditions and their values were normalized by the maximal depolarization caused by veratridine (VT) (Barros-Barbosa et al., 2015). These findings are clinically relevant taking into consideration that (1) during epileptic activity the extracellular Ca2+ concentration falls in parallel to increases in the amount of released ATP (Engel et al., 2012, Engelborghs et al., 2000, Jimenez-Pacheco et al., 2013), (2) neocortical nerve terminals from patients with mesial temporal lobe epilepsy (MTLE) refractory to medication express higher P2X7 receptor amounts than control individuals (Barros-Barbosa et al., 2016), and (3) a significant reduction in GABAergic nerve terminals (synaptosomes) was observed in the epileptic human neocortex (Rassner et al., 2016). As a matter of fact, our previous findings suggest that up-regulation of the P2X7 receptor bolsters ATP signals leading to the unbalance of GABAergic vs. glutamatergic neurotransmission by differentially interfering with GABA and Glu transport. In view of this, we and others hypothesized that targeting the P2X7 receptor may be a valuable alternative to control epileptogenesis (Barros-Barbosa et al., 2016, Engel et al., 2012).

Concerning the release of Glu operated by activation of the P2X7 receptor it has been proposed by some authors that the presynaptic P2X7 receptor pore mediates Ca2+ influx in sufficient amounts to trigger the exocytosis of Glu containing synaptic vesicles, while others argue that Glu may directly permeate the P2X7 dilated pore during prolonged periods of receptor activation (Cervetto et al., 2013, Di Cesare Mannelli et al., 2015, Marcoli et al., 2008). It is also possible that Glu may be released through the functional coupling between the P2X7 receptor and hemichannels containing connexins and/or pannexins (Di Cesare Mannelli et al., 2015, Orellana et al., 2015, Suadicani et al., 2012, Xu et al., 2012). On the other hand, little is known about the mechanism by which activation of the P2X7 receptor promotes GABA release. A study performed in a type-2 astrocytic cell line showed that the release of GABA triggered by activation of the P2X7 receptor occurs via a Cl/HCO3-dependent mechanism, probably through the action of a Cl channel/exchanger that is Ca2+- and Na+-independent (Wang et al., 2002).

Our study was designed to investigate in parallel and under the same experimental conditions the molecular mechanisms underlying [3H]GABA and [14C]Glu release from isolated nerve terminals (synaptosomes) of the rat cerebral cortex caused by the P2X7 receptor activation in the absence or in the presence of extracellular Ca2+. Performing experiments in the absence of extracellular Ca2+ aims at mimicking conditions of paroxysmal neuronal activity verified during and after physiological and pathological brain events (such as epilepic seizures) where extracellular Ca2+ concentration may fall up to 90% of the initial levels (Engelborghs et al., 2000). Although it is generally assumed that low extracellular Ca2+ conditions reduces vesicular exocytosis of neurotransmitters, an inverse correlation between Ca2+ concentration in the extracellular milieu and depolarization-evoked amino acid transmitters release has been observed (this study; see also Levi et al., 1980, Cunningham and Neal, 1981, Minc-Golomb et al., 1988, Santos et al., 1992, Rassner et al., 2016). Thus, the purinergic control, via the P2X7 receptor, of the mechanism(s) behind the balance between GABAergic inhibition and glutamatergic excitation may be important during epileptic seizures and may prevent epileptogenesis progression.

Section snippets

Drugs and solutions

GABA (γ-aminobutyric acid), BzATP (2′ (3′)-O-(4-benzoylbenzoyl)adenosine 5′-triphosphate triethylammonium salt), NMDG (N-methyl-d-glucamine), EGTA (ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid), aminooxyacetic acid, BAPTA-AM (1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis (acetoxymethyl ester), choline chloride and carbenoxolone were obtained from Sigma-Aldrich (St. Louis, MO). l-Glutamic acid, A-438079 (3-[[5-(2,3-dichlorophenyl)-1H-tetrazol-1-yl]

Activation of the P2X7 receptor triggers [3H]GABA and [14C]Glu release from rat cerebral cortex synaptosomes

Previous results from our group evidenced that the ionotropic P2X7 receptor is expressed predominantly on nerve terminals of the rat cerebral cortex and that the activation of P2X7 receptors in a Ca2+-free media promotes the release of GABA and Glu from rat cortical synaptosomes (Barros-Barbosa et al., 2015). In this study, we evaluated if in the presence of physiological extracellular Ca2+ concentrations the same occurred and whether the release of [3H]GABA and [14C]Glu triggered by the P2X7

Activation of the P2X7 receptor consistently increases Glu over GABA release from cortical nerve terminals, but the Glu/GABA ratio depends on extracellular Ca2+

It has been demonstrated that activation of the P2X7 receptor triggers the release of Glu from astrocytic cells (Duan et al., 2003) and neurons (Cho et al., 2010, Marcoli et al., 2008). Less is, however, known about the release of GABA induced by activation of the P2X7 receptor and whether this receptor affects differentially the release of both amino acids measured simultaneously. Therefore, this study was designed to evaluate, in parallel, the mechanisms by which the prototypic P2X7 receptor

Conflicts of interest

The authors declare that they have no conflict of interest.

Funding

This study was supported by University of Porto/Santander Totta, Liga Portuguesa Contra a Epilepsia (LPCE), Tecnifar and Fundação para a Ciência e a Tecnologia (FCT, projects PEst-OE/SAU/UI0215/2014 and UID/BIM/4308/2016). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. JMC was hired under the scope of FCT Portugal CIÊNCIA 2008 Programme (FSE-POPH-QREN, CONT_DOUT/117/ICBAS-UP/215/10824/2/2008); ABB was in receipt of a

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