GABAergic system and chloride cotransporters as potential therapeutic targets to mitigate cell death in ischemia.

Gamma aminobutyric acid (GABA) is a critical inhibitory neurotransmitter in the central nervous system that plays a vital role in modulating neuronal excitability. Dysregulation of GABAergic signaling, particularly involving the cotransporters NKCC1 and KCC2, has been implicated in various pathologies, including epilepsy, schizophrenia, autism spectrum disorder, Down syndrome, and ischemia. NKCC1 facilitates chloride influx, whereas KCC2 mediates chloride efflux via potassium gradient. Altered expression and function of these cotransporters have been associated with excitotoxicity, inflammation, and cellular death in ischemic events characterized by reduced cerebral blood flow, leading to compromised tissue metabolism and subsequent cell death. NKCC1 inhibition has emerged as a potential therapeutic approach to attenuate intracellular chloride accumulation and mitigate neuronal damage during ischemic events. Similarly, targeting KCC2, which regulates chloride efflux, holds promise for improving outcomes and reducing neuronal damage under ischemic conditions. This review emphasizes the critical roles of GABA, NKCC1, and KCC2 in ischemic pathologies and their potential as therapeutic targets. Inhibiting or modulating the activity of these cotransporters represents a promising strategy for reducing neuronal damage, preventing excitotoxicity, and improving neurological outcomes following ischemic events. Furthermore, exploring the interactions between natural compounds and NKCC1/KCC2 provides additional avenues for potential therapeutic interventions for ischemic injury.

Caffeine potentiates the release of GABA mediated by NMDA receptor activation: Involvement of A1 adenosine receptors.

Caffeine, a methylated derivative of xanthine and widely consumed psychoactive substance, acts in several targets in the nervous system. We investigated its role in retinal explants of chick embryo analyzing the role of purinergic receptors in [(3)H]-GABA release induced by d-aspartate (d-asp). d-Asp increases GABA-release 4.5-fold when compared to basal levels from 13-day-old chick embryo retinal explants. Caffeine 500µM elevated d-asp-induced GABA release in 60%. The release was inhibited in the presence of NNC-711, a GABA transporter-1 (GAT-1) blocker or by MK-801, an N-methyl-d-aspartate receptor (NMDAR) antagonist. Caffeine did not modify [(3)H]-GABA uptake carried out for 5, 10, 30 and 60min and did not increase the release of d-asp or glutamate at basal or stimulated conditions. The caffeine effect was mimicked by the adenosine A1 receptor antagonist DPCPX and by the adenylyl cyclase (AC) activator forskolin. It was also blocked by the protein kinase A (PKA) inhibitor H-89, tyrosine kinase inhibitor genistein or by the src family kinase (SFK) inhibitor PP1. Forskolin-stimulated cyclic adenosine monophosphate (cAMP) levels were reduced in the presence of the A1 receptor agonist CHA. Western blot analysis revealed that 500µM caffeine increased phosphoGluN2B expression levels in approximately 60% when compared to total GluN2B levels in embryonic E13 retina. The GluN2B subunit-containing NMDAR antagonist ifenprodil inhibited the caffeine effect. Our results suggest that caffeine potentiates d-asp-induced GABA release, which is mediated by GAT-1, via inhibition of adenosine A1 receptor and activation of the PKA pathway. Regulation of NMDAR by phosphorylation of GluN2B subunit by a SFK may also be involved in the effect promoted by caffeine.

Characterization of a GABAergic neurotransmission in adult Schistosoma mansoni.

The neuromuscular systems of parasitic helminths are targets that are particularly amenable for anthelmintics. In this study, we describe a GABAergic neurotransmission in adult Schistosoma mansoni, the trematode responsible for high levels of morbidity in people living in developing countries. GABA immunoreactivity (GABA-IR) was detected in nerve cells and fibres of the cerebral ganglia and longitudinal nerve cords and the nerve plexuses ramifying throughout the parenchyma of male adult worms. In addition, strong GABA-IR was also found associated with the oral and ventral suckers as well as in testes indicating a role for GABA in fixation to the host vascular wall and spermatogenesis. The capacity to synthesize GABA from glutamate was confirmed by measurement of a glutamate decarboxylase (GAD) activity. Supporting these data, a single band with an apparent molecular weight of about 67 kDa was detected using an antibody raised against mammalian GAD. In vivo studies revealed that picrotoxin, a non-competitive antagonist of the GABAA receptor, produced a modification of the motility and locomotory behaviour of adult worms, suggesting that GABAergic signalling pathway may play a physiological role in the motonervous system of S. mansoni and could be considered as a potential target for the development of new drugs.

Inhibition of choline acetyltransferase by excitatory amino acids as a possible mechanism for cholinergic dysfunction in the central nervous system.

Choline acetyltransferase (ChAT) activity was reduced by more than 85% in cultured retina cells after 16 h treatment with 150 microM kainate (T(1/2) : 3.5 h). Glutamate, AMPA and quisqualate also inhibited the enzyme in equivalent proportion. Cell lesion measured by lactate dehydrogenase (LDH) release, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide - thiazolyl blue (MTT) reduction and microscopic observation was not detected even after 48 h with kainate. Other retina neurochemical markers were not affected by kainate and full recovery of the enzyme was achieved 9 days after kainate removal. Moreover, hemicolinium-3 sensitive choline uptake and hemicolinium-3 binding sites were maintained intact after kainate treatment. The immunoblot and immunohistochemical analysis of the enzyme revealed that ChAT molecules were maintained in cholinergic neurons. The use of antagonists showed that ionotropic and group 1 metabotropic receptors mediated the effect of glutamate on ChAT inhibition, in a calcium dependent manner. The quisqualate mediated ChAT inhibition and part of the kainate effect (30%) was prevented by 5 mM N(G)-nitro-L-arginine methyl ester (L-NAME). Veratridine (3 microM) also reduced ChAT by a Ca(2+) dependent, but glutamate independent mechanism and was prevented by 1 microM tetrodotoxin.

Atypical effect of dopamine in modulating the functional inhibition of NMDA receptors of cultured retina cells.

Cultured retina cells released accumulated [3H]GABA (gamma-aminobutyric acid) when stimulated by L-glutamate, N-methyl-D-aspartate (NMDA) and kainate. In the absence of Mg2+, dopamine at 200 microM (IC50 60 microM), inhibited in more than 50% the release of [3H]GABA induced by L-glutamate and NMDA, but not by kainate. This effect was not blocked by the D1-like dopamine receptor antagonist, R-(+)-7-chloro-8-hydroxy-3-methyl- -phenyl-2,3,4,5-tetrahydro- H-3-benzazepine hydrochloride (SCH 23390), neither by haloperidol nor spiroperidol (dopamine D2-like receptor antagonists). The dopamine D1-like receptor agonist R(+)-1-phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,diol hydrochloride (SKF 38393) at 50 microM, but not its enantiomer, also inhibited the release of [3H]GABA induced by NMDA, but not by kainate; an effect that was not prevented by the antagonists mentioned above. (+/-)-6-Chloro-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepin e hydrobromide (SKF 812497) had no effect. Neither 8BrcAMP (5 mM) nor forskolin (10 microM) inhibited the release of [3H]GABA. Our results suggest that dopamine and (+)-SKF 38393 inhibit the glutamate and NMDA-evoked [3H]GABA release through mechanisms that seem not to involve known dopaminergic receptor systems.

Aspartate as a selective NMDA receptor agonist in cultured cells from the avian retina.

Although glutamate is considered the natural neurotransmitter that mediates excitatory function in the CNS, other active natural compounds can also drive the functional activation of excitatory amino acid receptors (EAAR). L-aspartate is the most likely neurotransmitter to mimic the actions of glutamate. Here we show that L-aspartate promotes the release of GABA acting selectively on the NMDA receptor subtype. Retina cell cultures, when exposed to excitatory amino acids (EAA), release [3H] GABA previously incorporated by the cells. Both L-glutamate and L- and D-aspartate at 100 microM concentration, promote the release which can be mimicked by kainate and NMDA. While aspartate-induced release of [3H] GABA occurs in the presence of 1 mM Mg2+, NMDA (100 microM) promotes the release only when Mg2+ is omitted from the superfusing medium. However, in the absence of Mg2+ the efficacy of 1- and d-aspartate (100 microM) to activate [3H] GABA release increases by a factor of 2 when compared to the release observed in the presence of 1 mM Mg2+. NMDA and aspartate induced release of [3H] GABA is completely inhibited by 10 microM MK-801 and is not affected by CNQX (100 microM). In the presence of Mg2+, aspartate-induced release of [3H] GABA is also completely inhibited by MK-801 (10 microM) and is not significantly affected by CNQX (100 microM). The [3H] GABA release induced by kainate (100 microM) is fully inhibited by CNQX (100 microM) and is not affected by MK-801 (10 microM). Our results indicate that in the retina, l-aspartate modulates its excitatory function on a set of GABAergic cells via the selective activation of NMDA receptors. The fact that L- and D-aspartate (but not D-glutamate) induce the release of GABA even in the presence of Mg2+ suggests that the electrogenic uptake of aspartate is required to lower the affinity of the NMDA channel for Mg2+. The observation that D-glutamate (200 microM), which is not taken up by the cells, activates the efflux of GABA only when Mg2+ is omitted from the incubating medium, supports this possibility.

Transient coupling of NMDA receptor with ip3 production in cultured cells of the avian retina.

The mobilization of inositol triphosphate ip3 by N-methyl D-aspartate (NMDA) and kainate, two excitatory amino acid EAA receptor agonists, was studied in cultured chick retina cells as a function of culture differentiation. Kainate (EC50 = 30 microM) stimulated from 6 to 9-fold the production of [3H]ip3 between E8C3 (embryonic day 8 plus 3 days in vitro) and E8C13. The kainate response was blocked by CNQX (100 microM) by more than 80% until stage E8C9. MK-801, however, was totally ineffective in preventing the kainate induced ip3 generation. [3H]ip3 production evoked by NMDA was increased 4-fold above basal levels at E8C3. As cultures differentiated, [3H]ip3 production promoted by NMDA decreased to 2.5-fold at E8C6 to 1.6-fold the basal levels in cultures at later stages of differentiation. The removal of Mg2+ from the incubating medium at E8C3 increased the NMDA mediated [3H]ip3 production by 80%. However, at more differentiated stages of the cultures, when cells were not responsive to NMDA, removal of Mg2+ plus the addition of 1 mM glycine did not change the pattern of the response. Although NMDA mediated ip3 production is almost absent in more differentiated cultures, NMDA is able to induce [3H]GABA release in E8C3 and E8C13 cultures with characteristics that reflect typical NMDA receptor activation: it is highly potentiated by the absence of Mg2+ and by the presence of glycine. The NMDA induced production of [3H]ip3 at E8C3 was entirely blocked by MK-801 (100 microM) and APV (100 microM) but not by CNQX.(ABSTRACT TRUNCATED AT 250 WORDS)