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NEUROPHARMACOLOGY

GABA Modulates Presynaptic Signalling Mediated by Dinucleotides on Rat Synaptic Terminals

Departamento de Bioquímica, Facultad de Veterinaria, Universidad Complutense de Madrid, Madrid, Spain

Received October 14, 2003; accepted November 25, 2003.

	Abstract

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Diadenosine pentaphosphate (Ap₅A) elicits Ca²⁺ transients in isolated rat midbrain synaptic terminals acting through specific ionotropic dinucleotide receptors. The activation of GABA_B receptors by baclofen changes the sigmoidal concentration-response curve for Ap₅A (EC₅₀ = 44 µM) into biphasic curves. Thus, when GABA_B receptors are activated, the curve shows a high-affinity component in the picomolar range (EC₅₀ = 77 pM) and a low-affinity component in the micromolar range (EC₅₀ = 17 µM). In addition, in the presence of GABA or baclofen, Ap₅A calcium responses are increased up to 50% over the control values. Saclofen, a specific antagonist of GABA_B receptors, blocks the potentiatory effect of baclofen. As occurs with Ap₅A, GABA_B receptors are also capable to modulate diguanosine pentaphosphate (Gp₅G)-induced calcium responses. The combination of immunocytochemical and microfluorimetric techniques carried out on single synaptic terminals have shown that in the presence of baclofen, 64% of the terminals responding to 100 µM Ap₅A are also able to respond to 100 nM Ap₅A. This value is close to the percentage of synaptic terminals responding to Ap₅A and labeled with the anti-GABA_B receptor antibody (69%). The activity of cyclic AMP-dependent protein kinase (PKA) seems to be involved in the potentiatory effect of GABA_B receptors on Ap₅A calcium responses, because PKA activation by forskolin or dibutiryl cyclic AMP blocks the potentiatory effect of baclofen, whereas PKA inhibition facilitates calcium signaling mediated by Ap₅A. These results demonstrate that the activation of presynaptic GABA_B receptors is able to modulate dinucleotide responses in synaptic terminals.

Considering that dinucleotide receptors are receptor-operated calcium channels, they might be involved in facilitatory presynaptic mechanisms in central neurons. In this way, the dinucleotide receptor activation induces the release of excitatory neurotransmitters such as acetylcholine or glutamate from cholinergic and glutamatergic synaptic terminals, respectively (Díaz-Hernández et al., 2002b; Gualix et al., 2003). Ap_nA are also able to evoke GABA release from GABAergic terminals (Gómez-Villafuertes et al., 2001). This fact together with the results reported previously by other groups suggests a close relationship between GABAergic and purinergic neurotransmitter systems in the central nervous system (Inoue et al., 1999; Jo and Schlichter, 1999; Hugel and Schlichter, 2000). In the brain of vertebrates, GABA is the main inhibitory neurotransmitter, being able to activate two different mechanisms: one ionotropic, GABA_A receptors and other metabotropic, G-coupled GABA_B receptors. Presynaptic GABA_B receptors are widely distributed on brain nerve terminals where they act mediating the inhibition of neurotransmitter release by direct inhibition of voltage-dependent calcium channels. This effect is inhibited by pertussis toxin, indicating the involvement of a G_i/G₀ protein-coupled receptor. Additionally, GABA_B receptor activation has been associated with the opening of G protein-coupled inwardly rectifying potassium channels and the modulation of adenylyl cyclase (Wu and Saggau, 1997; Filippov et al., 2000; Billinton et al., 2001). Keeping in view that diadenosine polyphosphates are able to induce GABA release form nerve terminals, and, consequently, both transmitters can coexist extracellularly in synaptosomal preparations, the aim of this work was to analyze the effect of presynaptic GABA receptor activation on the Ca²⁺ responses elicited by Ap₅A in rat synaptic terminals.

	Materials and Methods

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Chemicals. Ap₅A, baclofen, bovine serum albumin (BSA), GABA, Gp₅G, muscimol, paraformaldehyde (PFA), saclofen, mouse anti-synaptophysin, rabbit anti-GABA_A receptor 3 subunit, guinea pig anti-GABA_BR1 receptor, rabbit anti-glial fibrillar acidic protein (GFAP), rabbit anti-post-synaptic density marker protein (PSD-95), fluorescein isothiocyanate-coupled goat anti-mouse IgG, tetramethylrhodamine isothiocyanate (TRITC)-conjugated goat anti-rabbit IgG and TRITC-conjugated rabbit anti-guinea pig IgG were all purchased from Sigma-Aldrich (St. Louis, MO). Fura-2 acetoxymethyl ester (Fura-2 AM) and Fura-2 were obtained from Molecular Probes (Leiden, The Netherlands). Forskolin and PKA inhibitory peptide were supplied by Calbiochem (San Diego, CA). Dibutiryl cyclic AMP (DiBucAMP) was obtained from Sigma/RBI (Natick, MA). SKF 89976A was from Tocris (Madrid, Spain). Other analytical grade reagents were purchased from Merck (Darmstadt, Germany).

Synaptosomal Preparation. Synaptosomes were obtained from the rat midbrain of cervically dislocated and decapitated adult male Wistar rats as indicated previously (Gómez-Villafuertes et al., 2001). All the experiments carried out at the Universidad Complutense de Madrid were performed according to the guidelines of the International Council for Laboratory Animal Science. The isolation procedure differed, depending on the proposed use of the synaptosomal preparation. Synaptosomes used for the determination of [Ca²⁺]_i levels in synaptosomal populations were isolated following the method of Pintor et al. (1992). Briefly, freshly isolated midbrain was homogenized in 0.32 M sucrose containing 5 mM TES and 0.5 mM EDTA, pH 7.4, and centrifuged at 900g for 5 min. The supernatant was centrifuged at 17,000g for 10 min, and the resulting P2 fraction was resuspended in 0.25 M sucrose containing 5 mM TES pH 7.4. Protein determination was carried out by the Biuret's method. Synaptosomes used to estimate [Ca²⁺]_i in single synaptic terminals, and those needed for immunocytochemical studies were purified on discontinuous Percoll gradients according to the procedure described by Dunkley et al. (1986). In brief, P2 fraction was layered on top of a Percoll density gradient composed of 3, 10, and 23% Percoll prepared in a 1.6 M sucrose medium containing 0.5 M EDTA and 0.1 M dithiothreitol. Centrifugation was carried out at 25,000g for 10 min. Synaptosomes were collected from the 10%/23% Percoll interface, resuspended in HBM (composition 140 mM NaCl, 5 mM KCl, 5 mM NaHCO₃, 1.2 mM NaH₂PO₄, 1 mM MgCl₂, 10 mM glucose, 10 mM HEPES, pH 7.4) and centrifuged at 20,000g for 3 min. After the last centrifugation step, the supernatant was discarded, and the pellet containing the synaptosomes was stored on ice. Under these conditions, the synaptosomes remain fully viable for at least 4 to 6 h as judged by the extent of the KCl-evoked [Ca²⁺]_i increase.

Determining Intrasynaptosomal Ca²⁺ in Synaptosomal Populations. Synaptosomal pellets containing 1 mg of protein were resuspended in 1 ml of incubation medium (composition 122 mM NaCl, 3.1 mM KCl, 0.4 mM KH₂PO₄, 5 mM NaHCO₃, 1.2 mM MgSO₄, 10 mM glucose, and 20 mM TES buffer, pH 7.4). The cytosolic free calcium concentration was determined using Fura-2 as described by Grynkiewicz et al. (1985). Briefly, 5 min after resuspension, 1.33 mM CaCl₂ and 5 µM Fura-2 AM were added to the synaptosomes. After incubation for 25 min, the synaptosomes were pelleted (centrifuged at 15,700g for 1 min), washed twice, and resuspended in fresh medium containing 1.33 mM CaCl₂. Fluorescence was measured in a PerkinElmer spectrofluorimeter LS-50 and monitored at _ex 340 nm and _em 510 nm. Data were collected at 0.5-s intervals.

Synaptosomes were preincubated for different times (from 30 s to 6 min) with 100 µM GABA, muscimol (GABA_A receptor agonist) or baclofen (GABA_B receptor agonist) before assaying Ap₅A. Once the optimal preincubation time was established, concentration-response curves for Ap₅A and Gp₅G ranging from 10^-4 to 10^-14 M were obtained in the presence of 100 µM baclofen. As controls, independent concentration-response curves (10^-4 to 10^-6 M) were prepared for Ap₅A and Gp₅G.

In some experiments, synaptosomes were preincubated for 2 min with 100 µM saclofen (a GABA_B receptor antagonist), 10 µM forskolin (an adenylate cyclase activator), or 100 µM DiBucAMP before Ap₅A application. Because PKA inhibitory peptide (PKA-IP; 25 nM) does not cross the plasma membrane, it was added to the homogenization buffer during synaptosomal preparation to allow its access to the intrasynaptosomal media (Pintor et al., 1997b). Finally, preincubation with 10 µM SKF 89976A (a specific GABA-uptake inhibitor) was performed for 2 min before Ap₅A addition to increase the extrasynaptosomal GABA concentration.

Intrasynaptosomal calcium levels are expressed as the mean ± S.E.M. of at least three determinations performed in triplicate in different synaptosomal preparations. Concentration-response curves were fitted using Fig P version 6.0 software (Biosoft, Cambridge, UK). EC₅₀ and Hill values are expressed as EC₅₀ ± S.D. and n_H ± S.D. respectively. Comparisons were performed by one-way analysis of variance. Bonferroni post test analysis was only applied when a significant (p < 0.05) main effect was indicated by the analysis of variance. Where appropriate, single experimental traces are shown in the figures; these represent at least three determinations performed in triplicate with equivalent results.

Immunocytochemical Procedures. Synaptosomal pellets containing 0.5 mg of protein were resuspended in 1 ml of phosphate-buffered saline (composition 137 mM NaCl, 2.6 mM KCl, 1.5 mM KH₂PO₄, 8.1 mM Na₂HPO₄, pH 7.4) and were allowed to attach to poly-L-lysine-coated coverslips for an hour. Then, the coverslips were fixed for 15 min in PBS containing 4% PFA (w/v). After several washes in PBS, synaptosomes were incubated for 1 h in PBS containing 5% normal goat serum (v/v), 3% BSA (w/v), and 0.1% Triton X-100 (v/v). They were then incubated for 1 h at 37°C with the appropriate primary antibody diluted in PBS containing 3% BSA (PBS/BSA) as follows: GABA_A receptor (1:500), GABA_B receptor (1: 5000), synaptophysin (1:200), glial fibrillar acid protein (GFAP, 1:100), and postsynaptic density marker (PSD-95, 1:500). The commercial anti-GABA_A receptor antibody was developed using a synthetic peptide (QGESRRQEPGDFVKQ) corresponding to amino acid sequence 1 to 15 of the extracellular N terminus domain of the rat GABA_A receptor 3 subunit. The commercial anti-GABA_B receptor antibody was raised against an amino acid sequence (PSEPPDRLSCDGSRVHLLYK) that is common to both GABA_BR1a and GABA_BR1b receptors.

After washing in PBS/BSA, synaptosomes were incubated for 1 h at 37°C with the appropriate secondary antibodies (see "Chemicals") diluted 1:200 in PBS/BSA. Finally, synaptosomes were washed in PBS and mounted with Prolong antifade kit from Molecular Probes. As controls for the immunochemical reactions, primary antibodies were omitted from the staining procedure.

Synaptosomes were viewed with a Nikon TE-200 microscope equipped with a 100x objective (oil, 1.3 numerical aperture), a mercury lamp light source and fluorescein-rhodamine Nikon filter sets. To quantify the number of terminals showing colocalization of two markers, fluorescein isothiocyanate- and TRITC-fluorescent images were subsequently obtained using an Ultrapix 2000 Mono charge-coupled device camera controlled by Ultraview PC software (PerkinElmer Life Sciences, Cambridge, UK). Images were analyzed using Lucida 3.0 software (Kinetic Imaging, Liverpool, UK). Data are presented as the mean ± S.E.M. corresponding to four to six visual fields from at least two different synaptosomal preparations.

Calcium Imaging in Single Synaptosomes. Synaptosomal pellets containing 0.5 mg of protein were resuspended in 1 ml of HBM and loaded with 5 µM Fura-2 AM and 1.33 mM CaCl₂ for 45 min at 37°C. The synaptosomal suspension was attached to a poly-L-lysine-coated coverslip for 45 min at room temperature. This time period is sufficient for both sticking on the substrate and hydrolysis of the intrasynaptosomal Fura-2 AM. Next, the coverslips were washed with fresh HBM containing 1.33 mM CaCl₂, and mounted in a small superfusion chamber on the stage of a Nikon TE-200 microscope. For [Ca²⁺]_i measurements, synaptosomes were stimulated by 30-s pulses (indicated in each graph by a bar) of 100 nM Ap₅A in the absence and presence of 100 µM baclofen. Successive pulses of 100 µM Ap₅A and 30 mM KCl were applied at the end of each experiment to confirm the functionality and viability of the synaptosomes under study.

Synaptosomes were excited alternately at 340 and 380 nm through a 100x objective (oil, 1.3 numerical aperture) with the aid of a monochromator (12-nm bandwidth, PerkinElmer Life Sciences). These wavelengths correspond to the fluorescence peaks of Ca²⁺-saturated and Ca²⁺-free Fura-2 solutions. The fluorescence emitted from the synaptosomes was isolated by a dichroic mirror (430 nm) and was collected through a band-pass filter centered at 510 nm (Omega Optical Inc., Brattleboro, VT). The video images were obtained using an Ultrapix 2000 Mono charge-coupled device camera operating at 12-bit digitization (4095 levels) and controlled by Ultraview PC software (PerkinElmer Life Sciences). The exposure time was 50 ms and wavelength changed over time in less than 15 ms. Images were continuously acquired at 1.06 Hz and buffered on a fast SCSI disk. Time course data represent the average light intensity in a small elliptical region within each identified synaptic terminal. Background and autofluorescence components were subtracted at each wavelength and the ratio 340/380 was calculated by 32-bit float arithmetic (real numbers). Ratios were calibrated into [Ca²⁺]_i values using the Grynkiewicz equation (Grynkievicz et al., 1985). The variables R_max, R_min and were calculated "in vitro" from the spectra of small Fura-2 droplets in Ca²⁺-saturated solution (composition 100 mM KCl, 10 mM NaCl, 1 mM MgCl₂, 10 mM Tris, 10 mM MOPS, 2 mM CaCl₂, and 100 µM Fura-2) and Ca²⁺-free solution (composition 100 mM KCl, 10 mM NaCl, 1 mM MgCl₂, 10 mM Tris, 10 mM MOPS, 2 mM CaCl₂, and 100 µM Fura-2), both determined empirically in our system.

	Results

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Effect of GABA Receptor Agonists on Ap₅A Calcium Responses in Rat Midbrain Nerve Terminals. Synaptosomal pellets from rat midbrain were preincubated with 100 µM GABA, baclofen (a selective GABA_B receptor agonist), or muscimol (a specific GABA_A receptor agonist) for 2 min before the addition of 100 µM Ap₅A. As previously reported by Gómez-Villafuertes et al. (2003), synaptosomes stimulation with GABA, baclofen, or muscimol in the absence of Ap₅A evoked no intrasynaptosomal calcium transient (data not shown). However, GABA and baclofen produced around a 55% increase in the calcium response induced by Ap₅A (Fig. 1A, second and third traces, respectively), compared with the control (Fig. 1A, top trace). In contrast, muscimol failed to modify the intrasynaptosomal calcium increase induced by Ap₅A (Fig. 1A, bottom trace). To establish the optimal preincubation period leading to greatest potentiation of the response to Ap₅A, synaptosomes were preincubated with 100 µM baclofen or muscimol for different time periods. Figure 1B shows the preincubation periods corresponding to 30 s, 2 min, 4 min, and 6 min. Potentiation of the Ap₅A calcium responses evoked by baclofen reached their maximum after 2 min of incubation, whereas muscimol failed to significantly modify the Ap₅A effect at each of the times assayed. Thus, a preincubation time of 2 min was fixed as the most appropriate to analyze baclofen effect on calcium responses induced by dinucleotides.

View larger version (15K): [in this window] [in a new window]   Fig. 1. Effects of GABA receptor agonists on calcium responses induced by 100 µMAp5A. A, single experimental traces of the effect of Ap5A in the absence (upper trace) and presence of GABA (second trace), baclofen (third trace), or muscimol (lower trace), all added at 100 µM. The traces presented in the figure represent three determinations performed in triplicate with equivalent results. B, determination of the effects of baclofen and muscimol on Ap5A-induced calcium at different incubation periods. Each bar represents the mean ± S.E.M. of three determinations performed in triplicate. *, p < 0.05 versus control; **, p < 0.01 versus control.

To analyze in depth the modulatory effect of presynaptic GABA receptors on Ap₅A calcium responses, the distribution of these receptors on synaptic terminals was studied using immunocytochemical techniques (Fig. 2). Antibodies raised against 3 subunit have been extensively employed as markers for GABA_A receptors (Benke et al., 1991). In this way, the absence of immunolabeling against 3 subunit in rat midbrain synaptic terminals correlates well with the lack of effect of muscimol on Ap₅A calcium responses (Fig. 2, A-C). On the contrary, the analysis of roughly 1475 synaptosomes from six fields showed that 62.1 ± 6.9% of them exhibit GABA_B receptors (Fig. 2, D-F), supporting the modulatory role that these receptors are playing on Ap₅A calcium responses at the presynaptic level. The same preparation did not show any GFAP or PSD-95 labeling, thus indicating the absence of glial and postsynaptic contamination in our synaptic terminal preparations (data not shown).

View larger version (50K): [in this window] [in a new window]   Fig. 2. Colocalization of synaptophysin and GABA receptors in nerve terminals from rat midbrain. Synaptic terminals were fixed onto poly-L-lysine-coated coverslips and double stained using antibodies against synaptophysin (A and D) and GABAA receptor 3 subunit (B) or GABAB receptor (E). Merged panels are shown in C and F, respectively. Antibody localization was visualized with fluorescein filters for synaptophysin and rhodamine filter for GABA receptors. Bar, 5 µm.

Ap₅A Increases Intrasynaptosomal Ca²⁺ Concentration in Single Synaptic Terminals Containing GABA_B Receptors. To determine whether a colocalization between GABA_B receptors and Ap₅A calcium responses occurs, we combined microfluorimetric studies in single synaptic terminals with immunocytochemical assays. To carry out these studies, it was necessary to use microgrid coverslips (square size 55 µm) to relocate individual synaptosomes after immunostaining procedures. Thus, isolated rat midbrain synaptic terminals loaded with Fura-2 and stuck on microgrid coverslips were tested for their ability to mobilize Ca²⁺ after 100 µM Ap₅A superfusion (Fig. 3, A-C). The intrasynaptosomal Ca²⁺ increase after depolarization with 30 mM KCl was analyzed at the end of each experiment to confirm synaptosomal functionality. Thus, the number of synaptic terminals responding to KCl was always considered as 100% of the total functional synaptosomal population. Finally, synaptosomes were fixed and labeled with antibodies that recognize specifically the vesicular protein synaptophysin and the GABA_B receptor (Fig. 3, D and E).

View larger version (84K): [in this window] [in a new window]   Fig. 3. Responses to 100 µM Ap5A in single nerve terminals from rat midbrain containing GABAB receptors. Synaptic terminals glued onto a microgrid coverslip mounted in a superfusion chamber were loaded with Fura-2 AM to follow the changes [Ca2+]i induced by stimulation with 100 µM Ap5A. After these studies, synaptosomes were fixed in 4% PFA and labeled with anti-GABAB receptor and anti-synaptophysin antibodies. A, phase contrast image of synaptosomes where a detail of the grid is shown. B, fluorescence image of the same field reveals synaptosomes challenged with the calcium dye Fura-2. C, time course of Fura-2 intrasynaptosomal calcium transients recorded for the terminal labeled with an arrow in B, D, and E. D, same field viewed with fluorescein optics reveals anti-synaptophysin-fluorescein isothiocyanate immunolabeling. E, rhodamine optics shows anti-GABAB-TRITC-immunostained synaptosomes. Bar, 5 µm.

Terminals responding to 100 µM Ap₅A represented 26.3 ± 3.3% of the total functional synaptosomal population (2275 synaptosomes from six fields). As expected for the widespread distribution of GABA_B receptors in our presynaptic model, 69.2 ± 10.8% of synaptic terminals responding to Ap₅A were labeled with the anti-GABA_B antibody (Fig. 3E). This value is close to the distribution of GABA_B receptors in the total population of synaptosomes (62.1 ± 6.9%). In addition, 25.4 ± 5.2% of the total terminals expressing GABA_B receptors responded to Ap₅A application, which is similar to the percentage of distribution shown for Ap₅A calcium responses on the whole synaptosomal population (26.3 ± 3.3%). Both coincidences suggest that GABA_B receptors are not preferably distributed on Ap₅A responding terminals from rat midbrain.

Presynaptic GABA_B Receptor Activation Potentiates Ap₅A and Gp₅G Calcium Responses in Rat Midbrain Nerve Terminals. To corroborate the modulatory role of GABA_B receptors on Ap₅A calcium responses, a concentration-response curve for the dinucleotide in the presence of 100 µM baclofen was performed. As a control, a concentration-response curve for Ap₅A was obtained in the absence of additional compounds. The curve observed was monophasic with an EC₅₀ value of 43.6 ± 7.0 µM and a maximal calcium increase of 28.1 ± 2.6 nM, which corresponds to 100% of the control maximal effect (Fig. 4A). When synaptosomes were preincubated with 100 µM baclofen, a substantial change in the concentration-response curve for Ap₅A (from 10^-14 to 10^-4 M) was observed, because lower concentrations of Ap₅A, ineffective in the absence of the GABA_B agonist, provoked a significant effect in its presence. Moreover, a biphasic concentration-response curve was obtained with a high-affinity component in the picomolar range and a low-affinity component in the micromolar range (Fig. 4A). The low-potency component showed an increase of 52.0 ± 6.2% above the control curve value, changing the maximal Ap₅A-induced calcium increment from 28.1 ± 2.6 to 42.7 ± 3.4 nM. These results obtained with baclofen, a selective agonist of GABA_B receptors, strongly support the involvement of these receptors in the potentiation of Ap₅A calcium responses.

View larger version (14K): [in this window] [in a new window]   Fig. 4. Concentration-response curves for Ap5A and Gp5G in the presence of baclofen. Before the addition of Ap5A (A) or Gp5G (B), baclofen was preincubated at a concentration of 100 µM for 2 min as described under Materials and Methods. The 100% effect corresponds to the maximal calcium transient elicited by 100 µM Ap5A or Gp5G in the absence of any other compound. Values are the mean ± S.E.M. of at least four experiments performed in triplicate (n = 4-8).

In control experiments, diguanosine polyphosphates (Gp_nG) seem to be as good agonist as Ap_nA acting through presynaptic-specific dinucleotide receptors in rat midbrain synaptic terminals (Pintor et al., 2000). To determine whether baclofen could enhance diguanosine polyphosphates effect, Gp₅G was assayed. The control curve obtained was monophasic with an EC₅₀ value of 20.8 ± 3.1 µM and a maximal calcium increase of 32.3 ± 3.2 nM, which corresponds to 100% of the control maximal effect (Fig. 4B). Moreover, the preincubation of synaptosomes with 100 µM baclofen also induced the appearance of a biphasic Gp₅G concentration-response curve (Fig. 4B). Note that Hill values observed for the high-potency component of Ap₅A or Gp₅G concentration-response curves were <1, whereas Hill numbers obtained for the low-potency component were always >1. However, the biphasic curve for Gp₅G differed from the two-step Ap₅A concentration-response curve in both the maximal calcium responses and the affinity of the high-potency step. Table 1 provides the EC₅₀ values, maximal responses and Hill numbers obtained in each experimental condition.

Effect of Baclofen on Ap₅A-Induced Intrasynaptosomal Ca²⁺ Concentrations Determined in Single Synaptic Terminals. After analyzing the potentiatory effect of baclofen in synaptosomal populations, we sought to study the effect of GABA_B receptor activation on Ap₅A calcium responses in single synaptic terminals from rat midbrain. To carry on this assay, synaptosomes were tested for their ability to mobilize Ca²⁺ after 100 nM Ap₅A superfusion in the absence or presence of 100 µM baclofen. The Ap₅A concentration 100 nM was chosen because in synaptosomal populations it induced no calcium response in the absence of baclofen but evoked a significant effect when synaptosomes were preincubated with the GABA_B agonist (Fig. 4A). Thus, synaptic terminals mounted in a superfusion chamber, were first challenged with 100 nM Ap₅A and then superfused with 100 µM baclofen for 2 min. Successive stimulation was achieved with 100 nM Ap₅A in the presence of baclofen immediately followed by 100 µM Ap₅A. As usual, a pulse of 30 mM KCl was applied at the end of each experiment.

The analysis of roughly 2450 synaptosomes from six fields showed that four different populations could be identified according to their response to Ap₅A. The first group was formed by a significant percentage of synaptosomes that failed to respond to 100 nM Ap₅A in the absence of baclofen, but responded to this stimulus in its presence (Fig. 5A). This group represented 16.8 ± 2.9% of the synaptic terminals responding to 30 mM K⁺, which were always considered as 100% of the total functional synaptosomal population. A second synaptosomal population, accounting for 9.5 ± 3.9% of the total, showed no response to 100 nM Ap₅A in the presence of baclofen, only responding to 100 µM Ap₅A (Fig. 5B). These results indicated that baclofen is able to modulate the effect of low Ap₅A concentrations in approximately 64% of the synaptosomes responding to high, but not to low, dinucleotide concentrations. Note the presence of a third group of synaptic terminals that showed an intense response to low Ap₅A concentrations (100 nM) in the absence of baclofen but did not enhance calcium entrance when further stimulated with Ap₅A (Fig. 5C). This group represented 16.1 ± 3.1% of the total rat midbrain synaptic terminals. Finally, the fourth population corresponded to synaptic terminals that do not respond to Ap₅A (data not shown).

View larger version (31K): [in this window] [in a new window]   Fig. 5. Potentiation of Ap5A calcium responses by baclofen in single synaptosomes from rat midbrain. Synaptic terminals glued into a coverslip mounted in a superfusion chamber were loaded with Fura-2 AM to monitor the changes in intrasynaptosomal Ca2+ concentration induced by 100 nM and 100 µM Ap5A, in the absence and presence of 100 µM baclofen. Representative traces of the intrasynaptosomal calcium transient time course are shown. Bars indicate the time of application of each substance.

The Potentiatory Effect of Baclofen on Ap₅A Calcium Responses Is Mediated by PKA Inhibition. To corroborate the involvement of GABA_B receptors in the potentiation of Ap₅A calcium responses, we analyzed the effect of 100 µM saclofen, a GABA_B receptor antagonist. This compound blocked the potentiatory effect mediated by baclofen, producing an approximate calcium increase of 24.2 nM that corresponds to a percentage of 86.0 ± 12.3% compared with the control value (Fig. 6). The incubation of the synaptosomal preparation with the GABA-uptake inhibitor SKF 89976A (10 µM), which increase the extrasynaptosomal GABA concentration, was able to enhance the Ap₅A calcium response to 36.2 nM, corresponding to 28.9 ± 7.7% over the control response (Fig. 6).

View larger version (37K): [in this window] [in a new window]   Fig. 6. Modulation Ap5A-evoked Ca2+ responses by PKA and GABAB receptor effectors in rat midbrain synaptic terminals. The GABAB receptor antagonist saclofen (100 µM) inhibited the potentiation evoked by baclofen on the Ap5A calcium response. The specific GABA uptake inhibitor SKF 89976A (10 µM) caused a significant enhancement of the Ca2+ response elicited by Ap5A. The preincubation of synaptosomes for 2 min with the adenylate cyclase activator forskolin (10 µM) and the PKA activator DiBucAMP (100 µM) significantly blocked the potentiation induced by baclofen on the calcium response elicited by Ap5A. PKA-IP on its own enhanced the response induced by Ap5A. Each bar represents the mean ± S.E.M. of at least three determinations performed in triplicate (n = 3-6). *, p < 0.05 versus control; **, p < 0.01 versus control; , p < 0.01 versus baclofen.

Previous results have shown that the activation of GABA_B receptors by baclofen mediates a decrease in intracellular cAMP levels in a variety of cell systems, including rat midbrain synaptic terminals (Gómez-Villafuertes et al., 2003). In this way, we adopted two experimental approaches to analyze the involvement of PKA in modulating the Ap₅A receptors in our model: one was designed to activate and the other to inhibit the activity of this enzyme (Fig. 6). PKA activation was indirectly induced using 10 µM forskolin and directly achieved with 100 µM DiBucAMP. As described for saclofen, preincubation of synaptosomes with forskolin or DiBucAMP completely inhibited the potentiatory effect mediated by baclofen, producing calcium increases of 24.5 and 26.1 nM, respectively, which correspond to values of 87.2 ± 8.5% and 93.1 ± 10.9% compared with the control (100%). PKA-IP (25 nM) was also assayed to check whether this kinase was implicated in the potentiation observed. This inhibitory peptide enhanced by its own the Ap₅A calcium response to 38.6 nM, corresponding to an increase of 37.5 ± 6.1% over the control value. Collectively, these findings are consistent with an inhibitory action of PKA on dinucleotide receptors.

	Discussion

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Presynaptic terminals have a large variety of receptors, including G protein-coupled receptors and multisubunit ion channels, which can be activated by the transmitters released from the same or adjacent terminals. There are many reports of presynaptic G_i/G₀-coupled receptors able to modulate the activity of ion channels, including metabotropic GABA_B receptors (Boehm and Kubista, 2002). GABA_B receptor activation decreases neurotransmitter release at GABAergic and other synapses via the inhibition of Ca²⁺conductance, presumably at N-type channels (Takahashi et al., 1998; Filippov et al., 2000). The present work provides the first demonstration of a presynaptic interaction between GABA_B receptors and ligand-gated calcium channels activated by dinucleotides. This result is supported by the broad distribution of metabotropic GABA_B receptors in synaptic terminals, in agreement with previous studies (Billinton et al., 2001; Möhler et al., 2001). On the contrary, synaptosomes did not show positive inmunostaining against 3 subunit, which is among the most strongly expressed GABA_A receptor subunit in the central nervous system (Benke et al., 1991), and muscimol has no effect on Ap₅A calcium responses. Indeed, a presynaptic localization of GABA_A receptors has been reported only in rat suprachiasmatic nucleus (Belenky et al., 2003).

The relevance of GABA_B receptors on Ap₅A calcium responses can be inferred from the abundance of single terminals that respond to Ap₅A and exhibit positive immunostaining against GABA_B receptors. Besides, GABA_B receptors activation in synaptosomal populations results in a dramatic change on the Ap₅A concentration-response curve. Thus, the initially sigmoidal curve, with micromolar EC₅₀ value, becomes biphasic with two saturation steps and EC₅₀ values in the picomolar and micromolar range. Moreover, maximal calcium response induced by Ap₅A is significantly increased compared with the control. The two-step curve could be analyzed taking into account that about two-thirds of the terminals responding to Ap₅A contain GABA_B receptors. In this population, the prestimulation with baclofen induces an increase in the affinity and maximal calcium response of the dinucleotide receptor. The remaining population (approximately one-third of the total) keeps the dinucleotide receptor in its low-affinity state, which also corresponds to one-third of the Ap₅A control curve.

Recently, it has been described that diguanosine polyphosphates are, together with diadenosine polyphosphates, naturally occurring substances in chromaffin granules (Jankowski et al., 2003). The present work shows that, as occurs with Ap₅A, GABA_B receptors are also capable to modulate Gp₅G calcium responses in synaptosomal populations, inducing the appearance of a two-step concentration-response curve. Because P2X are not activated by GTP or GDP (Pintor et al., 2000), the existence of specific dinucleotide receptors equally sensitive to both adenine and guanine dinucleotides is required. The high-potency step of Gp₅G curve shows a significant reduction in the affinity and maximal calcium response compared with the biphasic curve obtained for Ap₅A. Although these data could be analyzed under the scope of the existence of various dinucleotide receptors, it is also possible that in control situation Ap₅A and Gp₅G behave as full agonists and induce similar effects on the dinucleotide receptor, but after GABA modulation they do not keep that behavior, being Gp₅G a partial agonist with lower affinity than Ap₅A. It is to notice that the two steps of Ap₅A or Gp₅G concentration-response curves exhibit cooperativity. The negative cooperativity showed by the high-potency component (n_H < 1) indicates that the binding of one agonist molecule decreases the intrinsic affinity of another vacant binding site. This behavior results in a broad concentration range around the EC₅₀ value before reaching the maximal response and could be a way to control dinucleotides-induced presynaptic events. It is also possible that the low Hill slopes reveal the appearance of additional binding sites or affinity states of the dinucleotide receptor when GABA_B receptors are activated. Concerning the low-potency component of the curves, micromolar dinucleotide concentrations, which probably are only reached in the borderline of physiological/pathological conditions, are required. In this situation, a positive cooperativity (n_H > 1) could be a way to face excitotoxic events in neurons.

Microfluorimetric experiments carried out on single synaptic terminals confirm that baclofen is able to potentiate the effect of low Ap₅A concentrations in approximately 64% of the synaptosomes responding to high, but not to low, dinucleotide concentrations. This value is closely related to the widespread distribution of GABA_B receptors in synaptic terminals (62%), as well as to the percentage of synaptic terminals responding to Ap₅A and labeled with the anti-GABA_B antibody (69%). In addition, 25% of the terminals expressing GABA_B receptors responded to Ap₅A application, which is similar to the percentage of distribution shown for Ap₅A calcium responses on the whole synaptosomal population (26%). Both coincidences indicate that GABA_B receptors are not preferably distributed on Ap₅A responding terminals from rat midbrain. As reported previously in synaptosomal populations, these results do not exclude the presence of more than one type of receptor responding to Ap₅A, but the biphasic curves obtained could be explained by the presence or absence of GABA_B and dinucleotide receptors in the same terminal (Fig. 7). The modulation of dinucleotide receptor activity by presynaptic G protein-coupled receptors has also been described for adenosine A₁ and A_2A receptors, both producing the appearance of biphasic concentration-response curves for Ap₅A when activated (Díaz-Hernández et al., 2002a). Notice that microfluorimetric studies on single synaptic terminals require a continuous superfusion with fresh buffer, which removes from the synaptosomes' surrounding area a variety of compounds able to negatively modulate presynaptic receptors. This situation can explain why even in the absence of baclofen, the application of 100 nM Ap₅A induces an increase in the [Ca²⁺]_i in a significant percentage of synaptosomes. This type of receptor activated by Ap₅A, which is not susceptible to study in synaptosomal populations, has been reported here for the first time and will require a deeper functional approach in the future.

View larger version (32K): [in this window] [in a new window]   Fig. 7. Schematic diagram of synaptic terminals exhibiting dinucleotide receptors. Diadenosine polyphosphates, GABA, and other neurotransmitter are stored within secretory vesicles and are released to the extracellular medium, activating presynaptic receptors. Extrasynaptic or nonexocytotic release of ApnA and GABA is also possible. A, in nerve terminals coexpressing GABAB and dinucleotide receptors, GABA is able to facilitate ApnA calcium signaling increasing the maximal calcium entry and the affinity of dinucleotide receptors. B, in synaptic terminals that do not coexpress both receptors, dinucleotide receptors are in a state of low affinity for ApnA and the calcium influx through the channel is reduced. The absence of GABAB receptors in this synaptosomal population does not exclude the existence of other Gi/G0 protein-coupled receptors, such as A1 or A2A adenosine receptors, able to modulate ApnA calcium responses. Nts, neurotransmitters.

Direct phosphorylation processes have been proposed as a regulatory mechanism for different ionotropic neurotransmitter receptors such as nicotinic, GABA_A, N-methyl-D-aspartate, -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, P2X, and dinucleotide receptors (Pintor et al., 1997b; Chen and Bobin, 1998; Swope et al., 1999; Paukert et al., 2001). GABA_B receptors are negatively coupled to adenylate cyclase, decreasing intracellular cAMP levels when activated. Previous studies in synaptic terminals have demonstrated that the activation of presynaptic GABA_B receptors potentiates ATP responses acting through P2X receptors and blocks forskolin-stimulated cAMP formation (Gómez-Villafuertes et al., 2003). Based on these results, it seems reasonable to suggest that a decreased cAMP concentration reduces PKA activity, leading to a lower degree of phosphorylation of the dinucleotide receptor that could modify its affinity for Ap₅A. Notice that the dinucleotide receptor has not been cloned yet, but ionotropic nucleotide receptors, such as P2X₂, contain phosphorylation sites for PKA in the C-terminal region (Chen and Bobin, 1998; Chow and Wang, 1998). To verify the phosphorylation hypothesis, we analyzed the effect of activators or inhibitors of the AC-PKA cascade on the potentiation of Ap₅A responses induced by GABA. Thus, PKA activation significantly inhibits the potentiation induced by baclofen, whereas inhibition of PKA mimics the effect of baclofen on the Ap₅A-induced Ca²⁺ responses. The inhibition of GABA uptake inhibitor enhances the Ca²⁺ influx evoked by Ap₅A, indicating that endogenous GABA is able to provoke the potentiation observed. This potentiation is dependent on GABA_B receptor activation, because the antagonist saclofen causes a return to control values.

These studies expand on the variety of interactions that occur between GABAergic and other neurotransmitter systems. Recently, it has been proposed that GABA_B receptors are playing an important role in pain perception modulation at the cerebral cortex level (Jasmin et al., 2003). Moreover, GABAergic pathways and interneurons are highly abundant in the midbrain and have been related to the physiological control of sleep induction in the anterior hypothalamus and voluntary movement in basal ganglia (Kim et al., 1997; Kriem et al., 1998). The mentioned physiological effects carried out by GABA_B receptors, are postulated to occur mainly via G_i/G₀ protein through the modulation of voltage operated Ca²⁺ and K⁺ channels, which exhibit a broad presence in neural cells (Filippov et al., 2000; Billinton et al., 2001). The relevance of the findings here reported relies on the effect of GABA_B receptor activation on other variety of ion channels, but this time being ligand-operated Ca²⁺/Na⁺ channels that exhibit a much more restricted distribution. It is particularly relevant the location of ionotropic dinucleotide receptors in aminergic terminals from rat basal ganglia and in midbrain cholinergic and GABAergic terminals (Giraldez et al., 2001; Díaz-Hernández et al., 2002b; Gómez-Villafuertes et al., 2001). It is to notice that in brain perfusates from neostriatum of conscious rat submitted to strong brain stimulation, the extracellular amounts of dinucleotides increases dramatically, reaching nanomolar concentrations (Pintor et al., 1995). The longer half-life of dinucleotides compared with ATP made these compounds to be able to exert their effects in more distant areas, inducing presynaptic stimulation when GABA_B receptors are activated (Mateo et al., 1997). It is too early to fully understand the physiopathological implications of these findings, but they should be taken into account when considering the modulatory actions of GABA_B receptors and the regulation of presynaptic ligand-gated ion channels.

	Footnotes

 
This work was supported by research grants from the Spanish Ministry of Science and Technology BFI 2002-03626 and CAM 08.5/0004.1/2003. R.G.-V. is a research fellow of the Spanish Ministry of Science and Technology BFI 2002-03626 (postdoctoral fellowship).

DOI: 10.1124/jpet.103.061564.

ABBREVIATIONS: Ap₅A, P¹,Pⁿ-di(adenosine-5') pentaphosphate; Gp₅G, P¹,Pⁿ-di(guanosine-5') pentaphosphate; PKA, cyclic AMP-dependent protein kinase; BSA, bovine serum albumin; PFA, paraformaldehyde; GFAP, glial fibrillar acidic protein; TRITC, tetramethylrhodamine isothiocyanate; DiBucAMP, dibutyryl cyclic AMP; [Ca²⁺]_i, intracellular calcium concentration; PSD-95, postsynaptic density protein; PKA-IP, cyclic AMP-dependent protein kinase inhibitory peptide; PBS, phosphate-buffered saline; SKF 89976A, N-(4,4-diphenyl-3-butenyl)-3-piperidinecarboxylic acid.

Address correspondence to: Dr. M. T. Miras-Portugal, Departamento de Bioquímica, Facultad de Veterinaria, Universidad Complutense de Madrid, Av. Puerta de Hierro s/n, 28040 Madrid, Spain. E-mail: mtmiras{at}vet.ucm.es

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