Interactions between Glycine and Glutamate through Activation of Their Transporters in Hippocampal Nerve Terminals.

Evidence supports the pathophysiological relevance of crosstalk between the neurotransmitters Glycine and Glutamate and their close interactions; some reports even support the possibility of Glycine-Glutamate cotransmission in central nervous system (CNS) areas, including the hippocampus. Functional studies with isolated nerve terminals (synaptosomes) permit us to study transporter-mediated interactions between neurotransmitters that lead to the regulation of transmitter release. Our main aims here were: (i) to investigate release-regulating, transporter-mediated interactions between Glycine and Glutamate in hippocampal nerve terminals and (ii) to determine the coexistence of transporters for Glycine and Glutamate in these terminals. Purified synaptosomes, analyzed at the ultrastructural level via electron microscopy, were used as the experimental model. Mouse hippocampal synaptosomes were prelabeled with [3H]D-Aspartate or [3H]Glycine; the release of radiolabeled tracers was monitored with the superfusion technique. The main findings were that (i) exogenous Glycine stimulated [3H]D-Aspartate release, partly by activation of GlyT1 and in part, unusually, through GlyT2 transporters and that (ii) D-Aspartate stimulated [3H]glycine release by a process that was sensitive to Glutamate transporter blockers. Based on the features of the experimental model used, it is suggested that functional transporters for Glutamate and Glycine coexist in a small subset of hippocampal nerve terminals, a condition that may also be compatible with cotransmission; glycinergic and glutamatergic transporters exhibit different functions and mediate interactions between the neurotransmitters. It is hoped that increased information on Glutamate-Glycine interactions in different areas, including the hippocampus, will contribute to a better knowledge of drugs acting at "glycinergic" targets, currently under study in relation with different CNS pathologies.

Enhanced Function and Overexpression of Metabotropic Glutamate Receptors 1 and 5 in the Spinal Cord of the SOD1G93A Mouse Model of Amyotrophic Lateral Sclerosis during Disease Progression.

Glutamate (Glu)-mediated excitotoxicity is a major cause of amyotrophic lateral sclerosis (ALS) and our previous work highlighted that abnormal Glu release may represent a leading mechanism for excessive synaptic Glu. We demonstrated that group I metabotropic Glu receptors (mGluR1, mGluR5) produced abnormal Glu release in SOD1G93A mouse spinal cord at a late disease stage (120 days). Here, we studied this phenomenon in pre-symptomatic (30 and 60 days) and early-symptomatic (90 days) SOD1G93A mice. The mGluR1/5 agonist (S)-3,5-Dihydroxyphenylglycine (3,5-DHPG) concentration dependently stimulated the release of [3H]d-Aspartate ([3H]d-Asp), which was comparable in 30- and 60-day-old wild type mice and SOD1G93A mice. At variance, [3H]d-Asp release was significantly augmented in 90-day-old SOD1G93A mice and both mGluR1 and mGluR5 were involved. The 3,5-DHPG-induced [3H]d-Asp release was exocytotic, being of vesicular origin and mediated by intra-terminal Ca2+ release. mGluR1 and mGluR5 expression was increased in Glu spinal cord axon terminals of 90-day-old SOD1G93A mice, but not in the whole axon terminal population. Interestingly, mGluR1 and mGluR5 were significantly augmented in total spinal cord tissue already at 60 days. Thus, function and expression of group I mGluRs are enhanced in the early-symptomatic SOD1G93A mouse spinal cord, possibly participating in excessive Glu transmission and supporting their implication in ALS. Please define all abbreviations the first time they appear in the abstract, the main text, and the first figure or table caption.

Colocalization of neurotransmitter transporters on the plasma membrane of the same nerve terminal may reflect cotransmission.

There is increasing evidence for the neuronal coexistence of classical transmitters. Implications in favor of cotransmission have often been represented by the identification, in the same neuron, of the putative cotransmitters, their synthetic enzymes and/or their vesicular transporters. In contrast, coexpression of neurotransmitter transporters on the plasma membrane of the same nerve terminal, although a potentially important indication for cotransmission, has received poor attention. We here used preparations of isolated nerve endings to functionally identify transporters coexpressed on the plasma membrane of the same terminal, in order to verify if such transporter coexpression indeed exists in neuronal systems in which cotransmission has already been established or reasonably suspected through other technical approaches. We could observe that functional transporters for glycine and glutamate are coexpressed on nerve terminals in the cerebellum; transporters for dopamine and GABA coexist on striatal terminals; transporters for glycine and GABA, previously found to coexist as cotransmission markers on nerve terminals of spinal cord and cerebellum, are not coexpressed in neocortex and hippocampus, where cotransmission has not been proposed to occur; transporters for GABA, glycine and glutamate are colocalized on nerve terminals of the spinal cord. Confocal microscopy experiments were performed to substantiate functional data, highlighting the presence of the co-existing transporters under study on MAP-2 positive synaptosomes. It is concluded that investigating the colocalization of functional neurotransmitter transporters on the plasma membrane of nerve terminals can provide useful information on the possibility of cotransmission.

Advances in understanding the functions of native GlyT1 and GlyT2 neuronal glycine transporters.

Glycine can be substrate for two transporters: GlyT1, largely expressed by astrocytes but also by some non-glycinergic neurons, and GlyT2, most frequently present in glycine-storing nerve endings. In morphological studies, GlyT2 expression had been found to be restricted to caudal regions, being almost undetectable in neocortex and hippocampus. Here, we compared the uptake activities of GlyT1 and GlyT2 in synaptosomes purified from mouse spinal cord, cerebellum, neocortex and hippocampus. Although, as expected, [(3)H]glycine uptake was significantly lower in telencephalic than in caudal regions, selective GlyT2-mediated uptake could be evaluated in all areas. Appropriately, [(3)H]glycine selectively taken up into hippocampal synaptosomes through GlyT2 could be subsequently released by exocytosis. Native GlyT2, which did not contribute to basal release from cerebellum or spinal cord nerve terminals, could mediate release of [(3)H]glycine by transporter reversal in synaptosomes exposed to veratridine. Moreover, GlyT2 transporters could perform Na(+)-dependent homoexchange in response to externally added glycine. In conclusion, transporters of the GlyT2 type exhibited significant uptake also in telencephalic regions, probably because of the elevated driving force related to their stoichiometry. Although glycine release through GlyT2 had been predicted to be a very difficult process, GlyT2 expressed on isolated glycinergic nerve terminals can perform both release by transporter reversal and homoexchange.

Multiple functions of neuronal plasma membrane neurotransmitter transporters.

Removal from receptors of neurotransmitters just released into synapses is one of the major steps in neurotransmission. Transporters situated on the plasma membrane of nerve endings and glial cells perform the process of neurotransmitter (re)uptake. Because the density of transporters in the membranes can fluctuate, transporters can determine the transmitter concentrations at receptors, thus modulating indirectly the excitability of neighboring neurons. Evidence is accumulating that neurotransmitter transporters can exhibit multiple functions. Being bidirectional, neurotransmitter transporters can mediate transmitter release by working in reverse, most often under pathological conditions that cause ionic gradient dysregulations. Some transporters reverse to release transmitters, like dopamine or serotonin, when activated by 'indirectly acting' substrates, like the amphetamines. Some transporters exhibit as one major function the ability to capture transmitters into nerve terminals that perform insufficient synthesis. Transporter activation can generate conductances that regulate directly neuronal excitability. Synaptic and non-synaptic transporters play different roles. Cytosolic Na(+) elevations accompanying transport can interact with plasmalemmal or/and mitochondrial Na(+)/Ca(2+) exchangers thus generating calcium signals. Finally, neurotransmitter transporters can behave as receptors mediating releasing stimuli able to cause transmitter efflux through multiple mechanisms. Neurotransmitter transporters are therefore likely to play hitherto unknown roles in multiple therapeutic treatments.

High-affinity GABA uptake by neuronal GAT1 transporters provokes release of [(3)H]GABA by homoexchange and through GAT1-independent Ca(2+)-mediated mechanisms.

High-affinity uptake of GABA into nerve terminals may have functions other than recapture of the neurotransmitter. Synaptosomes purified from mouse cerebellum were prelabelled with [(3)H]GABA and then superfused with GABA and drugs selective for some presynaptic targets. Influx of GABA through GAT1 transporters stimulated efflux of [(3)H]GABA in a concentration-dependent manner (EC50 ∼ 3 µM). The efflux of the transmitter occurred in part by GAT1 reversal through the so called homoexchange. The ion fluxes (particularly Na(+) influx) accompanying GABA uptake triggered intraterminal Ca(2+) signals through both plasmalemmal Na(+)/Ca(2+) exchangers, sensitive to KB-R7943 or to ifenprodil and mitochondrial Na(+)/Ca(2+) exchangers, sensitive to CGP37157. These Ca(2+) signals likely facilitated GABA release from nerve terminals via niflumic acid- and NPPB-sensitive anion channels. The results show that GABA, at concentrations corresponding to the high-affinity uptake, can evoke GABA release which occurs in part by the expected GAT1-mediated homoexchange, while the transporter-independent component of the GABA uptake-evoked GABA release takes place by hitherto unsuspected mechanisms which include Na(+)/Ca(2+) exchangers and anion channels. The significance of the novel function of the GABA high-affinity uptake here identified deserves further multidisciplinary investigation.

Benefits of high-dose N-acetylcysteine to exacerbation-prone patients with COPD.

BACKGROUND: Although high-dose N-acetylcysteine (NAC) has been suggested to reduce COPD exacerbations, it is unclear which category of patients with COPD would benefit most from NAC treatment. The objective of this study was to compare the effect of high-dose NAC (600 mg bid) between high-risk and low-risk Chinese patients with COPD. METHODS: Patients with spirometry-confirmed stable COPD were randomized to treatment with either NAC 600 mg bid or placebo in addition to their usual treatments. Patients were followed up every 16 weeks for a total of 1 year. Further analysis was performed according to each patient's exacerbation risk at baseline as defined by the current GOLD (Global Initiative for Chronic Obstructive Lung Disease) strategy to analyze the effect of high-dose NAC in high-risk and low-risk patients. RESULTS: Of the 120 patients with COPD randomized (men, 93.2%; mean age, 70.8 ± 0.74 years; prebronchodilator FEV1, 53.9 ± 2.0%; baseline characteristics comparable between treatment groups), 108 (NAC, 52; placebo, 56) completed the 1-year study. For high-risk patients (n = 89), high-dose NAC compared with placebo significantly reduced exacerbation frequency (0.85 vs 1.59 [P = .019] and 1.08 vs 2.22 [P = .04] at 8 and 12 months, respectively), prolonged time to first exacerbation (P = .02), and increased the probability of being exacerbation free at 1 year (51.3% vs 24.4%, P = .013). This beneficial effect of high-dose NAC vs placebo was not significant in low-risk patients. CONCLUSIONS: High-dose NAC (600 mg bid) for 1 year reduces exacerbations and prolongs time to first exacerbation in high-risk but not in low-risk Chinese patients with COPD. TRIAL REGISTRY: ClinicalTrials.gov; No.: NCT01136239; URL: www.clinicaltrials.gov.

GABA release provoked by disturbed Na(+), K(+) and Ca(2+) homeostasis in cerebellar nerve endings: roles of Ca(2+) channels, Na(+)/Ca(2+) exchangers and GAT1 transporter reversal.

GABA release provoked by ion dysregulations typical of some neuropathological conditions was analyzed in purified cerebellar synaptosomes pre-labeled with [(3)H]GABA and exposed in superfusion to KCl, 4-aminopyridine (4-AP) or veratridine. The overflows caused by relatively low concentrations of the releasers were almost totally external Ca(2+)-dependent. Higher concentrations of KCl or veratridine, but not 4-AP, involved also external Ca(2+)-independent mechanisms. The GABA overflows evoked by veratridine and, less so, the overflow evoked by high K(+), occurred in part by reversal of the GAT1 transporter. None of the depolarizing agents activated store-operated or transient receptor potential or L-type Ca(2+) channels. Only the overflow caused by 4-AP occurred in part by N- and P/Q-type voltage-sensitive calcium channel-dependent exocytosis. Significant portions of the external Ca(2+)-dependent overflows evoked by the three releasers involved reversal of plasmalemmal Na(+)/Ca(2+) exchangers. The overflows evoked by high K(+) or veratridine, but not by 4-AP were evoked by Ca(2+) originated through mitochondrial Na(+)/Ca(2+) exchangers. Ca(2+)-induced Ca(2+) release mediated by inositoltrisphosphate receptors participated exclusively in the GABA release stimulated by high KCl which also occurred in a modest portion through anion channels. Important differences could be observed between the release mechanisms of GABA here described and those previously reported for glycine, in spite of the abundant vesicular co-localization of the two transmitters in cerebellar interneurons.

Twice daily N-acetylcysteine 600 mg for exacerbations of chronic obstructive pulmonary disease (PANTHEON): a randomised, double-blind placebo-controlled trial.

BACKGROUND: Increased oxidative stress and inflammation has a role in the pathogenesis of chronic obstructive pulmonary disease (COPD). Drugs with antioxidant and anti-inflammatory properties, such as N-acetylcysteine, might provide a useful therapeutic approach for COPD. We aimed to assess whether N-acetylcysteine could reduce the rate of exacerbations in patients with COPD. METHODS: In our prospective, randomised, double-blind, placebo-controlled, parallel-group study, we enrolled patients aged 40-80 years with moderate-to-severe COPD (post-bronchodilator forced expiratory volume in 1 s [FEV1]/forced vital capacity <0·7 and FEV1 of 30-70% of predicted) at 34 hospitals in China. We stratified patients according to use of inhaled corticosteroids (regular use or not) at baseline and randomly allocated them to receive N-acetylcysteine (one 600 mg tablet, twice daily) or matched placebo for 1 year. The primary endpoint was the annual exacerbation rate in patients who received at least one dose of study drug and had at least one assessment visit after randomisation. This study is registered with the Chinese Clinical Trials Registry, ChiCTR-TRC-09000460. FINDINGS: Between June 25, 2009, and Dec 29, 2010, we screened 1297 patients, of whom 1006 were eligible for randomisation (504 to N-acetylcysteine and 502 to placebo). After 1 year, we noted 497 acute exacerbations in 482 patients in the N-acetylcysteine group who received at least one dose and had at least one assessment visit (1·16 exacerbations per patient-year) and 641 acute exacerbations in 482 patients in the placebo group (1·49 exacerbations per patient-year; risk ratio 0·78, 95% CI 0·67-0·90; p=0·0011). N-acetylcysteine was well tolerated: 146 (29%) of 495 patients who received at least one dose of N-acetylcysteine had adverse events (48 serious), as did 130 (26%) of 495 patients who received at least one dose of placebo (46 serious). The most common serious adverse event was acute exacerbation of COPD, occurring in 32 (6%) of 495 patients in the N-acetylcysteine group and 36 (7%) of 495 patients in the placebo group. INTERPRETATION: Our findings show that in Chinese patients with moderate-to-severe COPD, long-term use of N-acetylcysteine 600 mg twice daily can prevent exacerbations, especially in disease of moderate severity. Future studies are needed to explore efficacy in patients with mild COPD (GOLD I). FUNDING: Hainan Zambon Pharmaceutical.

A new function for glycine GlyT2 transporters: Stimulation of γ-aminobutyric acid release from cerebellar nerve terminals through GAT1 transporter reversal and Ca(2+)-dependent anion channels.

Glycine GlyT2 transporters are localized on glycine-storing nerve endings. Their main function is to accumulate glycine to replenish synaptic vesicles. Glycine was reported to be costored with γ-aminobutyric acid (GABA) in cerebellar interneurons that may coexpress glycine and GABA transporters, and this is confirmed here by confocal microscopy analysis showing coexpression of GAT1 and GlyT2 transporters on microtubule-associated protein-2-positive synaptosomes. It was found that GABA uptake elicited glycine release from cerebellar nerve endings by various mechanisms. We investigated whether and by what mechanisms activation of glycine transporters could mediate release of GABA. Nerve endings purified from cerebellum were prelabeled with [3H]GABA and exposed to glycine. Glycine stimulated [3H]GABA release in a concentration-dependent manner. The glycine effect was insensitive to strychnine or to 5,7-dichlorokynurenate but it was abolished when GlyT2 transporters were blocked. About 20% of the evoked release was dependent on external Ca2+ entered by reversal of plasmalemmal Na+/Ca2+ exchangers. A significant portion of the GlyT2-mediated release of [3H]GABA (about 50% of the external Ca(2+)-independent release) occurred by reversal of GABA GAT1 transporters. Na+ ions, reaching the cytosol during glycine uptake through GlyT2, activated mitochondrial Na+/Ca2+ exchangers, causing an increase in cytosolic Ca2+, which in turn triggered a Ca(2+)-induced Ca2+ release process at inositoltrisphosphate receptors. Finally, the increased availability of Ca2+ in the cytosol allowed the opening of anion channels permeable to GABA. In conclusion, GlyT2 transporters not only take up glycine to replenish synaptic vesicles but can also mediate release of GABA by reversal of GAT1 and permeation through anion channels.

Neuropeptide S inhibits release of 5-HT and glycine in mouse amygdala and frontal/prefrontal cortex through activation of the neuropeptide S receptor.

Neuropeptide S (NPS) is a neurotransmitter/neuromodulator that has been identified as the natural ligand of G protein-coupled receptors termed NPS receptors (NPSRs). The NPS-NPSR system is involved in the control of numerous centrally-mediated behaviours, including anxiety. As several classical transmitters play a role in fear/anxiety, we here investigated the regulation by NPS of the exocytotic release of 5-hydroxytryptamine (5-HT) and glycine in nerve terminals isolated from mouse frontal/prefrontal cortex and amygdala. Synaptosomes, prelabelled with the tritiated neurotransmitters, were depolarized in superfusion with 12-15 mM KCl and exposed to varying concentrations of NPS. The evoked release of [(3)H]5-HT in frontal/prefrontal cortex was potently inhibited by NPS (maximal effect about 25% at 0.1 nM). Differently, the neuropeptide exhibited higher efficacy but much lower potency in amygdala (maximal effect about 40% at 1 µM). NPS was an extremely potent inhibitor of the K(+)-evoked release of [(3)H]glycine in frontal/prefrontal nerve endings (maximal effect about 25% at 1 pM). All the inhibitory effects observed were counteracted by the NPSR antagonist SHA 68, indicating that the neuropeptide acted at NPSRs. In conclusion, NPS can inhibit the exocytosis of 5-HT and of glycine through the activation of presynaptic NPSRs situated on serotonergic and glycinergic terminals in areas involved in fear/anxiety behaviours. The possibility exists that the NPSRs in frontal/prefrontal cortex are high-affinity receptors involved in non-synaptic transmission, whereas the NPSRs on amygdala serotonergic terminals are low-affinity receptors involved in axo-axonic synaptic communication.

High-dose N-acetylcysteine in stable COPD: the 1-year, double-blind, randomized, placebo-controlled HIACE study.

BACKGROUND: The mucolytic and antioxidant effects of N-acetylcysteine (NAC) may have great value in COPD treatment. However, beneficial effects have not been confirmed in clinical studies, possibly due to insufficient NAC doses and/or inadequate outcome parameters used. The objective of this study was to investigate high-dose NAC plus usual therapy in Chinese patients with stable COPD. METHODS: The 1-year HIACE (The Effect of High Dose N-acetylcysteine on Air Trapping and Airway Resistance of Chronic Obstructive Pulmonary Disease-a Double-blinded, Randomized, Placebo-controlled Trial) double-blind trial conducted in Kwong Wah Hospital, Hong Kong, randomized eligible patients aged 50 to 80 years with stable COPD to NAC 600 mg bid or placebo after 4-week run-in. Lung function parameters, symptoms, modified Medical Research Council (mMRC) dyspnea and St. George's Respiratory Questionnaire (SGRQ) scores, 6-min walking distance (6MWD), and exacerbation and admission rates were measured at baseline and every 16 weeks for 1 year. RESULTS: Of 133 patients screened, 120 were eligible (93.2% men; mean age, 70.8±0.74 years; %FEV1 53.9±2.0%). Baseline characteristics were similar in the two groups. At 1 year, there was a significant improvement in forced expiratory flow 25% to 75% (P=.037) and forced oscillation technique, a significant reduction in exacerbation frequency (0.96 times/y vs 1.71 times/y, P=.019), and a tendency toward reduction in admission rate (0.5 times/y vs 0.8 times/y, P=.196) with NAC vs placebo. There were no significant between-group differences in mMRC dypsnea score, SGRQ score, and 6MWD. No major adverse effects were reported. CONCLUSION: In this study, 1-year treatment with high-dose NAC resulted in significantly improved small airways function and decreased exacerbation frequency in patients with stable COPD. TRIAL REGISTRY: ClinicalTrials.gov; No.: NCT01136239; URL: www.clinicaltrials.gov.

Glycine release is regulated by metabotropic glutamate receptors sensitive to mGluR2/3 ligands and activated by N-acetylaspartylglutamate (NAAG).

The presence of metabotropic glutamate receptors (mGluRs) of group II modulating glycine exocytosis from glycinergic nerve endings of mouse spinal cord was investigated. Purified synaptosomes were selectively prelabeled with [(3)H]glycine through the neuronal transporter GlyT2 and subsequently depolarized by superfusion with 12 mM KCl. The selective mGluR2/3 agonist LY379268 inhibited the K(+)-evoked overflow of [(3)H]glycine in a concentration-dependent manner (EC(50) about 0.2 nM). The effect of LY379268 was prevented by the selective mGluR2/3 antagonist LY341495 (IC(50) about 1 nM). N-acetylaspartylglutamate (NAAG) inhibited [(3)H]glycine overflow with extraordinary potency (EC(50) about 50 fmol). In contrast, glutamate was ineffective up to 0.1 nM, excluding that glutamate contamination of commercial NAAG samples is responsible for the reported activity of NAAG at mGluR3. LY341495 antagonized the NAAG inhibition of [(3)H]glycine release. The effect of a combination of maximally effective concentrations of LY379268 and NAAG exhibited no additivity. The non-hydrolysable NAAG analogue N-acetylaspartyl-ß-linked glutamate (ß-NAAG) antagonized NAAG and LY379268. In conclusion, our results show that glycinergic nerve endings in spinal cord are endowed with group II mGluRs mediating inhibition of glycine exocytosis. NAAG can activate these presynaptic receptors with extremely high affinity and with characteristics compatible with the reported mGluR3 pharmacology. This article is part of a Special Issue entitled 'Metabotropic Glutamate Receptors'.

GABA transporters mediate glycine release from cerebellum nerve endings: roles of Ca(2+)channels, mitochondrial Na(+)/Ca(2+) exchangers, vesicular GABA/glycine transporters and anion channels.

GABA transporters accumulate GABA to inactivate or reutilize it. Transporter-mediated GABA release can also occur. Recent findings indicate that GABA transporters can perform additional functions. We investigated how activation of GABA transporters can mediate release of glycine. Nerve endings purified from mouse cerebellum were prelabeled with [(3)H]glycine in presence of the glycine GlyT1 transporter inhibitor NFPS to label selectively GlyT2-bearing terminals. GABA was added under superfusion conditions and the mechanisms of the GABA-evoked [(3)H]glycine release were characterized. GABA stimulated [(3)H]glycine release in a concentration-dependent manner (EC(50) = 8.26 µM). The GABA-evoked release was insensitive to GABA(A) and GABA(B) receptor antagonists, but it was abolished by GABA transporter inhibitors. About 25% of the evoked release was dependent on external Ca(2+) entering the nerve terminals through VSCCs sensitive to ω-conotoxins. The external Ca(2+)-independent release involved mitochondrial Ca(2+), as it was prevented by the Na(+)/Ca(2+) exchanger inhibitor CGP37157. The GABA uptake-mediated increases in cytosolic Ca(2+) did not trigger exocytotic release because the [(3)H]glycine efflux was insensitive to clostridial toxins. Bafilomycin inhibited the evoked release likely because it reduced vesicular storage of [(3)H]glycine so that less [(3)H]glycine can become cytosolic when GABA taken up exchanges with [(3)H]glycine at the vesicular inhibitory amino acid transporters shared by the two amino acids. The GABA-evoked [(3)H]glycine efflux could be prevented by niflumic acid or NPPB indicating that the evoked release occurred essentially by permeation through anion channels. In conclusion, GABA uptake into GlyT2-bearing cerebellar nerve endings triggered glycine release which occurred essentially by permeation through Ca(2+)-dependent anion channels. Glial GABA release mediated by anion channels was proposed to underlie tonic inhibition in the cerebellum; the present results suggest that glycine release by neuronal anion channels also might contribute to tonic inhibition.

Glycine release provoked by disturbed Na⁺, Na⁺ and Ca²âº homeostasis in cerebellar nerve endings: roles of Ca²âº channels, Na⁺/Ca²âº exchangers and GlyT2 transporter reversal.

Glycine release provoked by ion dysregulations typical of some neuropathological conditions was analyzed in cerebellar synaptosomes selectively pre-labelled with [³H]glycine through GlyT2 transporters and exposed in superfusion to KCl, 4-aminopyridine (4-AP) or veratridine. The overflows caused by relatively low concentrations of the releasers were largely external Ca²âº-dependent. Higher concentrations of KCl (50 mM) or veratridine (10 µM), but not of 4-AP (1 mM), involved also external Ca²âº-independent mechanisms. GlyT1-mediated release could not be observed; only the external Ca²âº-independent veratridine-evoked overflow occurred significantly by GlyT2 reversal. None of the three depolarizing agents activated store-operated or transient receptor potential or L-type Ca²âº channels. The overflows caused by KCl or 4-AP occurred in part by N- and P/Q-type voltage-sensitive calcium channel-dependent exocytosis. Significant portions of the external Ca²âº-dependent overflow evoked by KCl or 4-AP (and all that caused by veratridine) were mediated by reverse plasmalemmal Na⁺/Ca²âº exchangers. Significant contribution to the overflows evoked by KCl or veratridine came from Ca²âº originated through mitochondrial Na⁺/Ca²âº exchangers. Ca²âº-induced Ca²âº release (CICR) mediated by inositoltrisphosphate receptors (InsP3Rs) represents the final trigger of the glycine release evoked by high KCl. The overflows evoked by 4-AP or, less so, by veratridine also involved InsP3R-mediated CICR and, in part, CICR mediated by ryanodine receptors. To conclude, ionic dysregulations typical of ischemia and epilepsy caused glycine release in cerebellum by multiple differential mechanisms that may represent potential therapeutic targets.

Ionic dysregulations typical of ischemia provoke release of glycine and GABA by multiple mechanisms.

Energy deprivation during ischemia causes dysregulations of ions, particularly sodium, potassium and calcium. Under these conditions, release of neurotransmitters is often enhanced and can occur by multiple mechanisms. The aim of this work was to characterize the modes of exit of glycine and GABA from nerve endings exposed to stimuli known to reproduce some of the ionic changes typical of ischemic conditions. Their approach was chosen instead of application of ischemic conditions because the release evoked during ischemia is mechanistically too heterogeneous. Mouse hippocampus and spinal cord synaptosomes, pre-labeled with [(3)H]glycine or [(3)H]GABA, were exposed in superfusion to 50 mM KCl or to 10 microM veratridine. The evoked overflows differed greatly between the two transmitters and between the two regions examined. Significant portions of the K(+)- and the veratridine-evoked overflows occurred by classical exocytosis. Carrier-mediated release of GABA, but not of glycine, was evoked by high K(+); GABA and, less so, glycine were released through transporter reversal by veratridine. External calcium-dependent overflows were only in part sensitive to omega-conotoxins; significant portions occurred following reversal of the plasmalemmal Na(+)/Ca(2+) exchanger. Finally, a relevant contribution to the overall transmitter overflows came from cytosolic calcium originating through the mitochondrial Na(+)/Ca(2+) exchanger. To conclude, ionic dysregulations typical of ischemia cause neurotransmitter release by heterogeneous mechanisms that differ depending on the transmitters and the CNS regions examined.

Functional 'glial' GLYT1 glycine transporters expressed in neurons.

Glycine transporter 1 (GLYT1) and GLYT2 are the glycine transporters in CNS. While GLYT2 is largely expressed in glycinergic neurons, GLYT1 has long been considered to be exclusively present in glial cells. There is increasing evidence that significant amounts of the 'glial' transporter also exist on neurons, particularly on pre-synaptic nerve endings of glutamatergic neurons. The functions of 'neuronal GLYT1' may be manifold and are discussed in this review. Of major interest are the interactions between neuronal GLYT1 and glutamatergic receptors of the NMDA type the activity of which is modulated not only by astrocytic GLYT1 but also by neuronal GLYT1. Pathophysiological roles and therapeutic implications of neuronal GLYT1 are emerging from recent studies with genetically modified mice, particularly with animals lacking forebrain neuron-specific GLYT1 transporters. These mutant mice exhibit promnesic phenotypes reflecting enhancement of NMDA receptor function, as it occurs following administration of GLYT1 inhibitors. Inactivation of neuronal GLYT1 in the forebrain may represent an effective therapeutic intervention for the treatment of schizophrenia.

The GABA B receptor antagonists CGP35348 and CGP52432 inhibit glycine exocytosis: study with GABA B1- and GABA B2-deficient mice.

GABA(B) receptors mediate inhibition of neurotransmitter exocytosis from nerve endings. Unexpectedly, the well known GABA(B) receptor antagonist CGP35348 and, in part, the compound CGP52432, are now found to inhibit on their own the K(+)-evoked exocytosis of glycine when added at low micromolar concentrations to superfused mouse glycinergic nerve endings prelabelled with [(3)H]glycine through GLYT2 transporters. CGP35348 inhibited [(3)H]glycine release both in spinal cord and in hippocampus, but was also able to prevent the inhibitory effect of (-)-baclofen; CGP52432 exhibited intrinsic activity only in the hippocampus; in spinal cord, it behaved exclusively as a silent orthosteric antagonist by blocking the release inhibition brought about by (-)-baclofen. The intrinsic activity of CGP35348 in spinal cord was not prevented by CGP52432, indicating that CGP35348 is not a partial GABA(B) agonist in this experimental system. CGP54626, an extremely potent antagonist, exhibited only a minimal intrinsic activity. SCH50911, a GABA(B) antagonist belonging to a different chemical class, was devoid of significant activity, while phaclofen was effective only at 100-300 microM. In synaptosomes purified from the spinal cord or the hippocampus of mice lacking either the GABA(B1) (GABA(B1-/-) mice) or the GABA(B2) (GABA(B2-/-) mice) subunit, the evoked exocytosis of [(3)H]glycine was no longer inhibited by (-)-baclofen, whereas the intrinsic activity of CGP35348 and CGP52432 was not decreased. Activation of unknown sites on glycinergic terminals is likely to be involved. These unexpected effects should not be ignored when interpreting results obtained with the above GABA(B) receptor antagonists.