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NEUROPHARMACOLOGY

Rapid Stimulation of Presynaptic Serotonin Transport by A₃ Adenosine Receptors

Departments of Pharmacology (C.-B.Z., J.A.S., W.A.H., R.D.B.) and Psychiatry (W.A.H., R.D.B.), and Center for Molecular Neuroscience (R.D.B.), Vanderbilt University School of Medicine, Nashville, Tennessee; and Department of Physiology (J.L.M., L.C.D.), University of Texas Health Science Center, San Antonio, Texas

Received February 22, 2007; accepted April 23, 2007.

	Abstract

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The inactivation of synaptic serotonin (5-hydroxytryptamine, 5-HT) is largely established through the actions of the presynaptic, antidepressant-sensitive 5-HT transporter (SERT, SLC6A4). Recent studies have demonstrated post-translational regulation of SERT mediated by multiple Ser/Thr kinases, including protein kinases C and G (PKC and PKG) and p38 mitogen-activated protein kinase (MAPK), as well as the Ser/Thr phosphatase PP2A. Less well studied are specific surface receptors that target these signaling pathways to control SERT surface expression and/or catalytic rates. Using rat basophilic leukemia 2H3 cell line (RBL-2H3), we previously established that activation of A₃ adenosine receptors (A₃AR) stimulates SERT activity via both PKG and p38 MAPK (Zhu et al., 2004a). Whether A₃ARs regulate SERT in the central nervous system (CNS) is unknown. Here we report that the A₃AR agonist N⁶-(3-iodobenzyl)-N-methyl-5'carbamoyladenosine (IB-MECA) rapidly (10 min) and selectively stimulates 5-HT transport in mouse midbrain, hippocampal, and cortical synaptosomes. IB-MECA-induced stimulation of 5-HT uptake is blocked by the selective A₃AR antagonist 3-ethyl-5-benzyl-2-methyl-phenylethynyl-6-phenyl-1,4(±)dihydropyridine-3,5-dicarboxylate (MRS1191) and is absent from synaptosomes prepared from A₃AR knockout mice. Kinetic analyses demonstrate that IB-MECA induces an increase of 5-HT transport V_max with no significant change in K_m. As in RBL-2H3 cells, IB-MECA stimulation of synaptosomal 5-HT uptake can be blocked by preincubation with PKG antagonists N-[2-(methylamino)ethy]-5-isoquinoline-sulfonamide (H8) and DT-2 (YGRKKRRQRRRPPLRK₅H), as well as by the p38 MAPK inhibitor SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole]. Chronoamperometry studies in the anesthetized rat hippocampus support a role for A₃ARs in SERT regulation in vivo. Together, these results identify a novel, region-specific action of CNS A₃ARs in the modulation of SERT-mediated 5-HT transport that may be relevant for the etiology and/or therapy of 5-HT-linked brain disorders.

Previous studies demonstrate that SERTs are tightly controlled by multiple signaling pathways, including G-protein-coupled receptor-linked pathways (Blakely et al., 2005). Activation of protein kinase C triggers a decrease in both SERT activity and surface expression in cultured cell lines (Ramamoorthy and Blakely, 1999), platelets (Jayanthi et al., 2005; Carneiro and Blakely, 2006), and nerve terminals (Samuvel et al., 2005) linked to changes in membrane distribution and destabilization of SERT-associated protein complexes (Carneiro and Blakely, 2006). In contrast, protein kinase G (PKG)-linked and p38 mitogen-activated protein kinase (MAPK)-linked pathways support a rapid increase in SERT surface expression and function (Miller and Hoffman, 1994; Zhu et al., 2004a,b, 2005). There is only limited information on specific receptors that use these pathways in vivo. Chronoamperometry studies suggest a role of presynaptic 5-HT₁ receptors in amplifying 5-HT clearance in rat hippocampus (Daws et al., 2000) through as yet unknown pathways. Ansah et al. (2003) demonstrated activity of presynaptic 2-adrenergic receptors in suppression of SERT activity in mouse brain synaptosomes and slices. Histamine receptor stimulation has been reported to up-regulate 5-HT uptake via PKG activation in platelets (Launay et al., 1994); evidence that this pathway regulates SERT in the CNS is lacking. Miller and Hoffman (1994) first noted activity of adenosine receptors (ARs) in stimulating SERT activity via a cGMP-linked pathway in rat basophilic leukemia (RBL-2H3) cells (Miller and Hoffman, 1994) in vitro. Interestingly, an in vivo microdialysis study has noted the ability of AR-targeted drugs to alter extracellular 5-HT levels in rat hippocampus (Okada et al., 1999), suggesting that CNS parallels may exist for AR modulation of SERT activity. Recently, we used RBL-2H3 cells and AR/SERT-cotransfected Chinese hamster ovary (CHO) cells to further define an A₃AR-linked pathway that supports up-regulation of SERT activity, establishing involvement of two distinct pathways: 1) a PKG-dependent pathway linked to enhanced SERT surface trafficking and 2) a PKG-dependent pathway supported by p38 MAPK, associated with a trafficking-independent process that enhances SERT catalytic activity by reducing the 5-HT K_m (Zhu et al., 2004a, 2005; Blakely et al., 2005). Whereas the latter pathway has been established to be targeted by inflammatory cytokines in CNS preparations (Zhu et al., 2006), receptors that initiate PKG-dependent SERT up-regulation in the brain are unknown.

A₃ARs are expressed in brain (Dixon et al., 1996) and implicated in a variety of conditions, including anxiety and depression (Fedorova et al., 2003). Because we have shown A₃AR modulation of RBL-2H3 cell SERT, we sought evidence for a similar activity in brain preparations, using in vitro studies of 5-HT uptake in brain synaptosomes and in vivo studies of clearance of pulse-applied 5-HT as assessed by chronoamperometry. Consistent with results in RBL-2H3 cells, the A₃AR-selective agonist IB-MECA induces rapid and significant stimulation of 5-HT uptake in mouse midbrain and hippocampal synaptosomes. These effects are blocked by MRS1191, a selective A₃AR antagonist, as well as by PKG and p38 MAPK inhibitors. IB-MECA stimulation of SERT is lost in synaptosomes from A₃AR knockout (A₃AR^-/-) mice. Finally, chronoamperometry studies demonstrate activity of A₃ARs in control of 5-HT clearance in vivo, revealing a pathway that may participate in or be used to ameliorate 5-HT-linked brain disorders.

	Materials and Methods

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Reagents. IB-MECA, NECA, H8, R-PIA, and MRS1191 were purchased from Sigma-Aldrich (St. Louis, MO), SB203580 and SB202474 were obtained from Alexis Biochemicals (San Diego, CA). DT-2 and the nonmembrane-permeant analog W45 were kind gifts from Dr. Wolfgang Dostmann (University of Vermont, Burlington, VT) (Dostmann et al., 2000). [³H]5-HT (107 Ci/mmol) was purchased from GE Healthcare (Piscataway, NJ). All other biochemical reagents were of the highest grade possible and obtained from Sigma-Aldrich. A₃AR^-/- mice (Salvatore et al., 2000), generously provided by Dr. Marlene Jacobson (Merck, West Point, PA), were maintained on a C57BL/6 background and were housed and bred in the Vanderbilt University Vivarium with water and food provided ad libitum.

Synaptosomal Studies. C57BL/6 mice (A₃AR^+/+; Harlan, Indianapolis, IN) and A₃AR^-/- mice were used in these studies following an approved protocol of the Vanderbilt University Institutional Animal Care Use Committee. Synaptosomes were prepared as described previously (Zhu et al., 2005). In brief, mouse midbrain, hippocampus and striatum were dissected on ice after sacrifice and homogenized in 0.32 M D-glucose at 400 rpm using a Teflon-glass tissue homogenizer (Wheaton Instruments, Millville, NJ). The homogenized tissue was centrifuged at 800 x g for 10 min at 4°C. The supernatant was transferred to new centrifuge tubes and centrifuged at 10,000 x g for 15 min at 4°C. Synaptosomal pellets were resuspended in Krebs-Ringer's HEPES buffer containing 130 mM NaCl, 1.3 mM KCl, 2.2 mM CaCl₂, 1.2 mM MgSO₄, 1.2 mM KH₂PO₄, 1.8 g/l D-glucose, 10 mM HEPES, pH 7.4, 100 µM pargyline, and 100 µM ascorbic acid. Synaptosomal suspensions were assessed for protein content (Bio-Rad, Hercules, CA) and maintained on ice until dispensed for 5-HT transport assays.

5-HT Transport Assays. [³H]5-HT transport activity was assayed as described previously (Zhu et al., 2006). In brief, 30 to 50-µg synaptosomes per sample (in a total volume of 200 µl) were preincubated at 37°C in a shaking water bath for 10 min. Vehicle or modifiers were then added for 10 to 20 min. After a 5-min incubation with [³H]5-HT (20 nM), [³H]DA (50 nM; PerkinElmer Life and Analytical Sciences, Boston, MA), or[³H]GABA (50 nM; PerkinElmer Life and Analytical Sciences) at 37°C, uptake was terminated by filtration through (polyethyleneimine-coated) GF/B Whatman filters using a Brandel Cell Harvester (Brandel, Gaithersburg, MD). Filters were washed three times with ice-cold Krebs-Ringer's HEPES buffer and immersed in scintillation liquid for 8 h, and radioactivity accumulated was quantitated by scintillation spectrometry (Beckman Coulter, Fullerton, CA). Counts obtained from the filtered samples were corrected for nonspecific uptake using parallel samples incubated at 37°C with paroxetine (1 µM for SERT assays) or GBR 12935 (1 µM for dopamine transporter assays) or using incubation on ice (GABA transport assays).

Assays for 5-HT Level from Whole Tissue of the Brain Regions. To assess levels of 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) in dissected brain regions (hippocampus, midbrain, striatum, and frontal cortex), tissues were dissected on ice and quickly frozen in liquid nitrogen and stored at -80°C until HPLC analysis. On the day of analysis, brain regions were homogenized in 100 to 750 µl of 0.1 M trichloroacetic acid containing 10^-2 M sodium acetate, 10^-4 M EDTA, and 10.5% methanol, pH 3.8. Samples were centrifuged at 10,000 x g for 20 min. Supernatants were stored at -80°C, and pellets were processed for protein analysis (Bio-Rad). Supernatants were analyzed for biogenic monoamine levels by HPLC [Phenomenex Nucleosil (5µ, 100A) C18 HPLC column (150 x 4.60 mm); Phenomenex, Torrance, CA] using an Antec Decade II (oxidation, 0.5; Antec Leyden, Hubbardston, MA) electrochemical detector in the Center for Molecular Neuroscience Neurochemistry Core (Nashville, TN). Data were converted into picomoles per milligram protein, with comparisons between wild-type and A₃AR^-/- samples performed using a Student's unpaired t test.

In Vivo Chronoamperometry Studies. Male Sprague-Dawley rats (Harlan), weighing 280 to 380 g, were used for all chronoamperometry experiments as approved by the University of Texas San Antonio Health Sciences Center Institutional Animal Care and Use Committee and were in strict accordance with the Institute of Laboratory Animal Resources (1996). Rats were anesthetized by intraperitoneal injection of chloralose (85 mg/kg) and urethane (850 mg/kg). A tube was inserted into the trachea to facilitate breathing, and the animal was placed into a stereotaxic frame. The electrode micropipette recording assembly was lowered into the CA3 region of the dorsal hippocampus (anterior-posterior, -4.1; midline, +3.5; dorsalventral, -3.6) (Paxinos and Watson, 1986). Body temperature was maintained at 37 ± 1°C by a water-circulated heating pad (Seabrook, Cincinnati, OH). High-speed chronoamperometric recordings were made using the FAST-12 system (Quanteon, Lexington, KY), as described previously (Daws et al., 2000). In brief, carbon fiber electrodes (tip diameter, 30 µm; Quanteon) were coated with Nafion solution (5% solution; Aldrich Chemical Co., Milwaukee, WI) to prevent interference from anionic substances in the extracellular fluid. Electrodes were tested for sensitivity to the 5-HT metabolite, 5-HIAA (250 µM), and calibrated with six increasing concentrations of 5-HT ranging from 0.5 to 3.0 µM. Only electrodes displaying a selectivity ratio for 5-HT over 5-HIAA of >1000:1 and a linear response (r² 0.997) to 5-HT (0.5-3.0 µM) were used. The detection limit for the measurement of 5-HT levels was defined as the concentration that produced a response with a signal-to-noise ratio of 3. The electrochemical recording assembly consisted of a Nafion-coated, single carbon fiber electrode attached to a four- or seven-barreled micropipette. The assembly was constructed, such that the electrode and micropipette tips were separated by 300 ± 20 µm. The tip diameter of each barrel of the multibarreled micropipette was between 10 and 15 µm. Barrels were filled with 5-HT (200 µM), the A₃AR agonist IB-MECA (200, 400, or 800 nM), or vehicle. All drugs were dissolved in 0.1 M phosphate-buffered saline with 100 µM ascorbic acid added as an antioxidant. 5-HT was pressure-ejected (5-25 p.s.i. for 0.25-3 s) at 3 to 10-min intervals until a reproducible signal was obtained (usually three or four applications). The mean ± S.E.M. number of picomoles of 5-HT delivered was 10 ± 1 in a volume of 50 ± 3 nl as measured by determining the amount of fluid displaced from the micropipette using a dissection microscope fitted with an eyepiece reticule. Once the 5-HT signal was reproducible, vehicle or drug was applied 60 to 90 s before the next application of 5-HT to allow sufficient time for diffusion of drug to areas around the recording electrode. These solutions were pressure-ejected over 10 to 20 s to minimize disturbances to the baseline electrochemical signal. IB-MECA was ejected in a volume of 125 nl to deliver 25, 50, or 100 pmol. An equivalent volume of vehicle was ejected as a control.

Statistical Analyses. All data were derived from experiments replicated a minimum of three times. Statistical analyses, comparing baseline and compound-modified uptake, were performed with GraphPad Prism (GraphPad, San Diego, CA) using one- and two-way analyses of variance (ANOVA) with subsequent planned comparisons (Dunnett's, Bonferroni's), as well as Student t tests, as noted in the figure legends. P < 0.05 was taken as significant for all evaluations.

	Results

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Activation of A₃ARs in Mouse Brain Synaptosomes Induces an Increase in 5-HT Uptake. IB-MECA (Gallo-Rodriguez et al., 1994) was used to examine A₃AR-dependent regulation of SERT in mouse midbrain synaptosomes. IB-MECA exerted a concentration- and time-dependent stimulation of 5-HT transport activity (Fig. 1, A and B). IB-MECA pretreatment of synaptosomes for 10 min resulted in stimulation of 5-HT transport that peaked at 10 nM (130150% of control levels; Fig. 1A). Higher concentrations of IB-MECA did not further increase 5-HT transport; rather, reduced efficacy was apparent above 10 nM, and when exceeding 100 µM, IB-MECA even induced a decrease in 5-HT uptake (data not shown), effects not pursued in the present study. Using a concentration of 10 nM IB-MECA, we examined the time course of IB-MECA stimulation, where effects were observed to reach a maximum at 10 to 20 min post-treatment with less efficacious actions evident with longer treatments (Fig. 1B). A similar action was observed for IB-MECA in hippocampal synaptosomes (see below; Fig. 3). In agreement with our previous study using RBL-2H3 cells (Zhu et al., 2004a), IB-MECA treatments of midbrain synaptosomes significantly increased the SERT V_max for 5-HT (Fig. 1C). We could detect no significant IB-MECA-induced change in 5-HT K_m (Fig. 1C). Importantly, MRS1191, a selective antagonist for A₃AR, prevented the stimulatory actions of IB-MECA on 5-HT uptake (Figs. 1C and 3B). MRS1191 alone did not influence basal 5-HT transport in synaptosomes over a wide range of concentrations (0.01-100 µM; data not shown).

View larger version (17K): [in this window] [in a new window]   Fig. 1. Effect of A3AR agonist IB-MECA on 5-HT uptake in mouse midbrain synaptosomes. A, dose response. Synaptosomes were treated with IB-MECA at the indicated concentrations for 10 min at 37°C, followed by a 5-min incubation with [3H]5-HT (final concentration: 20 nM). Nonspecific uptake was assessed by incubation of synaptosomes with 10 µM paroxetine for 10 min before adding [3H]5-HT. B, time course of IB-MECA effects on SERT activity. Synaptosomes were incubated with IB-MECA (10 nM) for the indicated times at 37°C before 5-HT uptake assays. C, kinetics of IB-MECA stimulation. Synaptosomes were treated with vehicle or MRS1191 (1 µM) for 10 min followed by IB-MECA (10 nM) for 10 min before transport assays. Data were fit to a Michaelis-Menten equation (single site) to derive values for 5-HT, Km (vehicle: 0.92 ± 0.25 µM; IB-MECA, 0.80 ± 0.25 µM; MRS1191+IB-MECA, 0.82 ± 0.29 µM) and Vmax (vehicle, 1762 ± 173; IB-MECA, 2544 ± 281**; MRS1191+IB-MECA, 1791 ± 218 fmol/mg protein/min) [values are expressed as mean values (n = 3) ± S.E.M.; **, P < 0.01 versus. control (Student's t test)]. Values in A and B are expressed as the mean of five experiments ± S.E.M. *, P < 0.05, **, P < 0.01 versus controls (one-way ANOVA, Dunnett's).

View larger version (17K): [in this window] [in a new window]   Fig. 3. Loss of IB-MECA stimulation of 5-HT uptake in midbrain synaptosomes derived from A3AR-/- (A3KO) mice. Synaptosomes from midbrain (A) or hippocampus (B) of either wild type mouse (wt) or A3 adenosine receptor knockout mouse (A3KO) were treated with vehicle (open bars) or IB-MECA (solid bars, 10 nM) for 10 min at 37°C followed by incubation with [3H]5-HT (20 nM). The basal 5-HT uptake (femtomole/minute/microgram protein) in midbrain for A3AR+/+ and A3AR-/- are 1016.7 ± 66.3 and 1017.5 ± 213.5, respectively, and in hippocampus for A3AR+/+ and A3AR-/-, they are 976.1 ± 114.4 and 983.8 ± 126.1, respectively. Values are expressed as mean (n = 4) ± S.E.M. **, P < 0.01 versus. control (Student's t test).

NECA and R-PIA Do Not Stimulate Synaptosomal 5-HT Uptake. Multiple ARs are present in brain; thus, we wished to explore whether the actions of IB-MECA reflect a specific association of the A₃AR with neuronal SERT regulation. NECA is a nonselective AR agonist with a similar potency at A₁,A₂, and A₃ ARs (similar K_i for rat and mouse A₁,A_2A, and A₃ = 6.3, 10, and 113 nM, respectively, with values 3.5-26-fold higher in human and guinea pig preparations) (Feoktistov and Biaggioni, 1997; Maemoto et al., 1997). Whereas NECA stimulates SERT activity in RBL-2H3 cells (Miller and Hoffman, 1994; Zhu et al., 2004a), this agent fails to stimulate SERT activity in midbrain synaptosomes (Fig. 2A). At concentrations above 1 µM, NECA actually inhibited 5-HT transport, suggesting an underlying receptor complexity that may limit conclusions from neural tissues (compared with cell lines), with nonselective AR agonists. Furthermore, we previously found that treatment of SERT/A₁AR-cotransfected CHO cells with the A₁AR-selective agonist R-PIA (Zhu et al., 2004a) stimulated SERT activity. Because A₁ARs are abundantly expressed in brain and use similar signaling pathways as A₃ARs (Feoktistov and Biaggioni, 1997; Fredholm et al., 2005), we tested the activity of R-PIA for SERT regulation in midbrain synaptosomes. No impact of R-PIA on 5-HT transport was observed across a wide range of concentrations (1-1000 nM) (Fig. 2B).

View larger version (20K): [in this window] [in a new window]   Fig. 2. Effects of NECA and R-PIA on 5-HT uptake. Synaptosomes from mouse midbrain (C57BL/6) were treated with NECA, a nonspecific adenosine receptor agonist (A) or R-PIA (a A1AR agonist) (B) at the indicated concentrations for 10 min at 37°C before 5-HT uptake assays. Values are expressed as the mean of three experiments ± S.E.M. **, P < 0.01 versus controls (one-way ANOVA, Dunnett's).

Specificity of IB-MECA Stimulation of Synaptosomal SERT Activity. To verify that MRS1191-sensitive IB-MECA stimulation of synaptosomal SERT activity is mediated by A₃ARs, we compared modulation of 5-HT uptake in midbrain and hippocampal synaptosomes prepared from A₃AR^-/- mice (Salvatore et al., 2000) versus wild-type (A₃AR^+/+) mice of the same genetic background (Fig. 3). As expected, IB-MECA induced a significant increase (40-50%) in 5-HT transport with A₃AR^+/+ mice. In contrast, IB-MECA failed to stimulate SERT activity in A₃AR^-/- mice. Although SERT regulation by IB-MECA was lost in A₃AR^-/- preparations, basal SERT activity was equivalent in A₃AR^+/+ and ^-/- synaptosomes (see Fig. 3 legend), as were tissue 5-HT and 5-HIAA levels (Table 1). Furthermore, we explored the regional and substrate specificity of IB-MECA stimulation of SERT activity (Fig. 4). Unlike midbrain, hippocampal (Figs. 1, 2, 3, 4) and cortical synaptosomes (data not shown), striatal synaptosomes displayed IB-MECA-insensitive SERT activity (Fig. 4A). In striatal and midbrain preparations, we also assessed IB-MECA effects on DA and GABA transport activity. In assays where clear up-regulation of SERT was evident, we observed no stimulation for either DA or GABA uptake. These studies indicate that A₃ARs are not required to maintain 5-HT uptake or 5HT/5HIAA levels but are essential for the actions of IB-MECA in stimulation of midbrain and hippocampal SERT in vitro.

View larger version (21K): [in this window] [in a new window]   Fig. 4. Brain region- and neurotransmitter-specific stimulation of IB-MECA. Synaptosomes from mouse striatum (A) and midbrain (B) were treated with vehicle (open bars) or IB-MECA (solid bars) for 10 min before assays for 5-HT, DA, or GABA uptake. Values are expressed as mean (n = 5) ± S.E.M. **, P < 0.01 versus vehicle control (Student's t test).

A₃AR Stimulation of Synaptosomal SERT Involves PKG and p38 MAPK Signaling Pathways. A₃AR stimulation of SERT arises via activation of guanyl cyclase, production of cGMP, and activation of PKG, leading to insertion of intracellular SERT proteins into the plasma membrane (Miller and Hoffman, 1994; Zhu et al., 2004a). Furthermore, PKG activation triggers phosphorylation and activation of p38 MAPK, leading to catalytic activation of surface SERTs (Zhu et al., 2004a). To explore whether the same pathways are involved in the synaptosomal actions of IB-MECA reported above, we asked whether PKG and p38 MAPK antagonists attenuate basal and IB-MECA-stimulated SERT activity in midbrain synaptosomes. DT-2, a membrane-permeant peptide that potently (K_i = 12.5 nM) (Dostmann et al., 2000) and selectively inhibits PKG1, fails to affect basal 5-HT uptake until concentrations reach or exceed 5 µM (Fig. 5A). When tested at a concentration lacking actions on basal SERT activity, DT-2 (1.0 µM, Fig. 5B) blocked the ability of IB-MECA to stimulate SERT activity. In contrast, W45, a membrane-impermeable version of DT-2 (Dostmann et al., 2000), failed to block IB-MECA effect at 10 µM (Fig. 5B). Furthermore, the nonpeptide PKG inhibitor H8, when tested at a concentration lacking basal actions (0.1 µM), also blocked IB-MECA stimulation of SERT activity (Fig. 5B). The p38 MAPK inhibitor SB203580 attenuated basal SERT activity at concentrations at or above 5 µM (Fig. 5C). When tested at a concentration lacking actions on basal SERT activity, SB203580 (1.0 µM, Fig. 5D) blocked stimulation of SERT activity by IB-MECA. As a negative control for the effects of SB203580, we utilized SB202474 (10 µM), an analog of SB203580 that lacks actions at p38 MAPK, and observed no attenuation of IB-MECA-stimulated SERT activity (Fig. 5D). Together, these findings support a role for both PKG and p38 MAPK-linked pathways in the acute modulation of SERT by A₃ARs.

View larger version (28K): [in this window] [in a new window]   Fig. 5. A3AR agonist effects on 5-HT uptake are blocked by A3AR, PKG and p38 MAPK inhibitors. A, dose response of DT-2, a peptide-based PKG inhibitor. Synaptosomes from mouse midbrain were treated with DT-2 at the indicated concentrations for 10 min at 37°C followed by 5-HT uptake assays. B, inhibitors of PKG (H8, 0.1 µM; DT-2, 1.0 µM) and A3AR (MRS1191, 1.0 µM) blocked IB-MECA-stimulated SERT activity. Synaptosomes were incubated with vehicle, H8, DT-2, W45 (10 µM), or MRS1191 for 10 min at 37°C, followed by a 10-min treatment with IB-MECA (10 nM) before 5-HT uptake assay. C, dose response of SB203580, a p38 MAPK inhibitor. Synaptosomes were treated with SB203580 at the indicated concentrations for 10 min at 37°C followed by 5-HT uptake assays. D, p38 MAPK inhibitor abolishes IB-MECA-stimulated 5-HT uptake. Synaptosomes were incubated with vehicle, SB203580 (1.0 µM), or SB202474 (10 µM) for 10 min at 37°C, followed by a 10-min treatment with IB-MECA (10 nM) before 5-HT uptake assay. Values are expressed as the mean of three or more experiments ± S.E.M. *, P < 0.05; **, P < 0.01 versus controls (one-way ANOVA, Dunnett's for A and C; two-way ANOVA for B and D).

A₃AR-Modulate SERT-Mediated 5-HT Clearance in Vivo. To evaluate whether A₃ARs can regulate SERTs in vivo, we monitored SERT-mediated 5-HT clearance using high-speed chronoamperometry (Daws and Toney, 2006). Chronoamperometric recordings of the clearance rates of exogenously applied 5-HT were obtained from the CA3 region of hippocampus in anesthetized rats. After obtaining a stable baseline for the clearance of injected 5-HT, we injected varying doses of IB-MECA, followed by reapplications of 5-HT. The time to clear 5-HT by 80% (T₈₀) was quantified and plotted as a function of IB-MECA concentration (Fig. 6A) and time (Fig. 6B). Injection of vehicle was without effect across the recording time course. In contrast, we observed a significant reduction in T₈₀ (enhanced clearance rate) with IB-MECA, effects that were both dose- and time-dependent. Low (25 pmol) but not high (50-100 pmol) amounts of IB-MECA injected significantly enhanced 5-HT clearance rate when assessed at 20 min (Fig. 6A). As observed in vitro, the effect of IB-MECA (25 pmol) to enhance 5-HT clearance was not immediate but rather displayed a time-dependent course with a peak effect at 15 to 20 min followed by reversal to control levels (Fig. 6B). These findings support the ability of A₃ARs to modulate SERT in vivo as predicted from studies in vitro with rat-derived RBL-2H3 cell (Zhu et al., 2004a) and mouse synaptosomal studies (as described above).

View larger version (15K): [in this window] [in a new window]   Fig. 6. In vivo assessment of IB-MECA effects on 5-HT clearance (A). Effect of IB-MECA on 5-HT clearance in CA3 region of hippocampus of anesthetized rats as monitored by chronoamperometry. 5-HT was locally applied by pressure-ejection until a reproducible signal was obtained. IB-MECA was then locally applied, followed again by 5-HT at 2 and 5 min post-IB-MECA and then at 5-min intervals thereafter until 5-HT clearance parameters returned to predrug baseline levels. Data shown are for 20 min following drug administration. IB-MECA (25 pmol) significantly decreased the clearance time for 5-HT. B, time course for the effect of IB-MECA (25 pmol) to enhance 5-HT clearance in CA3 region of hippocampus of anesthetized rats. Serotonin was locally applied by pressure-ejection until a reproducible signal was obtained (triplicate replicate signals are averaged and represented by the data point at t =-2 min). At time = 0, IB-MECA was locally applied to the hippocampus, and then 5-HT was again ejected 2 and 5 min post-IB-MECA and at 5-min intervals thereafter until 5-HT clearance parameters returned to predrug baseline levels. **, P < 0.01 versus controls (one-way ANOVA, Dunnett's).

	Discussion

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In our previous study, we observed that A₃AR activation stimulates 5-HT uptake in RBL-2H3 cells via a PKG- and p38 MAPK-linked pathway (Zhu et al., 2004a). Although we (Zhu et al., 2004a, 2005) and others (Samuvel et al., 2005) established that PKG and p38 pathways regulate SERT in brain preparations, specific receptors linked to these pathways are unknown. Recently (Zhu et al., 2006), we demonstrated that interleukin-1 receptors couple via the p38 MAPK pathway to activate SERT. Here, we demonstrate that A₃ARs in the brain also participate in the modulation of CNS SERT activity. The actions of the A₃AR agonist IB-MECA to stimulate 5-HT transport occur across a narrow concentration and time window (10-30 nM for 20 min). This may reflect regulation of SERT by the multiple AR subtypes found in the brain (Fredholm et al., 2005), with temporal profiles of SERT activity shaped by the particular coupling kinetics of A₃ARs. Although IB-MECA is relatively potent and selective for the A₃AR (K_D = 1.1 nM), it can also activate A₁ and A₂A ARs (K_D = 50 nM in rat and mouse) (Feoktistov and Biaggioni, 1997; Maemoto et al., 1997). Simultaneous stimulation of multiple AR types in the brain could compromise detection of A₃AR-mediated effects on SERT activity. Possibly such issues relate to observations that the nonselective AR agonist NECA fails to stimulate 5-HT transport in synaptosomes, whereas it stimulates SERT in cultured RBL-2H3 cells. Furthermore, ARs exert their modulation on a variety of neurotransmitter systems (Fredholm et al., 2005), and one AR subtype can interact functionally with other AR subtypes (Okada et al., 1999). We did not observe an ability of the A₁AR-selective agonist R-PIA to stimulate 5-HT transport in brain, whereas we have found that cotransfected A₁AR can enhance 5-HT uptake via SERT in cotransfected cells (Zhu et al., 2004a). Overall, these findings indicate a selective relationship between A₃ARs and SERT, presumably due to their coexpression on serotonergic terminals. Unlike SERT, where gene expression is limited in the CNS to raphe neurons, gene expression for A₃ARs is widespread throughout the CNS, including hippocampus and midbrain (Dixon et al., 1996). Colocalization studies with antibodies specific for SERT and A₃ARs are needed to more finely establish the spatial relationships of these proteins. We also demonstrate that A₃AR-mediated 5-HT transport enhancement can be blocked by H8 and DT-2 (general and selective inhibitors of PKG, respectively) or SB203580 (a specific p38 MAPK inhibitor), consistent with the participation of both PKG and p38 MAPK in rapid enhancements of SERT activity. As our kinetic studies of A₃AR-mediated stimulation of SERT activity in midbrain synaptosomes demonstrated an increase of V_max, enhanced trafficking of SERT proteins as demonstrated in RBL-2H3 and transfected CHO cells is probable (Zhu et al., 2004a, 2006). SERT has been identified on the plasma membrane as well as intracellular vesicles of platelets and CNS-serotonergic terminals (Huang and Pickel, 2002; Carneiro and Blakely, 2006). Our findings suggest that intracellular stores of SERT may be mobilized by A₃AR-triggered PKG activation. Alternatively, SERT recycling rates may be accelerated to permit an increase in transporter surface expression (Loder and Melikian, 2003). A₃AR stimulation also leads to a p38 MAPK-dependent pathway engaged in stimulation of 5-HT transport. Our prior studies suggest that this mode of regulation relates to changes in SERT catalytic activity (Blakely et al., 2005).

To further validate the A₃AR-targeted actions of IB-MECA on SERT, we took advantage of A₃AR^-/- mice (Salvatore et al., 2000). Although basal SERT activity, as well as 5-HT and 5-HIAA levels, is normal in A₃AR^-/- mice, IB-MECA stimulation of SERT was abolished. Previous behavioral studies with A₃AR^-/- mice demonstrate an increase in locomotor behavior in the open field test, an increase in open arm entries on the elevated plus maze test, and increased transitions in the light/dark box test (Fedorova et al., 2003), suggesting a reduction in the level of anxiety or fearfulness. Furthermore, A₃AR affects immobility in the Porsolt swim test and the tail suspension test, two behaviors typically offset by acute selective serotonin reuptake inhibitor administration (Fedorova et al., 2003). Although the inherent limitations of knockout studies preclude more definitive conclusions, these findings suggest a functional relationship between A₃AR activation, SERT activity, and 5-HT signaling. It is possible that the loss of A₃ARs may prevent environment-triggered increases in SERT activity that could be anxiogenic, and over time, such deficits would result in increased exploratory behavior and diminished fearfulness. At times when environmental stress should be translated into behavioral activation, failure to activate SERT may prevent normal struggling responses in swim and tail-suspension tests. Temporally controlled A₃AR gene ablation or knockdown restricted to raphe neurons should be helpful to test such ideas.

The actions of IB-MECA in vitro seem relatively selective for SERT as neither DA nor GABA transport was modulated in the same preparations where 5-HT uptake was increased. These findings indicate that SERT modulation does not arise from a nonspecific action of IB-MECA on ion gradients or membrane potential that commonly support the transport of each of these substrates, and they are consistent with our implication of PKG and p38 MAPK pathways in SERT modulation. More interestingly, our results show that IB-MECA-induced SERT modulation is region-dependent, suggesting possible differences in A₃AR abundance, localization, or coupling as related to SERT expression. Alternatively, negative regulatory pathways unique to striatum or the presence of limited regulatory capacity may preclude detection of an ability of A₃ARs to modulate SERT. Striatal SERTs participate in actions of 5-HT in motor and reward pathways, whereas hippocampal and cortical SERTs constrain the actions of 5-HT in cognitive and mood circuits. Our findings of region-specific control of SERTs by A₃ARs support the development of medications that modify some but not all facets of 5-HT signaling. Clearly, extension of our findings for medication development requires additional in vivo studies. Important validation of a role of A₃AR activation in regulation of SERT activity in vivo arises from our chronoamperometry studies, where we assessed the clearance of pulse-applied 5-HT. Specifically, we observed a dose- and time-dependent effect of IB-MECA administration to increase 5-HT clearance, consistent with synaptosome studies. Our findings are also consistent with a prior in vivo dialysis study that reported an A₃AR-dependent reduction in extracellular 5-HT (Okada et al., 1999).

The results from our current study also raise the question regarding the source of endogenous adenosine that acts through A₃AR to stimulate SERT activity. In the CNS, adenosine is produced from at least three different sources. The most important source is believed to be the hydrolysis of 5'-AMP by 5'-nucleotidase. Extracellular 5'-AMP is derived partly from ATP, which is colocalized and coreleased with other neurotransmitters, including acetylcholine, norepinephrine, and dopamine (Salter et al., 1993). An additional source of adenosine arises as a byproduct of transmethylation reactions that are important for catabolism of catecholamines and histamine (via catecholamine O-methyltransferase and histamine N-methyltransferase). A third source involves the release of adenosine from neuronal and glial cells (Latini and Pedata, 2001), possibly by reversal of the transporters normally responsible for adenosine uptake. Adenosine in the CNS generally exerts a tonic inhibitory effect on neural excitability (Prince and Stevens, 1992) primarily via A₁ and A_2AARs. The concentration of adenosine and location of production are probably critical determining factors in the activation of AR subtypes. At low concentrations (nanomolar range), adenosine activates A₁AR and A_2AAR; at higher concentrations (micromolar range), adenosine activates A_2B and A₃AR (Peakman and Hill, 1994). Therefore, conditions that cause pronounced release of ATP/adenosine may result in stimulation of SERT activity via A₃ARs.

In summary, our study provides the first evidence that A₃AR activation stimulates SERT function in the brain. Moreover, they point to a conservation of SERT regulatory pathways from mast cells to brain 5-HT neurons. As a modulator of SERT function, inspection of the A₃AR gene for polymorphic variants may reveal genetic contributions to risk for 5-HT-linked disorders and/or antidepressant response. Furthermore, our results suggest that pharmacological targeting of A₃ARs may represent a viable route for development of novel 5-HT modulatory therapeutics. Specifically, agents that block A₃ARs may be able to selectively diminish elevations in SERT activity in a region-dependent manner without affecting basal 5-HT clearance or steadystate 5-HT levels. Such agents could limit unwanted elevations in 5-HT clearance that could trigger hyposerotonergic states and, as such, could prove useful for the treatment of mood disorders.

	Acknowledgements

 
We gratefully acknowledge excellent laboratory management and general technical support from Tammy Jessen, Jane Wright, Angela Steele, and Qiao Han. We thank Dr. Marlene Jacobson (Merck) for provision of A₃AR knockout mice and Wolfgang Dostmann (University of Vermont) for provision of DT-2 and W45. We also thank Raymond Johnson for help in HPLC test.

	Footnotes

 
This work was supported by the National Institutes of Health through Award DAO7390 (to R.D.B). and a National Alliance for Research on Schizophrenia and Depression Young Investigator Award (to C.-B.Z.).

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.107.121665.

ABBREVIATIONS: 5-HT, 5-hydroxytryptamine; A₃AR, A₃ adenosine receptor; PKG, protein kinase G; MAPK, mitogen-activated protein kinase; SERT, serotonin transporter; NET, norepinephrine transporter; RBL-2H3, rat basophilic leukemia 2H3; IB-MECA, N⁶-(3-iodobenzyl)-N-methyl-5'carbamoyladenosine; NECA, 5'-N-ethyl-carboxamidoadenosine; H8, N-[2-(methylamino)ethy]-5-isoquinoline-sulfonamide; R-PIA, (R)-N⁶-phenylisopropyladenosine; [³H]5-HT, 5-hydroxy[³H]tryptamine trifluoroacetate; MRS1191, 3-ethyl-5-benzyl-2-methyl-phenylethynyl-6-phenyl-1,4(±)dihydropyridine-3,5-dicarboxylate; [³H]DA, dihydroxyphenylethylamine, 3,4-[7-3H]; [³H]GABA, -[2,3-3H(N)]-aminobutyric acid; CHO, Chinese hamster ovary; CNS, central nervous system; 5-HIAAA, 5-hydroxyindoleacetic acid; HPLC, high-performance liquid chromatography; ANOVA, analysis of variance; DT-2, YGRKKRRQRRRPP-LRK₅H; SB203580, 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole; GBR 12935, 1-[2-(diphenylmethoxy)ethyl]-4-(3-phenylpropyl)piperazine dihydrochloride; W45, LRK₅H; SB202474, 4-(ethyl)-2-(4-methoxyphenyl)-5-(4-pyridyl)1H-imidazole.

Address correspondence to: Dr. Randy D. Blakely, Suite 7140, MRBIII, Center for Molecular Neuroscience, Vanderbilt University Medical Center, Nashville, TN 37232-8548. E-mail: randy.blakely{at}vanderbilt.edu

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