glycine transporters and anion channels

Neurochemistry International 61 (2012) 133–140

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GABA transporters mediate glycine release from cerebellum nerve endings: Roles of Ca2+channels, mitochondrial Na+/Ca2+ exchangers, vesicular GABA/glycine transporters and anion channels Cristina Romei a, Maurizio Raiteri a,b,c, Luca Raiteri a,b,c,⇑ a b c

Department of Experimental Medicine, Section of Pharmacology and Toxicology, University of Genoa, Italy Center of Excellence for Biomedical Research, University of Genoa, Italy National Institute of Neuroscience, Genoa, Italy

a r t i c l e

i n f o

Article history: Received 14 March 2012 Received in revised form 18 April 2012 Accepted 1 May 2012 Available online 9 May 2012 Keywords: GABA transporters Glycine release Mouse cerebellum Ca2+ signaling Na+/Ca2+ exchangers Anion channels

a b s t r a c t 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 [3H]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 [3H]glycine release were characterized. GABA stimulated [3H]glycine release in a concentration-dependent manner (EC50 = 8.26 lM). The GABA-evoked release was insensitive to GABAA and GABAB receptor antagonists, but it was abolished by GABA transporter inhibitors. About 25% of the evoked release was dependent on external Ca2+entering the nerve terminals through VSCCs sensitive to x-conotoxins. The external Ca2+-independent release involved mitochondrial Ca2+, as it was prevented by the Na+/Ca2+exchanger inhibitor CGP37157. The GABA uptake-mediated increases in cytosolic Ca2+ did not trigger exocytotic release because the [3H]glycine efflux was insensitive to clostridial toxins. Bafilomycin inhibited the evoked release likely because it reduced vesicular storage of [3H]glycine so that less [3H]glycine can become cytosolic when GABA taken up exchanges with [3H]glycine at the vesicular inhibitory amino acid transporters shared by the two amino acids. The GABA-evoked [3H]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 Ca2+-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. Ó 2012 Elsevier Ltd. All rights reserved.

Abbreviations: 2-APB, 2-aminoethoxydiphenyl borate; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N0 ,N0 -tetraacetic acid tetrapotassium salt; BoNT/A, Botulinum toxin A; BoNT/E, Botulinum toxin E; CGP37157, 7-chloro-5-(2-chlorophenyl)-1,5dihydro-4,1-benzothiazepin-2(3H)-one; CGP52432, 3-([[[(3,4-dichlorophenyl)methyl] amino]propyl] diethoxymethyl)phosphinic acid; CICR, Ca2+-induced Ca2+ release; InsP3, inositoltrisphosphate; GAT1, GABA transporter 1; KB-R7943, 2-[2-[4-(4-nitrobenzyloxy)phenyl] ethyl]isothiurea; NCX, Na+/Ca2+ exchanger; NFPS (also known as ALX 5407), N-[(3R)-3-([1,10 -biphenyl]-4-yloxy)-3-(4-fluorophenyl)propyl]-N-methylglycine hydro chloride; NPPB, 5-nitro-2-(3-phenylpropylamino)benzoic acid; Org25543,4-benzyloxy3,5-dimethoxy-N-[1-(dimethylaminociclopentyl)-methyl] benzamide; SSR504734, 2chloro-N-[(S)-phenyl [(2S)-piperidin-2-yl] methyl]-3-trifluoromethyl benzamide, monohydrochloride; SKF89976A, 1-(4,4-diphenyl-3-butenyl)-3-piperidinecarboxylic acid; SOC, store-operated channels; TeTx, Tetanus toxin; TRPC, transient receptor potential canonical; VIAAT, vesicular inhibitory amino acid transporter; VSCCs, voltage sensitive Ca2+ channels. ⇑ Corresponding author. Address: Department of Experimental Medicine, Pharmacology and Toxicology Section, Viale Cembrano 4, 16148 Genova, Italy. Tel.: +39 010 3532659; fax: +39 010 3993360. E-mail address: [email protected] (L. Raiteri). 0197-0186/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neuint.2012.05.005

1. Introduction GABA is the major inhibitory neurotransmitter in the adult mammalian CNS. Extracellular GABA can be captured into neurons and glial cells by specific transporters, to be inactivated or reutilized. In addition to this traditional view, other functions have emerged for GABA transporters. It is known that GABA transporters can, under some conditions, work in reverse and perform carriermediated release (reviewed in Adam-Vizi (1992), Attwell et al. (1993), Levi and Raiteri (1993), Vizi and Sperlágh (1999), and Richerson and Wu (2003)). Subtypes of GABA transporters have been identified in neurons and glia and their distinct functions are being characterized (Eulenburg and Gomeza, 2010; Madsen et al., 2010; Conti et al., 2011; Jin et al., 2011; Schousboe et al., 2011; Rowley

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et al., 2012). Because uptake of GABA is electrogenic, activation of GABA transporters by external GABA can result into intracellular events whose mechanisms are not well understood. In a very recent paper, Bagley et al. (2011) reported that GABA transporter 1 (GAT1) currents directly increased synaptic GABA release from nerve terminals in the periaqueductal grey during opioid withdrawal in rodents. Inhibition or deletion of GAT1 reduced withdrawal-induced GABA release. Doengi et al. (2009) described in some detail how GABA transporters may participate in intracellular Ca2+ signaling in glial cells. The authors reported that, in olfactory bulb astrocytes, GABA evoked Ca2+ transients which were prevented by blocking GABA transporters. The mechanisms underlying the GABA effect include cytosolic Na+ and Ca2+ concentration increases sufficient to trigger Ca2+-induced Ca2+ release (CICR) via inositoltrisphosphate (InsP3) receptors. Several years ago Raiteri et al. (1992) studied effects of GABA on the release of glycine in different rat CNS regions. GABA was found to stimulate the release of glycine in a way independent of GABA receptors, but sensitive to GABA transport inhibition. The releasing effect of GABA differed among CNS regions and was particularly pronounced in cerebellum. The mechanisms by which GABA stimulated the efflux of glycine were not investigated, however, also because some molecular targets were poorly known and selective pharmacological tools were not yet available. Glycine is an inhibitory transmitter when it activates strychnine-sensitive receptors especially in spinal cord, brain stem and cerebellum (Betz, 1992; Legendre, 2001). In addition, glycine exerts important excitatory functions throughout the CNS as an obligatory coagonist of glutamate at NMDA receptors (Johnson and Ascher, 1987). It is now well established that glycine can be substrate for two transporters: GlyT1, largely, though not exclusively, expressed by astrocytes (Zafra et al., 1995a,b) and GlyT2, essentially localized on glycinergic neurons (Zafra et al., 1995a,b; Poyatos et al., 1997; Gomeza et al., 2003). Clearly, during preincubation of crude CNS preparations with [3H]glycine (Raiteri et al., 1992), the transmitter was taken up into neuronal as well as into glial particles and possibly released from both. Discrimination between glycine uptake/release in synaptosomes vs. gliosomes is now possible because GlyT1 and GlyT2 are pharmacologically distinct and selective inhibitors permit preferential labelling through GlyT1 or GlyT2 (see López-Corcuera et al., 2001; Aragón and LópezCorcuera, 2005; Gomeza et al., 2006; Harsing et al., 2006, for reviews). The new information on synaptic targets and the availability of novel pharmacological tools prompted us to reconsider and to extend our old findings. Cerebellum was chosen also because, in this region, GABA and glycine are costored in several interneurons, including different types of Golgi cells and Lugaro cells (Simat et al., 2007) and may interact with each other at various levels. In the present work, purified mouse cerebellum synaptosomes were incubated with [3H]glycine in the presence of a selective GlyT1 inhibitor (Atkinson et al., 2001; Herdon et al., 2001), in order to obtain preferential uptake by GlyT2-bearing nerve terminals. Release of [3H]glycine was monitored under superfusion conditions know to minimize indirect effects, i.e. any effect of GABA on release should essentially be due to direct action of GABA at targets localized on glycine-storing nerve terminals (see Raiteri and Raiteri, 2000). The results show that GABA, acting on glycine-storing nerve terminals of adult mouse cerebellum, evoked release of [3H]glycine in a receptor-independent, transporter-mediated manner. Most important, the releasing effect could easily be observed at physiological concentrations of GABA as a GAT1 substrate. The release of [3H]glycine involved multiple mechanisms, including an unexpected permeation through anion channels.

2. Materials and methods 2.1. Animals Adult male Swiss mice (weighing 20–25 g; Charles River, Calco, Italy) were used. Animals were housed at constant temperature (22 ± 1 °C) and relative humidity (50%) under a regular light/dark schedule (light 7.00 a.m. to 7.00 p.m.). Food and water were freely available. The experimental procedures were approved by the Ethical Committee of the Pharmacology and Toxicology Section, Department of Experimental Medicine, in accordance with the European legislation (European Communities Council Directive of 24 November 1986, 86/609/EEC). All efforts were made to minimize animal suffering and to use the minimum number of animals necessary to produce reliable results. 2.2. Preparation of synaptosomes Animals were sacrificed and the cerebellum was quickly removed. The tissue was homogenized in 10 vol. of 0.32 M sucrose buffered at pH 7.4 with Tris–HCl, using a glass-Teflon tissue grinder (clearance 0.25 mm, 24 up-down strokes in about 2 min). The homogenate was centrifuged (5 min, 1000g at 4 °C) to remove nuclei and debris and the supernatant was gently stratified on a discontinuous PercollÒ gradient (2%, 6%, 10% and 20% (v/v) in Tris-buffered sucrose) and centrifuged at 33,500g for 5 min. The layer between 10% and 20% PercollÒ (synaptosomal fraction) was collected, washed by centrifugation and resuspended in a physiological medium (standard medium) having the following composition (mM): NaCl, 140; KCl, 3; MgSO4, 1.2; CaCl2, 1.2; NaH2PO4, 1.2; NaHCO3, 5; glucose, 10; HEPES, 10; pH adjusted to 7.4 with NaOH. All the above procedures were performed at 0–4 °C. 2.3. Glycine uptake measurements [3H]glycine uptake was measured according to the following procedure. The synaptosomal pellet was resuspended in standard medium. Aliquots (500 ll) of the synaptosomal suspension (about 25 lg protein) were incubated for 2 min at 37 °C with [3H]glycine (0.15 lM) in the absence (control samples) or in the presence of varying concentrations of NFPS or Org25543. At the end of the incubation period, samples were rapidly filtered on Whatman glass fibre filters (GF/B; VWR International, Milan, Italy). Each sample was washed three times with 5 ml aliquots of standard medium and filters were counted for radioactivity. Blank values were obtained by maintaining the samples in an ice water bath. 2.4. Experiments of release Synaptosomes were incubated at 37 °C for 15 min with [3H]glycine (0.15 lM) in the presence of the selective GlyT1 transporter blocker NFPS (0.3 lM). At the end of incubation, aliquots of the synaptosomal suspension (about 25 lg protein) were distributed on microporous filters placed at the bottom of a set of parallel superfusion chambers maintained at 37 °C (Superfusion System, Ugo Basile, Comerio, Varese, Italy) and superfused with standard medium at a rate of 0.5 ml/min (Raiteri and Raiteri, 2000). After 36 min of superfusion with standard medium, to equilibrate the system, four 3 min fractions were collected. Synaptosomes were exposed to GABA at the end of the first fraction collected (t = 39 min). In a set of experiments synaptosomes were exposed to muscimol or ()baclofen at t = 39 min of superfusion. x-Conotoxin GVIA and x-conotoxin MVIIC were added to the superfusion medium 4 min before GABA; bicuculline, CGP52432, SKF89976A, SKF100330A, nipecotic acid, KB-R7943, dantrolene, 2-APB, NFPS,

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SSR504734, Org25543, niflumic acid and NPPB were introduced 9 min before GABA and CGP37157 was added 19 min before GABA. In a set of experiments, synaptosomes had been incubated for 90 min at 37 °C in the absence (control synaptosomes) or in the presence of BoNT/A, BoNT/E or TeTx; [3H]glycine (0.15 lM) was present during the last 15 min of incubation. In another set of experiments, synaptosomes had been incubated for 30 min in the presence or in the absence of bafilomycin A1 that was added 15 min before and during labelling with [3H]glycine. Membrane impermeant BAPTA was entrapped into synaptosomes by homogenizing brain tissue in presence of the Ca2+ chelator (see Raiteri et al., 2000). We preferred not to use the membrane-permeable BAPTA-AM in order to avoid possible effects on K+ channels reported to be produced by BAPTA-AM (Watkins and Mathie, 1996). When appropriate, Ca2+ was omitted from the superfusion medium at t = 20 min of superfusion. The Ca2+-free medium contained 8.8 mM MgCl2, substituting for an isoosmotic amount of NaCl. Fractions collected and superfused filters were counted for radioactivity by liquid scintillation counting.

GlyT1 transporter blocker NFPS, to achieve preferential labelling of glycinergic terminals through GlyT2. In these conditions, [3H]glycine uptake (3.31 ± 0.27 pmol/mg protein; n = 7) was totally sensitive to Org25543, since the GlyT2 transporter blocker inhibited [3H]glycine uptake by about 55%, 90% and 99% when added at 0.1, 1 and 3 lM, respectively (0.1 lM Org25543: percent inhibition 53.93 ± 4.51, n = 7; 1 lM Org25543: percent inhibition 90.89 ± 6.57, n = 7; 3 lM Org25543: percent inhibition 98.89 ± 7.38, n = 7). After incubation, synaptosomes were exposed in superfusion to different concentrations of GABA. Fig. 1 shows that exogenous GABA concentration-dependently increased the spontaneous release of tritium; the calculated EC50 amounted to 8.26 ± 0.91 lM and the maximal potentiation of release, reached at 30 lM, was about 230% over basal. Authentic [3H]glycine accounted for >85% of the radioactivity released, identified by thin-layer chromatography as previously reported (Raiteri et al., 1992). Therefore, in the remainder of the article, we refer to the GABA-evoked tritium release as [3H]glycine release.

2.5. Calculations

3.2. Effects of bicuculline, CGP52432, muscimol, ()baclofen, SKF89976A, SKF100330A and nipecotic acid on the release of [3H]glycine evoked by GABA

Neurotransmitter released in each fraction collected was expressed as a percentage of the radioactivity content of synaptosomes at the start of the respective collection period (fractional rate  100). Drug effects were evaluated by calculating the ratio between the efflux in the third fraction collected (in which the maximum effect of GABA was generally reached) and that of the first fraction. This ratio was compared to the corresponding ratio obtained under control conditions. Appropriate controls were always run in parallel. 2.6. Statistics All data are given as means ± SEM. Statistical comparison of data was performed by one-way ANOVA followed by Dunnett’s test. Differences were regarded as statistically significant for p < 0.05. 2.7. Materials

To identify the targets of GABA on glycine-releasing nerve terminals, the effect of 10 lM GABA was tested in the presence of 10 lM bicuculline or 3 lM CGP52432, antagonists of GABAA and GABAB receptors, respectively. As shown in Fig. 2, the effect of GABA was not significantly affected by the two compounds. Moreover, the effect of GABA was not mimicked by GABAA and GABAB receptor agonists: as shown in Fig. 2, muscimol or ()baclofen, each added at 10 lM, did not increase [3H]glycine release. The two compounds were inactive up to 100 lM (not shown). Fig. 3 shows that the effect of 10 lM GABA was almost halved by 0.3 lM of the GAT1 transporter blocker SKF89976A and it was strongly (85%) or completely prevented when the compound was added at 1 or 3 lM, respectively. SKF100330A, another GAT1 transporter blocker, similarly inhibited the GABA-evoked [3H]glycine release (percent inhibition 50% and 95% with 0.3 and 3 lM, respectively; Fig. 3). The effect of 10 lM GABA was also

[3H]glycine (specific activity: 1,65  1015 Bq/mol) was purchased from Perkin Elmer (Boston, MA, USA). PercollÒ, GABA, muscimol, bicuculline, SKF89976A, nipecotic acid, niflumic acid, BAPTA, BoNT/A and TeTx were from Sigma Chemical Co. (St. Louis, MO, USA). ()Baclofen, CGP52432, x-conotoxin GVIA, x-conotoxin MVIIC, KB-R7943, dantrolene, 2-APB, NFPS (ALX 5407), CGP37157, bafilomycin A1 and NPPB were from Tocris Bioscience (Bristol, UK). BoNT/E was from Wako (Osaka, Japan). SKF100330A was a gift from Smith Kline & French Labs, Philadelphia. SSR504734 and Org25543 were kind gifts from Dr. Bernard Scatton (Sanofi-Synthelabo, Bagneaux, France) and from Dr. Thijs de Boer (Organon Laboratories Ltd., Newhouse, Scotland), respectively. Some of these drugs, in particular niflumic acid, NPPB and CGP37157, when added to the superfusion medium at relatively high concentrations (see Section 3), exhibited unspecific releasing activities on their own and could be utilized to establish their maximal inhibitory potential. 3. Results 3.1. Effects of GABA on the release of [3H]glycine from cerebellar nerve endings Purified mouse cerebellar synaptosomes were incubated with [3H]glycine (0.15 lM) in the presence of 0.3 lM of the selective

Fig. 1. Release of [3H]glycine evoked by GABA from purified mouse cerebellar synaptosomes. Synaptosomes were exposed in superfusion to varying concentrations of GABA. The effect of GABA was evaluated by performing the ratio between the efflux in the third fraction collected and that of the first fraction. This ratio was compared to the corresponding ratio obtained under control conditions. Results are expressed as percent potentiation with respect to the basal efflux. Data are means ± SEM of 4–7 experiments in triplicate (three superfusion chambers for each experimental condition).

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Fig. 2. Effects of the GABA receptor antagonists bicuculline and CGP52432 on the release of [3H]glycine evoked by 10 lM GABA from mouse cerebellar synaptosomes and effects of the GABA receptor agonists muscimol and ()baclofen. GABA, muscimol, or ()baclofen were added to the superfusion medium at the end of first fraction collected. Bicuculline and CGP52432 were introduced 9 min before GABA. Results are expressed as percent potentiation with respect to the basal efflux. Means ± SEM of three experiments performed in triplicate are reported.

Fig. 4. Effects of Ca2+ omission, x-conotoxin GVIA, x-conotoxin MVIIC, KB-R7943, dantrolene, 2-APB, NFPS, SSR504734 and Org25543 on the release of [3H]glycine induced by 10 lM GABA. When appropriate, Ca2+ was omitted from the superfusion medium 19 min before introduction of GABA. x-Conotoxin GVIA and x-conotoxin MVIIC were introduced 4 min before GABA. KB-R7943, dantrolene, 2-APB, NFPS, SSR504734 and Org25543 were introduced 9 min before GABA. Results are expressed as percent potentiation with respect to the basal release. Data are means ± SEM of 6–7 experiments performed in triplicate. ⁄p < 0.05; ⁄⁄p < 0.01 vs. the control value representing the effect of GABA in standard medium and in the absence of drugs (one-way ANOVA followed by Dunnett’s test).

Finally, Fig. 3 (inset) shows that the increase of [3H]glycine release induced by GABA was totally Na+-dependent: the effect of 10 lM GABA was abolished when Na+ concentration in the superfusion medium was decreased to 6.2 mM. 3.3. External Ca2+-dependent release: effects of x-conotoxin GVIA, x-conotoxin MVIIC, KB-R7943 and clostridial neurotoxins

Fig. 3. Effects of SKF89976A, SKF100330A and nipecotic acid on the release of [3H]glycine induced by 10 lM GABA. GABA was added to the superfusion medium at the end of first fraction collected. Drugs were introduced 9 min before GABA. Results are expressed as percent potentiation with respect to the basal release. Data are means ± SEM of 3–4 experiments performed in triplicate. ⁄p < 0.01 vs. the control value representing the effect of GABA in standard medium and in the absence of drugs (one-way ANOVA followed by Dunnett’s test). Inset: Na+dependence of the GABA-induced [3H]glycine release. N-methyl-D-glucamine (280 mM) substituting an isoosmotic concentration of NaCl was introduced 9 min before GABA. Data are means ± SEM of three experiments performed in triplicate. ⁄ p < 0.01 vs. the control value obtained using standard medium (two-tailed Student’s t-test).

strongly prevented by 10 lM nipecotic acid. The latter compound, which can also be a GABA transporter substrate, slightly increased the spontaneous efflux of [3H]glycine on its own (data not shown).

To investigate the mechanisms through which [3H]glycine exits from nerve terminals in response to GABA, we first assessed the external Ca2+-dependence of the GABA-evoked release. As shown in Fig. 4, the effect of 10 lM GABA was significantly (25%) prevented by omission of Ca2+ from the superfusion medium. The effect of 10 lM GABA, in the presence of external Ca2+, was inhibited by 20–25% by a mixture of x-conotoxin GVIA (100 nM) and x-conotoxin MVIIC (100 nM), blockers of N- and P/ Q-type voltage-sensitive Ca2+ channels (VSCCs), respectively. The compound KB-R7943, blocker of the plasmalemmal Na+/ Ca2+ exchanger (NCX) working in the ‘‘reverse’’ mode, exhibited only a slight, barely significant inhibitory effect when added at 3 lM (Fig. 4). When the drug was added concomitantly with the x-conotoxins, the inhibitory effect of the mixture was not significantly enhanced with respect to the effect of the toxins (percent inhibition around 25%; data not shown). To investigate the possible role of exocytosis, the effects of clostridial neurotoxins on the GABA-evoked [3H]glycine release were evaluated. As shown in Table 1, Tetanus Toxin (TeTx; 50 nM), Botulinum Toxin A (BoNT/A; 150 nM) and Botulinum Toxin E (BoNT/E; 200 nM) did not significantly affect neurotransmitter release evoked by 10 lM GABA.

C. Romei et al. / Neurochemistry International 61 (2012) 133–140 Table 1 Effects of the clostridial neurotoxins TeTx, BoNT/A and BoNT/E on the release of [3H]glycine evoked by GABA from mouse cerebellar synaptosomes. Drug GABA GABA GABA GABA

Percent potentiation (10 lM) (10 lM) + TeTx (50 nM) (10 lM) + BoNT/A (150 nM) (10 lM) + BoNT/E (200 nM)

141.1 ± 9.5(6) 127.3 ± 10.2(7) 120.3 ± 13.6(6) 137.5 ± 11.0(7)

Synaptosomes were incubated for 90 min at 37 °C in the presence or in the absence of the toxins; [3H]glycine (0.15 lM) was present during the last 15 min of incubation; GABA was added at t = 39 min of superfusion (see Methods for details). Data are means ± SEM of the number of experiments given in parentheses.

3.4. Absence of Ca2+-induced Ca2+ release processes To assess the possible role of Ca2+-induced Ca2+ release (CICR) processes, the involvement of ryanodine receptors and/or inositol (1,4,5) trisphosphate (InsP3) receptors was investigated. As illustrated in Fig. 4, the effect of 10 lM GABA was not significantly affected by the ryanodine receptor antagonist dantrolene or by the InsP3 receptor blocker 2-APB (both drugs added at 10 lM). The latter compound, added at 30–50 lM, exhibited only a slight, not quite significant tendency to inhibit [3H]glycine release. Higher concentrations of 2-APB (up to 100 lM) evoked tritium release on their own and could not provide reliable results (data not shown). 3.5. The glycine transporter inhibitors NFPS, SSR504734 and Org25543 do not affect the GABA uptake-mediated [3H]glycine release [3H]glycine release evoked by 10 lM GABA was unaffected by blocking GlyT1 transporters with 0.3 lM NFPS or with 0.3 lM SSR504734 (Fig. 4). The Figure also shows that neurotransmitter release was insensitive to the selective GlyT2 transporter blocker Org25543 (3 lM).

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GABA was strongly inhibited by niflumic acid (percent inhibition 50–55% and 70–75% when the drug was added at 30 and 100 lM, respectively) or by NPPB (percent inhibition 25–30% with 10 lM and 55–60% with 30 lM of the compound). Unfortunately, concentrations of niflumic acid and NPPB higher than 100 and 30 lM, respectively, enhanced basal release on their own so that their maximal inhibitory activities could not be evaluated. It has to be recalled that 10–15% of tritium release represents efflux of tritiated glycine metabolites. To further clarify the roles of external and internal Ca2+ ions we studied the effect of GABA on [3H]glycine release in nominal absence of external Ca2+. As shown in Fig. 6, the effect of 10 lM GABA was inhibited by niflumic acid in a concentration-dependent manner (percent inhibition about 35%, 65% and 90% when the drug was added at 10, 30 and 100 lM, respectively) and by NPPB (percent inhibition 40–45% with 10 lM and up to 65% with 30 lM of the compound). Thus, the sensitivity of the GABA uptake-mediated release did not seem to be diminished when external Ca2+ was removed. 3.7. Involvement of internal Ca2+ and of mitochondrial Na+/Ca2+ exchangers: effects of BAPTA and CGP37157 Finally, to assess the involvement of internal Ca2+ we tested the effect of the calcium chelator BAPTA. As reported in Fig. 6, [3H]glycine release evoked by 10 lM GABA in the absence of external Ca2+ was significantly (30–35%) reduced by entrapping the Ca2+ chelator BAPTA into synaptosomes (see Methods for details). Considering that CICR processes seem not to be involved, we investigated the possible role of mitochondria as a source of internal Ca2+. As shown in Fig. 6, the mitochondrial Na+/Ca2+ exchanger blocker CGP37157 (10 lM) inhibited the effect of 10 lM GABA, in the absence of external Ca2+, by 40%. Of note, 10 lM is the

3.6. Niflumic acid and NPPB inhibit the GABA uptake-evoked [3H]glycine release As reported above, the release of [3H]glycine evoked by GABA was only slightly external Ca2+-dependent; furthermore, release did not involve glycine transporter reversal. We then attempted to clarify the mechanisms underlying the residual portion of release induced by GABA. As shown in Fig. 5, the effect of 10 lM

Fig. 5. Effects of niflumic acid and NPPB on the release of [3H]glycine induced by 10 lM GABA. Niflumic acid and NPPB were introduced 9 min before GABA. Results are expressed as percent potentiation with respect to the basal release. Data are means ± SEM of 5–6 experiments performed in triplicate. ⁄p < 0.05; ⁄⁄p < 0.01 vs. the effect of GABA in the absence of drugs (one-way ANOVA followed by Dunnett’s test).

Fig. 6. Effects of niflumic acid, NPPB, BAPTA, CGP37157 and bafilomycin A1 on the release of [3H]glycine induced by 10 lM GABA in nominal absence of external Ca2+. Ca2+ was omitted from the superfusion medium 19 min before addition of GABA. Niflumic acid and NPPB were added 9 min before GABA. BAPTA had been entrapped into synaptosomes during tissue homogenization (see Methods for details). CGP37157 was introduced 19 min before GABA. In the experiments with bafilomycin A1, synaptosomes had been incubated for 30 min in the presence or in the absence of the drug which was present from 15 min before and during labelling with [3H]glycine. Results are expressed as percent potentiation with respect to the basal release. Data are means ± SEM of 6–7 experiments performed in triplicate. ⁄ p < 0.05; ⁄⁄p < 0.01 vs. the control value representing the effect of GABA in the absence of drugs (one-way ANOVA followed by Dunnett’s test).

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maximal concentration of CGP37157 permitted in our system. Higher concentrations provoke tritium release on their own and therefore the maximal inhibitory activity of CGP37157 cannot be established. Finally, pre-treatment of synaptosomes with 0.1 lM of the vesicle membrane V-type H+-ATPase inhibitor bafilomycin A1 significantly reduced the effect of GABA (percent inhibition: 30–35%; Fig. 6).

4. Discussion GABA stimulated the basal release of glycine in nerve terminals isolated and purified from adult mouse cerebellum. The evoked release occurred from glycinergic nerve endings because synaptosomes were prelabeled with [3H]glycine in the presence of the GlyT1 blocker NFPS to permit selective uptake into nerve endings endowed with transporters of the GlyT2 type, which are reliable markers for glycine-storing terminals (Poyatos et al., 1997; Gomeza et al., 2003). The effect of GABA can be attributed to direct action of GABA on glycine-storing nerve endings because of the characteristics of the superfusion technique used to monitor transmitter release (see, for details, Raiteri and Raiteri, 2000; Popoli et al., 2012). The targets for GABA were first investigated. It is known that, although GABA receptors of the GABAA type generally mediate inhibition, depolarizing GABAA receptors have been described not only in the developing brain (Cherubini et al., 1991), but also in some adult systems (Kohling, 2002). The stimulatory effect of GABA on the release of glycine was however insensitive to the GABAA receptor antagonist bicuculline and was not mimicked by the GABAA receptor agonist muscimol, excluding the involvement of presynaptic GABAA receptors. GABA did not act at GABAB receptors either, because its effect was insensitive to the GABAB receptor antagonist CGP52432, while the GABAB receptor agonist ()baclofen was ineffective. The augmentation of the basal release of glycine evoked by GABA could be abolished by GAT1 transporter blockers, including SKF89976A. The characteristics of the inhibitors tested were very similar to those calculated from uptake experiments (Sitte et al., 2002), indicating that the releasing effect of GABA was entirely dependent on GABA uptake through GAT1. Differently, the GABA uptake-dependent Ca2+ signaling observed by Doengi et al. (2009) in olfactory bulb astrocytes was prevented by SNAP5114, but not by SKF89976A, consistent with the involvement of GABA transporters of the murine GAT4 type. The concentrations of GABA able to evoke functional responses in glycinergic neurons of the cerebellum deserve attention. The apparent EC50 value for the GABA-induced glycine release amounted to about 8 lM, in keeping with the high-affinity values calculated from GABA uptake studies in many laboratories. The finding suggests that activation of GAT1 to elicit release of glycine may have important pathophysiological implications in the adult mammalian CNS. A significant portion (about 25%) of the GABA(10 lM)-evoked glycine release was dependent on external Ca2+ ions. Entry of extracellular Ca2+ into the cytosol can occur by multiple modes including (i) influx through VSCCs activated by depolarizing stimuli and (ii) reversal of plasmalemmal Na+/Ca2+ exchangers (NCXs; Annunziato et al., 2004; Török, 2007) that can occur when the membrane is depolarized and cytosolic Na+ concentrations increase to facilitate entry of Ca2+ by NCX reversal. Because the stoichiometry of GAT1 is 2Na+/1Cl/GABA (Keynan and Kanner, 1988), GABA uptake is accompanied by an influx of Na+ ions which could produce localized membrane depolarization triggering activation of VSCCs and changing plasmalemmal NCX function. Exposure of synaptosomes to a mixture of x-conotoxins able to block N- and P/Q-type VSCCs produced a significant inhibition (about 25%) of the GABA effect on glycine release. Based on an

abundant literature, one would have expected dependence on external Ca2+ entering nerve endings through VSCCs to reflect release by vesicular exocytosis. This does not seem to be the case, however, because treatment of synaptosomes with Tetanus toxin, Botulinum toxin A or Botulinum toxin E did not change the effect of GABA on glycine release. A modest, though not significant, portion of the GABA uptake-mediated glycine release appeared sensitive to KB-R7943, an inhibitor of NCXs working in the reverse mode (Iwamoto et al., 1996). When x-conotoxins and KB-R7943 were added together, the effect of the mixture on the depolarizationevoked release of glycine did not differ significantly from that of the toxins alone. Thus, Ca2+ entry through reverse NCXs is likely to give little contribution to the releasing effect of GABA. The InsP3 receptor inhibitor 2-APB (Maruyama et al., 1997) did not affect the GABA-uptake dependent glycine release, thus excluding the involvement of InsP3 receptors. Of note, 2-APB is not selective for InsP3 receptors as it can also inhibit store-operated (SOC; Kukkonen et al., 2001; Bootman et al., 2002) and transient receptor potential (TRPC; Xu et al., 2005) Ca2+ channels. The lack of effect of 2-APB on the GABA uptake-mediated glycine release tends therefore to exclude involvement not only of InsP3 receptors, but also of SOCs and TRPCs. Ryanodine receptors did not play a role either, because the GABA effect was insensitive to the ryanodine receptor blocker dantrolene (Frandsen and Schousboe, 1992). The concentrations of GABA used by Doengi et al. (2009) provoked cytosolic Ca2+ increases that could be monitored by confocal Ca2+ imaging. In our system, elevations in cytosolic Ca2+ occurring in nerve endings when exposed to 10 lM GABA could not be measured by fluorescence techniques; actually Ca2+ signals could barely be detected when adding 100 lM GABA (data not shown). However, the involvement of intraterminal Ca2+ is indicated by the inhibition of the GABA uptake-evoked release of glycine observed in synaptosomes previously entrapped with the Ca2+ chelator BAPTA and then exposed to 10 lM GABA, in the absence of external Ca2+ ions. Efflux of glycine evoked by GABA uptake might have occurred by transporter reversal (Adam-Vizi, 1992; Attwell et al., 1993; Levi and Raiteri, 1993; Vizi and Sperlágh, 1999). Because labelling of synaptosomal preparations was performed in the presence of the GlyT1 inhibitor NFPS, which causes long lasting block of GlyT1 function (Aubrey and Vandenberg, 2001), the lack of effect of added NFPS could be expected. However, these authors also reported that NFPS could inhibit glycine uptake but seemed unable to affect from inside release by GlyT1 reversal. We therefore tested a structurally different, competitive and reversible GlyT1 inhibitor, SSR504734 (Depoortère et al., 2005) which also was unable to prevent the GABA uptake-evoked glycine release, indicating that the evoked glycine overflow was not by GlyT1 reversal. Glycine release by reversal of GlyT1 may occur in other systems, as recently reported by Harsing et al. (2012). As to GlyT2, its stoichiometry (3Na+/Cl/glycine), together with a reported kinetic constraint for reverse transport (Roux and Supplisson, 2000), made a carriermediated release unlikely. Accordingly, the selective GlyT2 inhibitor Org25543 (Caulfield et al., 2001) was unable to affect the GABAevoked glycine release. Thus, the mechanisms considered so far could not explain the mode of exit of glycine produced by activation of GAT1. A possible solution came from a recent paper (Lee et al., 2010). This work aimed to understand the mechanisms of GABA release underlying a tonic form of synaptic inhibition that occurs throughout the CNS. The authors found that tonic inhibition in the cerebellum is due to GABA being released from glial cells by permeation through Bestrophin 1 (Best1) anion channels sensitive to niflumic acid and NPPB. Permeation through anion channels was described in the case of anionic compounds like glutamate in astrocytes (see, for instance, Malarkey and Parpura, 2008; Milanese et al., 2010).

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According to Lee et al. (2010), ‘even though GABA is predominantly zwitterionic, the amount of GABA in the anionic form is sufficient to carry considerable current’. It seemed then appropriate to investigate if glycine also could permeate through similar channels in nerve terminals and the inhibition of the GABA-evoked glycine release by niflumic acid and NPPB here observed supports this view. Activation of some anion channels is dependent on intracellular Ca2+ (see, for a review, Duran et al., 2010). As reported above, only about 25% of the evoked release was dependent on external Ca2+. Moreover, the Ca2+ ions entering the nerve terminals through VSCCs did not trigger exocytosis and did not participate in CICR processes at ryanodine and InsP3 receptors. These results tend to exclude that external Ca2+ ions are mandatory for the activation of anion channels, although they may well contribute to the process. On the other hand, the glycine release occurring in the absence of external Ca2+ (about 75% of the total) was abolished by 100 lM of niflumic acid, indicating that permeation through anion channels is by far the major mode of exit of glycine from glycinestoring nerve endings exposed to 10 lM GABA. How activation of anion channels can occur in the absence of external Ca2+? Assuming that the process is Ca2+-dependent, mobilization of Ca2+ from internal stores other than those localized on the endoplasmic reticulum (Berridge, 1998; Rizzuto and Pozzan, 2006) was investigated. Influx of Na+ ions with GABA uptake could have stimulated mitochondrial Na+/Ca2+exchangers, thus promoting entry of mitochondrial Ca2+ into the cytosol (Rizzuto and Pozzan, 2006). Accordingly, the release of glycine evoked by GABA, in the absence of external Ca2+, was significantly inhibited by the mitochondrial Na+/Ca2+ exchanger blocker CGP37157 (Hernández-SanMiguel et al., 2006), suggesting that the influx of Na+ accompanying the uptake of 10 lM GABA was sufficient to trigger Ca2+ signaling from intraterminal mitochondria. The glycine release evoked by GABA uptake, in the absence of external Ca2+, also was inhibited in synaptosomes that had been labelled with [3H]glycine in the presence of bafilomycin, a drug able to prevent vesicular storage of neurotransmitters. Inhibition of neurotransmitter release caused by bafilomycin is generally considered to reflect inhibition of vesicular exocytosis. However, the lack of effect of clostridial toxins on the GABA-evoked glycine release militates against this view. A more likely explanation originates from some peculiar characteristics of GABAergic/glycinergic neurons. It is known that, in neurons in which GABA and glycine are colocalized, the two cotransmitters can be stored together in the same vesicles because they share the vesicular inhibitory amino acid transporter termed VIAAT (Todd et al., 1996; Chaudry et al., 1998; Jonas et al., 1998). To be able to exit from nerve endings through the anion channels, glycine stored in vesicles needs first to become cytosolic. This is likely to occur when GABA taken up by GAT1 facilitates, by VIAAT-mediated heteroexchange, the entry of vesicular glycine into the cytosol. Pretreatment of synaptosomes with bafilomycin would decrease vesicular storage of glycine and would subsequently decrease the amount of glycine that reaches the cytosol and becomes available to permeation via anion channels. The GABA uptake-dependent glycine release occurring in the absence of external Ca2+ was largely prevented by niflumic acid or by NPPB. In particular, when niflumic acid was added at 100 lM (the maximal concentration permitted in our system, see Section 3), the releasing effect of GABA was completely abrogated, suggesting that, in the absence of external Ca2+, permeation through anion channels represents the only mode of exit of glycine provoked by GABA uptake. When external Ca2+ was present, the GABA-evoked glycine release also was largely sensitive to anion channel blockade, although the use of concentrations of niflumic acid and NPPB possibly able to abolish completely the GABA effect was not permitted.

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Based on the present results, it can be proposed that activation of GAT1 on glycine-storing cerebellar nerve endings by ‘physiological’ concentrations of GABA elicits release of glycine essentially by permeation through Ca2+-dependent anion channels. This final event originates from different interrelated processes: (1) electrogenic GABA uptake triggers opening of VSCCs; (2) entry of Na+ with GABA leads to further increase in cytosolic Ca2+ via activation of mitochondrial Na+/Ca2+ exchangers; (3) Ca2+ ions originated through VSCCs and mitochondria permit opening of anion channels and permeation of cytosolic glycine (4) GABA penetrated into nerve terminals exchanges with glycine at VIAATs thus increasing cytosolic glycine and glycine release. Tonic inhibition is a sustained form of synaptic inhibition that occurs throughout the CNS (Farrant and Nusser, 2005) and has been implicated in epilepsy, sleep, memory and cognition (Caraiscos et al., 2004; Jia et al., 2005; Cope et al., 2009). Tonic inhibition involves tonic release of GABA which is independent of action potentials and does not require vesicular exocytosis. In a recent publication (Lee et al., 2010) tonic inhibition in cerebellum was found to be due to GABA released from glial cells by permeation through Best1 anion channels. One could speculate that non-vesicular glycine release stimulated by ‘physiological concentrations’ of GABA from glycine-storing nerve endings endowed with anion channels might also contribute to tonic inhibition. Clearly, the ‘physiological’ GABA stimuli here investigated can become ‘pathological’ and participate in the transmitter dysregulations characteristic of neurodegenerative disorders, including Alzheimer’s disease. Disruptions in the GABAergic and glycinergic systems have been shown to occur in animal models of Alzheimer’s disease and stabilization of the levels of GABA and glycine following administration of proline-rich polypeptide-1 was recently reported (Yenkoyan et al., 2011). To conclude, the results available suggest that anion channels in glycine-storing nerve endings of the mouse cerebellum can allow glycine permeation following ‘physiological’ GABAergic stimuli and indicate that functions of GABA transporters other than uptake and carrier-mediated release deserve further attention.

Acknowledgements This work was supported by Grants from Italian MIUR. The authors wish to thank Mrs. Maura Agate for her expert editorial assistance.

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