Under stressful conditions activation of the ionotropic P2X7 receptor differentially regulates GABA and glutamate release from nerve terminals of the rat cerebral cortex
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
References (54)
- et al.
The participation of plasma membrane hemichannels to purinergic signaling
Biochim. Biophys. Acta
(2013) - et al.
P2X7 receptor activation downmodulates Na+-dependent high-affinity GABA and glutamate transport into rat brain cortex synaptosomes
Neuroscience
(2015) - et al.
Aluminium-induced impairment of Ca2+ modulatory action on GABA transport in brain cortex nerve terminals
J. Inorg. Biochem.
(2003) - et al.
Ca2+ regulation of the carrier-mediated ϒ-aminobutyric acid release from isolated synaptic plasma membrane vesicles
Neurosci. Res.
(2000) - et al.
Paradoxical nifedipine facilitation of 45Ca uptake into rat hippocampal synaptosomes
Eur. J. Pharmacol.
(2006) - et al.
Oxaliplatin evokes P2X7-dependent glutamate release in the cerebral cortex: a pain mechanism mediated by Pannexin 1
Neuropharmacology
(2015) - et al.
Regulation of connexin hemichannel activity by membrane potential and the extracellularcalcium in health and disease
Neuropharmacology
(2013) - et al.
Glutamate release through connexin 43 by cultured astrocytes in a stimulated hypertonicity model
Brain Res.
(2011) - et al.
Release of D-[3H]aspartate and [14C]GABA in rat hippocampus slices: effects of fatty acid-free bovine serum albumin and Ca2+ withdrawal
Brain Res.
(1988) - et al.
Connexin-based gap junction hemichannels: gating mechanisms
Biochim. Biophys. Acta
(2005)
Effect of depolarizing agents on the Ca2+-independent and Ca2+-dependent release of [3H]GABA from sheep brain synaptosomes
Biochem. Pharmacol.
P2X7 receptor: an emerging target in central nervous system diseases
Trends Pharmacol. Sci.
Veratridine-induced release of endogenous glutamate from rat brain cortex slices: a reappraisal of the role of calcium
Brain Res.
Effects of divalent cations, protons and calmidazolium at the rat P2X7 receptor
Neuropharmacology
Nonvesicular inhibitory neurotransmission via reversal of the GABA transporter GAT-1
Neuron
Reversal or reduction of glutamate and GABA transport in CNS pathology and therapy
Pflugers Arch.
Nicotine-induced and depolarisation-induced glutamate release from hippocampus mossy fibre synaptosomes: two distinct mechanisms
J. Neurochem.
Up-regulation of P2X7 receptor-mediated inhibition of GABA uptake by nerve terminals of the human epileptic neocortex
Epilepsia
The P2X7 receptor as a route for non-exocytotic glutamate release: dependence on the carboxyl tail
J. Neurochem.
P2X7 receptors enhance glutamate release in hippocampal hilar neurons
Neuroreport
On the mechanism by which veratridine causes a calcium-independent release of gamma-aminobutyric acid from brain slices
Br. J. Pharmacol.
Pannexins, distant relatives of the connexin family with specific cellular functions?
Bioessays
Mammalian P2X7 receptor pharmacology: comparison of recombinant mouse, rat and human P2X7 receptors
Br. J. Pharmacol.
P2X7 receptor-mediated release of excitatory amino acids from astrocytes
J. Neurosci.
P2X(7) receptors: properties and relevance to CNS function
Glia
The effects of Na+ replacement on intracellular pH and [Ca2+] in rabbit salivary gland acinar cells
J. Physiol.
P2X7 receptor in epilepsy; role in pathophysiology and potential targeting for seizure control
Int. J. Physiol. Pathophysiol. Pharmacol.
Cited by (14)
How is the P2X7 receptor signaling pathway involved in epileptogenesis?
2024, Neurochemistry InternationalP2X7 receptor-activated microglia in cortex is critical for sleep disorder under neuropathic pain
2023, Frontiers in NeuroscienceEmerging Role of Microglia-Mediated Neuroinflammation in Epilepsy after Subarachnoid Hemorrhage
2021, Molecular NeurobiologyAstrocytic and Oligodendrocytic P2X7 Receptors Determine Neuronal Functions in the CNS
2021, Frontiers in Molecular Neuroscience
- 1
These authors contribute equally to this work.