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NEUROPHARMACOLOGY

Calcium-Dependent Inhibition of Synaptosomal Serotonin Transport by the ₂-Adrenoceptor Agonist 5-Bromo-N-[4,5-dihydro-1H-imidazol-2-yl]-6-quinoxalinamine (UK14304)

Department of Pharmacology, Meharry Medical College, Nashville, Tennessee (T.A.A); Department of Physiology and Neuroscience, Medical University of South Carolina, Charleston, South Carolina (S.R.); Department of Psychiatry, University of Texas Health Science Center at San Antonio, San Antonio, Texas (S.M., L.C.D.); and Department of Pharmacology and Center for Molecular Neuroscience, Vanderbilt University School of Medicine, Nashville, Tennessee (R.D.B.)

Received for publication November 21, 2002
Accepted February 21, 2003.

	Abstract

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Termination of serotonergic transmission is the function of the plasma membrane 5-hydroxytryptamine (serotonin, 5-HT) transporter (SERT), which is also a high-affinity target in vivo for antidepressants, amphetamines, and cocaine. Studies show that SERT is regulated by protein kinase- and phosphataselinked pathways. In contrast, receptor-linked modulation of SERT is only minimally defined. Because noradrenergic stimulation is reported to influence 5-HT release, we explored possible presynaptic adrenoceptor-mediated regulation of SERT. In mouse forebrain synaptosomes, ₂-adrenoceptor agonists, particularly 5-bromo-N-[4,5-dihydro-1H-imidazol-2-yl]-6-quinoxalinamine (UK14304), triggered a concentration- and time-dependent decrease in 5-HT transport. In contrast, 5-HT uptake was unaffected by pharmacological ₁-adrenoceptor activation. Kinetically, UK14304 significantly decreased the apparent substrate affinity, K_m without altering transport capacity, V_max. At concentrations of UK14304 supporting maximal inhibition of SERT in synaptosomes, no effect on SERT in transfected cells was observed, suggesting that UK14304 acts indirectly to reduce SERT activity. The effect of UK14304 on 5-HT uptake was not shared by other Na⁺ and Cl^—-dependent transporters. UK14304-mediated inhibition of SERT function was yohimbine-sensitive, as was inhibition triggered by norepinephrine, and was abolished in the absence of added Ca²⁺. Moreover, UK14304 effects were attenuated by voltage-sensitive Ca²⁺ channel antagonists, consistent with a role for Ca²⁺ in UK14304 effects. In agreement with altered 5-HT transport activity in vitro, in vivo chronoamperometry studies revealed that UK14304 significantly prolonged 5-HT clearance. Our findings suggest that UK14304 modulates SERT function in vitro and in vivo via signaling pathways, possibly supported by an influx of Ca²⁺ through voltage-sensitive Ca²⁺ channels.

In the brain, clearance of released 5-HT in the synaptic cleft is mediated by the high-affinity 5-HT transporter (SERT) (Barker and Blakely, 1995). SERTs are also the primary target for antidepressant drugs, the serotonin-selective reuptake inhibitors as well as drugs of abuse such as 3,4-methylenedioxymethamphetamine (ecstasy), amphetamine, and cocaine (Rudnick and Wall, 1992; Barker and Blakely, 1995; Tatsumi et al., 1997). SERTs have been cloned from multiple species (Blakely et al., 1991; Hoffman et al., 1991; Ramamoorthy et al., 1993) and found to belong to the Na⁺/Cl^—-dependent transporter gene family (SLC6A) comprised of the transporters for -aminobutyric acid (GABA transporter 1), dopamine (DAT), norepinephrine (NET), glycine, taurine, proline, betaine, and creatine (Nelson, 1998). An emerging attribute among many of these transporters is acute regulation via activation of protein kinase C (Vaughan et al., 1997; Apparsundaram et al., 1998; Ramamoorthy et al., 1998; Beckman et al., 1999). Ca²⁺/calmodulin-dependent pathways have also been implicated in SERT regulation. Thus, studies have revealed that intracellular Ca²⁺ depletion (Yura et al., 1996), treatment with calmodulin antagonists (Jayanthi et al., 1994) or calmodulin kinase inhibitors decrease transport activity in native preparations (Yura et al., 1996) and cells in culture (Jayanthi et al., 1994). These pathways may also be modulated by direct or indirect effects of protein phosphatases, such as PP2A, whose catalytic subunit has been found in SERT complexes in transfected cells and native preparations (Bauman et al., 2000).

Whereas signaling pathways that modulate transporter trafficking or intrinsic activity are under active investigation, the endogenous trigger mechanisms responsible for transporter regulation are poorly understood. In non-neuronal RBL2H3 cells, 5-HT transport can be increased by adenosine receptor activation (Miller and Hoffman, 1994) and in platelets by histamine receptor activation (Launay et al., 1994). Using in vivo amperometric techniques, Daws et al. (2000) provided evidence that SERT activity in rat hippocampus can be enhanced by presynaptic 5-HT_1B receptor activation. Likewise, endogenous DAT activity has been reported to be enhanced by presynaptic dopamine D₂ receptor activation (Wu et al., 2002). Moreover, there is clear evidence that activation of protein kinase C-linked receptors (e.g., muscarinic receptors) down-regulate the functional capacity of NET (Apparsundaram et al., 1998) and GABA transporter 1 (Beckman et al., 1999), although whether these findings extend to SERT is unclear.

The current study was undertaken as part of an effort to explore the role of presynaptic receptor-mediated regulation of SERT in native preparations. Norepinephrine modulates many aspects of serotonergic transmission. For example, presynaptic ₂-adrenoceptor activation not only regulates 5-HT release (Maura et al., 1992; Gobbi et al., 1993a; Scheibner et al., 2001) but also 5-HT biosynthesis (Yoshioka et al., 1992). ₂-Adrenoceptor sensitivity is reported to be increased in the frontal cortex of suicide victims with mood disorders (Gonzalez-Maeso et al., 2002), a clinical condition that is associated with altered SERT function (Malison et al., 1998). The intimate connection between NE and 5-HT systems led us to investigate the effects of ₂-adrenoceptor agonists on 5-HT transport in brain synaptosomes in vitro and in the rat CNS in vivo by chronoamperometry. We report that acute treatment of synaptosomes with the ₂-adrenoceptor agonists UK14304, oxymetazoline, dexmedetomidine, and norepinephrine specifically down-regulates SERT activity. We also describe a dependence of UK14304 effects on external calcium, suggesting modulation of pathways supported by presynaptic calcium channels. The inhibition of 5-HT transport capacity was recapitulated through in vivo studies where we found UK14304 to significantly prolong 5-HT clearance.

	Materials and Methods

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Materials. 5-Bromo-N-[4,5-dihydro-1H-imidazol-2-yl]-6-quinoxalinamine (UK14304), oxymetazoline, L-norepinephrine, pargyline, polyethylenimine, verapamil, nifedipine, imipramine, ascorbic acid, yohimbine, and nicardipine were obtained from Sigma-Aldrich (St. Louis, MO). BAPTA-AM was bought from Calbiochem (San Diego, CA). S-(+)-Fenfluramine was purchased from Sigma/RBI (Natick, MA). Paroxetine was a gift from GlaxoSmithKline (Welwyn Garden City, Hertfordshire, UK). [³H]5-HT (100 Ci/mmol), [³H]dopamine (52 Ci/mmol), [³H]glycine (45 Ci/mmol), and [³H]glutamate (49 Ci/mmol) were purchased from Amersham Biosciences, Inc. (Piscataway, NJ). [³H]Citalopram (82 Ci/mmol) and [³H]-aminobutyric acid (70 Ci/mmol) were obtained from PerkinElmer Life Sciences (Boston, MA). All other reagents were of analytical purity and were obtained from standard sources.

Animals. Experimentally naive male mice (C57BL/6) weighing 20 to 30 g were obtained from Harlan (Indianapolis, IN). For in vivo chronoamperometry studies, male Sprague-Dawley rats (Harlan), weighing 250 to 350 g, were used. Animals were housed in groups of four (mice) or three (rats) per cage and maintained in a temperature-controlled room with a 12-h light/dark cycle (lights on at 7:00 AM) and allowed free access to food and water. All animal procedures were approved by the local institutional animal care and use committee and were in strict accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All efforts were made to minimize both the number of animals used and stress or discomfort to the animals during experimental procedures.

Preparation of Synaptosomes. Mice were sacrificed, and the forebrain was dissected out on ice and homogenized gently in 10 volumes (w/v) of ice-cold 0.32 M sucrose with a Teflon-glass homogenizer (Wheaton Science Products, Millville, NJ). After centrifugation of the homogenate at 1,000g for 10 min at 4°C, the supernatant was again centrifuged at 12,500g for 20 min. The final pellet was gently resuspended in 10 volumes of 0.32 M sucrose and used as a crude synaptosomal fraction (Yura et al., 1996). Protein was assayed using the Bradford method (Bio-Rad, Hercules, CA) with bovine serum albumin as the standard.

Synaptosomal 5-HT Uptake Assay. Aliquots (0.1 ml) of synaptosomal preparations (1 mg/ml) were preincubated at 37°C in a total volume of 0.5 ml of Krebs-Ringer bicarbonate (KRB) medium containing 118 mM NaCl, 4.8 mM KCl, 25 mM NaHCO₃, 1 mM NaH₂PO₄, 1.3 mM CaCl₂, 1.4 mM MgSO₄, 10 mM glucose, 0.1 mM pargyline, and 0.1 mM ascorbic acid, pH 7.4 (saturated with 95% O₂, 5% CO₂ for at least 30 min before use) and in the presence of modulating agents or appropriate vehicle as described in figure legends. After 10 min of preincubation, 5-HT transport assays (5 min at 37°C) were initiated by the addition of [³H]5-HT (100 Ci/mmol). In some experiments, synaptosomes were preincubated in Ca²⁺-free buffer at 37°C for 15 min. Modulators were added during the final 5 min of the preincubation period. The assays were terminated by immediate filtration over 0.3% polyethylenimine-coated glass fiber filters (Whatman GF/B; Whatman, Maidstone, UK) using a cell harvester (Brandel Inc., Gaithersburg, MD). The filters were washed three times with 1.5 ml of ice-cold KRB containing 1 mM imipramine and were incubated overnight in Ecoscint H (National Diagnostics, Atlanta, GA). Radioactivity bound to filters was counted using Beckman LS 6000 liquid scintillation counter. Nonspecific [³H]5-HT uptake, defined as the accumulation in the presence of 100 nM paroxetine was subtracted from total uptake to define SERT specific accumulation. All transport assays were performed in triplicate, and the mean values for specific uptake ± S.E.M. of at least seven separate experiments were determined. IC₅₀ values were derived using a nonlinear least-squares curve fit and nonlinear curve fits of saturation data used the Michaelis-Menten model V = V_max[S]ⁿ/[S]ⁿ + [K]ⁿ (Kaleidagraph; Synergy Software, Reading, PA).

Radioligand Binding Assay. To evaluate whether the inhibition of SERT function by UK14304 reflects direct effects of UK14304 on SERT, radioligand binding experiments were performed on membranes prepared from synaptosomes. Synaptosomes were resuspended in lysis buffer consisting of 10 mM Tris and 20 mM NaCl, pH 8.0, and kept on ice for 30 min. After passage (8–10 times) through a syringe with 27-gauge needle, the membrane preparation was centrifuged at 20,000g for 30 min. The pellet was resuspended in binding buffer consisting of 50 mM Tris and 100 mM NaCl, pH 8.0. The protein content of the membrane suspension was determined by the Bradford method (Bio-Rad). Binding assay was performed in triplicate with binding buffer, 0.1 mM ascorbic acid, 50 µg of protein, 5 nM [³H]citalopram for 1 h at 25°C. Nonspecific binding was determined in parallel incubations of membranes with 100 nM paroxetine. Membranes were collected on Brandel GF/B glass fiber filters, presoaked in 0.3% polyethylenimine, using a Brandel harvester. Filters were incubated overnight in Ecoscint H and emission from bound label determined in a liquid scintillation counter. The mean values for specific binding ± S.E.M. of at least seven separate experiments were determined. IC₅₀ values were derived using a nonlinear least-squares curve fit (Kaleidagraph; Synergy Software).

5-HT Release from Brain Slices. Animals were sacrificed, and the forebrain was removed and placed in ice-cold KRB medium containing 118 mM NaCl, 4.8 mM KCl, 25 mM NaHCO₃, 1 mM NaH₂PO₄, 1.3 mM CaCl₂, 1.4 mM MgSO₄, 10 mM glucose, 0.1 mM pargyline, and 0.1 mM ascorbic acid, pH 7.4 (saturated with 95% O₂, 5% CO₂ for at least 30 min before use). Slices (300 µm in thickness) were made using a McIlwain tissue chopper. To ascertain the effects of UK14304 on transporter-mediated 5-HT efflux, release experiments were performed as described by Reimann and Schneider (1998). Briefly, slices were preincubated with 50 nM [³H]5-HT at 37°C for 30 min and then transferred to glass superfusion chambers in a 2500 superfusion apparatus (Brandel Inc.) and superfused with KRB medium at a rate of 0.6 ml/min for 105 min at 37°C. Fractions of the superfusate were collected for consecutive periods of 5 min, starting 45 min after onset of superfusion. Paroxetine or UK14304 was added to the superfusion medium at 50 min and fenfluramine at 55 min after onset of superfusion. At the end of the experiment, the slices were solubilized in 1% SDS and the radioactivity in superfusates and slices was measured by liquid scintillation spectrometry. Release experiments were the mean values ± S.E.M. of five separate experiments.

Cell Culture. To determine whether the actions of UK14304 were a direct effect of UK14304 on SERT, heterologous expression of mouse SERT cDNA was used. Cells maintained in monolayer culture at 37°C, 5% CO₂ were plated at a density of 500,000 cells/well in six-well culture dishes as described previously (Ramamoorthy et al., 1998). Mouse SERT pcDNA3 (Chang et al., 1996) was introduced into HEK-293 or COS-7 cells (American Type Culture Collection, Manassas, VA) using FuGENE reagent (Roche Diagnostics, Indianapolis, IN) as described by the manufacturers for transient transfection studies.

5-HT Uptake in Cell Culture. Sixteen hours after transfections, cells were assayed for [³H]5-HT transport as described previously (Ramamoorthy et al., 1998). Briefly, cells were washed in assay buffer (130 mM NaCl, 1.3 mM KCl, 2.2 mM CaCl₂, 1.2 mM MgSO₄, 1.2 mM KH₂PO₄, and 10 mM HEPES, pH 7.4), followed by incubation in 37°C assay buffer containing 1.8 g/l glucose, 100 µM pargyline, and 100 µM ascorbic acid for 10 min with or without agonists. 5-HT uptake was initiated by the addition of [³H]5-HT (20 nM final concentration). The reaction (10 min at 37°C) was terminated by washing three times with ice-cold assay buffer. The cells were dissolved in OptiPhase scintillation fluid (PerkinElmer Wallac, Gaithersburg, MD) and [³H]5-HT accumulated determined by liquid scintillation spectrometry. Specific 5-HT uptake was determined by subtracting the amount accumulated in the presence of 100 nM paroxetine. All transport assays were performed in triplicate, and the mean values for specific uptake ± S.E.M. of eight separate experiments were determined.

Chronoamperometric Evaluation of 5-HT Clearance. To evaluate modulation of SERT function in vivo, high-speed chronoamperometric recordings were made using the FAST-12 system (Quanteon, Lexington, KY). Electrode preparation and in vitro calibration were carried out as previously described (Daws et al., 2000). Briefly, carbon fiber electrodes (30-µm tip diameter; Quanteon, Lexington, KY) were coated with Nafion (5% solution; Aldrich Chemical Co, Milwaukee, WI), to prevent interference from anionic substances in extracellular fluid. Only electrodes displaying a selectivity ratio for 5-HT over 5-hydroxyindoleacetic acid greater than 500:1 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 was defined as the concentration that produced a response with a signal-to-noise ratio of 3, and in these experiments averaged 48 ± 11 nM (n = 12). None of the drugs elicited an electrochemical signal themselves. Moreover, the drugs did not affect the signal produced by 5-HT in vitro, indicating that it is unlikely they change the surface chemistry of the electrode and hence its sensitivity to 5-HT. As described in Daws et al. (2000), rats were anesthetized by i.p. injection of chloralose (85 mg/kg) and urethane (850 mg/kg), a tube inserted into the trachea to facilitate breathing and placed into a stereotaxic frame. Body temperature was maintained at 37 ± 1°C by a water-circulated heating pad (Seabrook, Cincinnati, OH). The electrode micropipette recording assembly was lowered into the parietal cortex (AP, +1.2; ML, +4.6; DV, —3.0 to —3.3 from surface of brain) (Paxinos and Watson, 1986). This brain region was selected because it is an area that is relatively densely populated by both the SERT and ₂-adrenoceptors (Hensler et al., 1994; Winzer-Serhan and Leslie, 1999). The electrochemical recording assembly consisted of a Nafion-coated, single carbon fiber electrode attached to a four-barreled micropipette. The assembly was constructed such that the electrode and micropipette tips were separated by 250 to 350 µm. The tip diameter of each barrel of the multibarreled micropipette was between 10 and 15 µm. Barrels were filled with either 5-HT (200 µM), UK14304 (400 µM), yohimbine (400 µM), or vehicle. Serotonin and yohimbine were dissolved in 0.1 M phosphate-buffered saline (PBS) with 100 µM ascorbic acid added as an antioxidant. UK14304 was dissolved in 1.11% DMSO. The pH of all solutions was 7.2 to 7.4. At the conclusion of each experiment, an electrolytic lesion was made to mark the placement of the electrode tip and the histological localization of the electrode was verified. Only data from rats where the electrode tip was confirmed to be in the parietal cortex were included for data analysis. Chronoamperometry data were analyzed using three signal parameters: 1) the maximal amplitude of the signals resulting from local application of 5-HT; 2) t₈₀, the time (in seconds) for the signal to decline by 80% of the maximal amplitude; and 3) t_c, the slope of the decay curve from 20 to 60% of maximal signal amplitude, i.e., the most linear portion of the decay. This is used as an index of the clearance rate. Only oxidation currents were used for data analyses.

Statistics. The data presented represent means ± S.E.M. The percentage of change from vehicle control values was analyzed using nonparametric one-way ANOVA (Kruskal-Wallis) followed by Dunn's post-test. For comparisons between two treatment groups Mann-Whitney U tests were used. Where appropriate, statistical analysis between two groups was performed using the two-tailed, unpaired Student's t test. Analysis among multiple groups were conducted using ANOVA followed by Tukey's test. Amplitude and time course data from chronoamperometry studies were analyzed with paired, two-tailed t tests (pre- versus post-application of drug). The percentage of change from predrug value for these parameters was analyzed by Mann-Whitney U tests. A two-tailed probability level of p < 0.05 was accepted as statistically significant for all tests.

	Results

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UK14304 Inhibits SERT Activity. To explore whether ₂-adrenoceptor activation modulates SERT activity, [³H]5-HT uptake was assayed in mouse forebrain synaptosomes pretreated for 10 min with the specific ₂-adrenoceptor agonist UK14304. Figure 1A shows that UK14304 inhibited synaptosomal 5-HT transport activity in a concentration-dependent manner. At a concentration of 1 µM UK14304, there was a 20% decrease in transport activity. The inhibition increased to 70% at 5 µM and 85% at 10 µM. The IC₅₀ value for UK14304 inhibition of [³H]5-HT uptake was estimated at 3 µM. Oxymetazoline and dexmedetomidine, also known to act as agonists at ₂-adrenoceptors, could also be shown to inhibit 5-HT uptake, although less potently. Thus, a 30% reduction in transport activity was observed at 10 µM of either agent (Fig. 1A). In contrast, the ₁-adrenoceptor agonist phenylephrine was ineffective in this preparation (Fig. 1A). UK14304-mediated effects were sensitive to the ₂-adrenoceptor antagonist yohimbine (Fig. 1B) and support a receptor-mediated regulation. The experimental design for yohimbine preincubation involved longer overall incubation periods, which we observed decreased UK14304 effects (Fig. 1B). Nonetheless, a rightward shift in the UK14304 concentration-response curve was obtained. It is worth noting that pretreatment of synaptosomes in the presence of 10 µM yohimbine alone modestly but consistently augmented (10–15%) basal SERT activity (Fig. 3), suggesting the existence of possible endogenous ₂-adrenoceptor-mediated tonic inhibition of SERT. Further characterization revealed that the effects of UK14304 (10 µM) were rapid, reaching maximum effects in 5 min (Fig. 1C). Kinetically, UK14304 significantly decreased the apparent substrate affinity, K_m (control = 35.4 ± 8.4 nM; UK14304 = 134.4 ± 11.7 nM; p < 0.05, Student's t test) without altering transport capacity, V_max (control = 1554 ± 56 fmol/mg/min; UK14304 = 1495 ± 29 fmol/mg/min) (Fig. 1D). There was a fair amount of variability in the basal SERT activity, ranging from 350 to 650 fmol/mg of protein/min. This variability impacted the magnitude of the effects of UK14304. Greater effects of UK14304 were observed when basal activity was high. Moreover, we observed decreasing basal SERT activity with time of incubation. Although this reduction occurs in the absence of exogenous agonist, it may represent time-dependent release of endogenous norepinephrine, which we readily detected in our preparation (data not shown). However, the possibility of alteration in the integrity of the preparation cannot be ruled out.

View larger version (28K): [in this window] [in a new window]   Fig. 1. 2-Adrenoceptor agonists decrease 5-HT transport in synaptosomes. A, synaptosomes were preincubated with agonists or vehicle at 37°C for 10 min. 5-HT uptake was initiated by the addition of 20 nM [3H]5-HT. UK14304 was made up as 10 mM stock solution in DMSO. The final concentration of DMSO was 0.5%. 5-HT transport was carried out for 5 min, as described under Materials and Methods. To define specific 5-HT uptake via SERT, parallel uptake assays were performed in the presence of 100 nM paroxetine and subtracted from total accumulation. B, antagonism of the effects of UK14304 by yohimbine. Synaptosomes were pretreated with 10 µM yohimbine for 15 min. UK14304 (0.01–20 µM) was added during the last 10 min of the preincubation period. C, time-dependent inhibition of uptake by 10 µM UK14304. D, kinetics of [3H]5-HT uptake in the absence and in the presence of 10 µM UK14304. Data represent mean ± S.E.M. of determinations from eight independent experiments carried out in triplicate. Asterisks indicate significant changes in [3H]5-HT uptake compared with vehicle controls (Kruskal-Wallis followed by Dunn's test, p < 0.05).

View larger version (28K): [in this window] [in a new window]   Fig. 3. Norepinephrine decreases 5-HT transport in synaptosomes. Synaptosomes were preincubated with norepinephrine or vehicle at 37°C for 15 min in the presence of 1 µM desipramine. In parallel experiments, synaptosomes were pretreated with 2 µM yohimbine for 15 min. 5-HT uptake was initiated by the addition of 20 nM [3H]5-HT. Nonspecific uptake was defined by the presence of 100 nM paroxetine. Data represent mean ± S.E.M. of determinations from six independent experiments carried out in triplicate. Asterisks indicate significant changes in [3H]5-HT uptake compared with activity in the presence of respective norepinephrine concentrations. Number indicates significant change compared with vehicle control (Kruskal-Wallis followed by Dunn's test, p < 0.05).

Certainly, nonspecific effects of drugs or alterations in ion gradients could explain the inhibitory actions of UK14304 in synaptosomes. To investigate these possibilities, we ascertained the effect of UK14304 in transfected HEK-293 and COS-7 cells transiently expressing mSERT. At concentrations of UK14304 (10 µM) supporting nearly full inhibition of SERT in synaptosomes, no effect on SERT in transfected cells was observed (Fig. 2A). However, at 1 mM UK14304 there was a 35% decrease in [³H]5-HT uptake observed in transfected cells (data not shown). The differences in the inhibitory concentrations of UK14304 in synaptosomes and transfected cells expressing mSERT suggest that UK14304 does not act to reduce SERT in synaptosomes via a direct interaction with transporter protein. To explore this issue further, we determined the IC₅₀ of UK14304 for inhibition of [³H]citalopram binding in synaptosome plasma membranes. Although UK14304 inhibits [³H]citalopram binding, the IC₅₀ (115.8 µM) is about 40-fold greater than that found for [³H]5-HT uptake (2.5 µM) in synaptosomes assayed in parallel (Fig. 2B). To assess the selectivity of UK14304 effects, we also determined the impact of UK14304 on transport capacity of other members of the Na⁺/Cl^—-dependent transporter gene family (Nelson, 1998), which includes the closely related DAT and NET. The accumulation of [³H]dopamine, glycine, GABA, and glutamate were not inhibited by UK14304 (Fig. 2C). In fact, [³H]GABA uptake was stimulated. These data show that the sensitivity of SERT to UK14304 is not shared by other transporter systems and thus is unlikely to arise from modulation of Na⁺ or Cl^— gradients that support transport more generally (Fig. 2C).

View larger version (24K): [in this window] [in a new window]   Fig. 2. Specificity of UK14304-mediated effects. The specificity of UK14304 effects was assessed by determining its activity in synaptosomes, heterologous expression systems, and in radioligand binding assays. A, differential effects of UK14304 on [3H]5-HT uptake in synaptosomes, transiently transfected COS-7 cells, and HEK-293 cells expressing mSERT. Cells or synaptosomes were preincubated with UK14304 or vehicle at 37°C for 10 min. Assay of 5-HT transport was as described under Materials and Methods. Nonspecific uptake was defined as the uptake in the presence of 100 nM paroxetine and subtracted from the total accumulation to yield specific uptake. B, dissociation between inhibitory concentrations of UK14304 in synaptosomal [3H]5-HT uptake and [3H]citalopram (5 nM) binding in membranes prepared from synaptosomes. Data are expressed as percentage of control values obtained from parallel experiments in the presence of vehicle. Synaptosomes or membrane preparations were preincubated with UK14304 or vehicle for 10 min. UK14304 was present throughout the synaptosomal transport (37°C for 5 min) and radioligand binding (25°C for 1 h) assays. Nonspecific uptake or binding was defined as the activity in the presence of 100 nM paroxetine and subtracted from total activity. C, sensitivity of SERT to UK14304 is not shared by other transporter systems. [3H]5-HT (20 nM), dopamine (20 nM), glycine (100 nM), GABA (200 nM), and glutamate (100 nM) uptake into synaptosomes were carried out in parallel at 37°C for 5 min. Nonspecific uptake was defined by the presence of 100 nM paroxetine (5-HT), 30 nM GBR12909 (DA), or the absence of Na+ in the incubation buffer (glycine, GABA, glutamate). Data represent mean ± S.E.M. of determinations from nine independent experiments carried out in triplicate. Asterisks indicate significant changes in [3H]5-HT uptake compared with vehicle controls (Kruskal-Wallis followed by Dunn's test, p < 0.05).

We examined the effects of the endogenous ligand for ₂-adrenoceptors, norepinephrine to ascertain the physiological relevance of the observed presynaptic modulation of SERT activity by UK14304. The assays were performed in the presence of the NET inhibitor desipramine (1 µM) to prevent alterations in norepinephrine concentrations due to neuronal uptake. As observed with UK14304, norepinephrine produced a concentration-dependent decrease (ANOVA, p < 0.05) in 5-HT uptake in synaptosomes (Fig. 3). Moreover, pretreatment of synaptosomes with 2 µM yohimbine blocked the effects of norepinephrine, suggesting the involvement of adrenergic receptor mechanisms (Fig. 3).

To further probe the specificity of the actions of UK14304, we used another functional assay of SERT to test the effects of UK14304. Fenfluramine acts as a substrate for SERT and leads to 5-HT release by a process of transporter-mediated efflux (Rudnick and Wall, 1992). In these experiments, slices were preincubated with [³H]5-HT and transferred to superfusion chambers (for 55 min) before exposure to label-free medium containing 1 µM fenfluramine. The fenfluramineevoked outflow of tritium was paroxetine-sensitive, supporting a transporter-mediated efflux of 5-HT (Fig. 4A). In the presence of 10 µM UK14304, 5-HT efflux was decreased by 30%, whereas 10 µM yohimbine plus fenfluramine returned 5-HT efflux to control levels (Fig. 4B). Together, with our synaptosomal studies, these results support the existence of a yohimbine-sensitive modulatory process triggered by UK14304 on SERT activity in vitro.

View larger version (37K): [in this window] [in a new window]   Fig. 4. Transporter-mediated 5-HT release is altered by UK14304. A, fenfluramine-evoked [3H]5-HT efflux is paroxetine-sensitive. Slices preloaded with 50 nM [3H]5-HT were transferred to glass perfusion chambers and superfused with KRB medium at a rate of 0.6 ml/min for 105 min at 37°C. Fractions of the superfusate were collected for consecutive periods of 5 min, starting 45 min after onset of superfusion. Paroxetine (1 µM) was added to the superfusion medium at 50 min and fenfluramine (1 µM) at 55 min after onset of superfusion. Superfusion time denotes time after start of sample collection. Thus, data shown are fractional release of 5-HT between 50 and 95 min of superfusion. B, yohimbine attenuates UK14304-mediated inhibition of 5-HT efflux. UK14304 (10 µM) and yohimbine (10 µM) were added to the perfusion medium at 50 min and fenfluramine (1 µM) at 55 min after onset of superfusion. Data shown are fractional release of 5-HT at 50 and 95 min of superfusion. Data represent mean ± S.E.M. of determinations from five independent experiments. Asterisk indicates significant changes in [3H]5-HT efflux compared with vehicle controls, number symbol indicates significant changes in [3H]5-HT efflux compared with fenfluramine (unpaired Student's t test, p < 0.05).

UK14304 Prolongs Clearance of Exogenously Applied Serotonin. To demonstrate that the effects of UK14304 in synaptosomes are not due to artifact of tissue preparation, we assessed SERT function in vivo by measuring the clearance of 5-HT in rat parietal cortex. Serotonin (5 pmol) was pressure ejected into the parietal cortex until reproducible amperometric signals were obtained. The oxidation current, converted to micromolar levels using a calibration factor determined in vitro, is shown in Fig. 5A. Once a reproducible signal was obtained, two consecutive applications of 5-HT were followed by pressure ejection of drug or vehicle 60 to 90 s before the next application of 5-HT. It is important to note that none of the drugs or vehicles altered the rise time of the signal produced by 5-HT (the time from pressure ejection of 5-HT to its peak signal amplitude), indicating that they did not alter diffusion characteristics or surface chemistry of the electrode. In contrast, when UK14304 (80 ± 7 pmol) was pressure ejected into the parietal cortex, the time course of the subsequent 5-HT signal was prolonged and the clearance rate of 5-HT decreased (Fig. 5 and Table 1). These effects were maximal by 2 to 5 min post-drug, and 5-HT signal parameters returned to predrug values typically within 30 min after application of UK 14304. Signal amplitude was unaltered by UK14304. In contrast, pressure-ejection of an equivalent amount of yohimbine or vehicle (either PBS or 1.11% DMSO) had no effect on the electrochemical signals produced by local application of 5-HT into the parietal cortex (Fig. 5B; Table 1).

View larger version (29K): [in this window] [in a new window]   Fig. 5. Effect of UK 14304 and yohimbine on clearance of exogenously applied 5-HT in the parietal cortex. A, representative tracings of the effect of locally applied UK14304 (52 pmol in 130 nl) on the oxidation current produced by pressure ejection of 5-HT (5 pmol in 25 nl) into the parietal cortex of the anesthetized rat. Serotonin was pressure ejected at 5-min intervals until a reproducible response was obtained. This control response is denoted as "baseline". The overlying signal depicts the oxidation current of 5-HT 2 min after drug administration. B, summary of the effect of locally applied UK14304, yohimbine or vehicle on amplitude, t80, and clearance parameters of the 5-HT signal. There were no significant differences between PBS and DMSO control groups, so these data were pooled and are referred to above as vehicle. Data are mean ± S.E.M. values, expressed as a percentage of change from the baseline value. Asterisk indicates significant changes from vehicle and yohimbine treatments. (Mann-Whitney U test, p < 0.05).

Calcium Mediates the Effects of UK14304. ₂-Adrenoceptor activation is negatively coupled via pertussis toxin-sensitive G_i/G_o-subtype G proteins to adenylate cyclase, suppresses voltage-dependent Ca²⁺ currents, and enhances inwardly rectifying K⁺ currents (Bylund et al., 1994). Attempts were made to block the effects of UK14304 with pertussis toxin. No effects were observed after 1 h of preincubation of synaptosomes with pertussis toxin, possibly due to limited penetration of the toxin with acute application. Unfortunately, the effects of UK14304 were substantially reduced in more prolonged incubations so we did not pursue this line of investigation. We were intrigued by reports of Ca²⁺-dependent 5-HT transport that seemed to require voltage-dependent Ca²⁺ channel activation (Yura et al., 1996) and proceeded to evaluate the role of Ca²⁺ in UK14304-mediated inhibition of 5-HT uptake. Our first objective was to determine whether Ca²⁺ plays a role in the expression of basal SERT activity. Incubating synaptosomes in Ca²⁺-free buffer for 15 min resulted in a 75% reduction in 5-HT transport (Fig. 6A). Addition of the membrane-permeable Ca²⁺ chelator BAPTA-AM caused a further reduction in 5-HT uptake (Fig. 6A). Similar results were obtained with 1 mM EGTA. The decreased 5-HT uptake was not due to alterations in the integrity of the membranes because 90% of uptake activity could be recovered when Ca²⁺ was restored to the incubation medium (Fig. 6B). Notably, we were able to show that inhibition of 5-HT uptake by UK14304 was abolished in the absence of added Ca²⁺ (Fig. 6C), suggesting that Ca²⁺ is required for the actions of UK14304. The UK14304 effects shown here are lower than those in other experiments because the experimental paradigm required a 15-min preincubation period that generally decreased both basal SERT activity and UK14304 effects. Because studies have shown that UK14304 inhibits voltage-dependent Ca²⁺ channels (Chieng and Bekkers, 1999), we tested the possibility that UK14304 may be inhibiting 5-HT uptake by blocking voltage-dependent Ca²⁺ channels in synaptosomes. Consistent with external Ca²⁺ depletion effects, preincubation of synaptosomes in the presence of the L-type Ca²⁺ channel blockers verapamil, nicardipine, and nifedipine resulted in a concentration-dependent decrease in basal 5-HT transport (Fig. 7A). As with UK14304, we observed a dissociation between concentrations of verapamil inhibiting 5-HT uptake in synaptosomes (IC₅₀ = 1.89 µM) and that inhibiting [³H] citalopram binding in membranes prepared from synaptosomes (IC₅₀ = 84.3 µM) (Fig. 7B), suggesting that the effect of these agents on transport are not directly via SERT. Moreover, a combination of the Ca²⁺ channel blockers and UK14304 did not produce additive effects. Compared with vehicle control, 5-HT transport activity was 49.4 ± 0.85% in the presence of 5 µM UK14304, 76.2 ± 1.4% in the presence of 5 µM nicardipine, and 42.6 ± 1.1% when the two agents were combined. These data suggest that inhibition afforded by UK14304 and Ca²⁺ channel antagonists target a common mechanism. Indeed, as shown in Fig. 7C, when the effects of UK14304 are normalized to the effects of verapamil alone, we actually see a blunting of SERT inhibition. Together, these findings point to a UK14304-sensitive ₂-adrenoceptor modulation of SERT activity that is dependent on presynaptic Ca²⁺ influx. Due to marked decrease in activity associated with calcium-free buffer, caution in interpretation of the data is warranted. However, in view of similar blunting of UK14304 effects in the presence of multiple L-type calcium channel blockers, we feel that further consideration of calcium-dependent pathway is warranted.

View larger version (19K): [in this window] [in a new window]   Fig. 6. Ca2+ activation of 5-HT transport capacity. Nonspecific uptake was determined in parallel incubations of synaptosomes with 100 nM paroxetine. A, Ca2+ supports basal SERT activity. Ca2+ depletion decreases [3H]5-HT uptake. Synaptosomes were preincubated in Ca2+-free buffer and 50 µM BAPTA-AM for 15 min. [3H]5-HT uptake was determined as described under Materials and Methods. Asterisks indicate significant changes in [3H]5-HT uptake compared with vehicle controls (n = 5, ANOVA, followed by Tukey's test, p < 0.05). B, augmentation of [3H]5-HT uptake by Ca2+ addition. Synaptosomes were preincubated in Ca2+-free buffer for 15 min. Ca2+ (1.3 mM) was added during the last 5 min of the preincubation period. Asterisk indicates significant change in [3H]5-HT uptake compared with vehicle controls (n = 9, Mann-Whitney U test, p < 0.05). C, diminished UK14304 effects in Ca2+-free buffer. Synaptosomes were preincubated in Ca2+-free buffer for 15 min. 10 µM UK14304 was added during the last 5 min of the preincubation period. Asterisk indicates significant change in [3H] 5-HT uptake compared with vehicle controls (n = 9, Mann-Whitney U test, p < 0.05).

View larger version (24K): [in this window] [in a new window]   Fig. 7. Calcium via voltage-dependent Ca2+ channels mediate UK14304 effects. A, inhibition of voltage-sensitive Ca2+ channels decrease [3H]5-HT transport. Concentration-dependent reduction of [3H]5-HT uptake by verapamil, nicardipine, and nifedipine. Synaptosomes were pretreated with Ca2+ channel antagonists for 10 min. B, dissociation between inhibitory concentrations of verapamil in synaptosomal [3H]5-HT uptake and [3H]citalopram (5 nM) binding in membranes prepared from synaptosomes. Data are expressed as percentage of control values obtained from parallel experiments in the presence of vehicle. Synaptosomes or membrane preparations were preincubated with UK14304 or vehicle for 10 min. Verapamil was present throughout the synaptosomal transport (37°C for 5 min) and radioligand binding (25°C for 1 h) assays. Specific uptake and binding were defined by subtracting data from synaptosomes or membranes treated with 100 nM paroxetine. C, verapamil attenuates UK14304-mediated effects in a concentration-dependent manner. Synaptosomes were pretreated with verapamil for 10 min. UK14304 was added 5 min before the initiation [3H]5-HT uptake. Data are expressed as percentage of respective controls. Data represent mean ± S.E.M. of determinations from seven to nine independent experiments carried out in triplicate. Asterisks indicate significant changes in [3H]5-HT uptake compared with vehicle controls (Kruskal-Wallis followed by Dunn's test, p < 0.05).

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
Recent findings indicate that SERT is subject to rapid modulation by multiple signaling events that are triggered by, or correlated with, receptor activation (Miller and Hoffman, 1994; Launay et al., 1994; Daws et al., 2000), second messenger modulation (Jayanthi et al., 1994; Yura et al., 1996; Ramamoorthy et al., 1998), or transporter phosphorylation (Ramamoorthy et al., 1998). How these signaling events are integrated to alter SERT intrinsic activity, surface expression, or protein levels are yet to be determined. We have sought evidence for a defined neurochemical input that modulates neuronal SERT to permit a systematic evaluation of SERT regulatory mechanisms in vivo. Our studies focused on possible NE/5-HT interactions and show that ₂-adrenoceptor agonists, particularly UK14304, rapidly down-regulate functional SERT activity in synaptosomes and increase 5-HT clearance in vivo. Furthermore, UK14304-mediated modulation of SERT activity seems to involve Ca²⁺-dependent mechanisms.

The ₂-adrenoceptor agonists UK14304, oxymetazoline, and dexmedetomidine and norepinephrine concentration dependently decreased [³H]5-HT uptake. The concentrations of UK14304 are similar to those shown to inhibit KCl-evoked 5-HT release in the hippocampus (Numazawa et al., 1995) or that modulate ligand-induced Ca²⁺ mobilization in acutely dissociated locus coeruleus neurons (Chieng and Bekkers, 1999). In contrast, the ₁-adrenoceptor agonist phenylephrine at concentrations known to produce depolarization of dorsal raphe neurons in slice preparations (Pan et al., 1994) was without effect (Fig. 1A). The time frame of UK14304 effects (<5 min) is inconsistent with alterations in SERT gene expression but compatible with a regulatory pathway modulation that may involve G protein activation or second messenger-modulated pathway. The results of a number of experiments we performed lead us to believe that the effects of UK14304 are not due to nonspecific actions on the transporter. First, much higher concentrations of UK14304 are required to inhibit [³H]5-HT uptake in transfected HEK-293 and COS-7 cells transiently expressing mSERT than we observed in synaptosomal uptake assays. Second, there was approximately 40-fold difference in the concentrations of UK14304 required to inhibit [³H]citalopram binding in membrane preparations than that needed to inhibit [³H]5-HT uptake into synaptosomes. Third, alterations in ion gradients cannot be a generic contributing factor because UK14304 did not inhibit other Na⁺ and Cl^—-dependent transporters (Fig. 2C) that similarly depend on the stability of ion gradients. Thus, the effects of UK14304 seem to be selective for SERT.

It is acknowledged that our studies could benefit from additional pharmacological analysis of ₂-adrenoceptor modulation of SERT function. Our studies focused primarily on the activity of UK14304 due to its potency and efficiency in modulation of SERT. Of all the antagonists tested, we were most successful with yohimbine. Additional ₂-adrenoceptor agonists used included oxymetazoline, dexmedetomidine, the endogenous ligand norepinephrine, and clonidine (data not shown). Clonidine inhibition of SERT activity was small yet yohimbine-sensitive, but due to the fact that it has partial agonist properties, further studies were not pursued. Despite this supporting data, we find that the rank order of potencies from preliminary analysis does not seem to correlate well with that of ₂-adrenoceptor agonists at their receptors. This could be due to the preparation or alternatively, that other mechanisms may be involved.

Fenfluramine, a known substrate for SERT (Rudnick and Wall, 1992) induced the release of 5-HT in forebrain slices and can be used as a functional assay for SERT in brain slices. Consistent with the studies of Gobbi et al. (1993b), we showed that UK14304 pretreatment decreased fenfluraminetriggered 5-HT efflux. Moreover, the ₂-adrenoceptor antagonist yohimbine reversed the effect of UK14304 (Fig. 4B). In addition, the modulation of synaptosomal [³H]5-HT uptake was yohimbine-sensitive (Fig. 1B). Together, the effects of UK14304 on SERT points to a receptor-mediated response. The augmentation of basal SERT activity in the presence of yohimbine (Fig. 3) may suggest tonic inhibition of SERT by ₂-adrenoceptor agonists. Analysis of the synaptosomal preparations by high-performance liquid chromatography revealed the presence of norepinephrine (data not shown). Yohimbine could be blocking leaked or released norepinephrine. Yohimbine did not produce similar effects on its own in vivo, perhaps because leakage was less likely. The possibility of constitutive ₂-adrenoceptor activity regulating SERT function is intriguing. There is ample neuroanatomical (Baraban and Aghajanian, 1981), electrophysiological (Haddjeri et al., 1997), and functional (Maura et al., 1992; Gobbi et al., 1993a; Numazawa et al., 1995; Gobert et al., 1998) evidence that ₂-adrenergic heteroceptors are localized on serotonergic terminals and their activation leads to inhibition of 5-HT release. It has been suggested that these ₂-adrenergic heteroceptors differ pharmacologically from ₂-adrenergic autoreceptors (Maura et al., 1992). However, other studies do not support this view (Scheibner et al., 2001). Based on radioligand binding and molecular genetics, three subtypes of ₂-adrenoceptors have been distinguished as _2A, _2B, and _2C (Bylund et al., 1994). Each receptor has a distinct tissue distribution in the brain. The _2A-adrenoceptors are the predominant presynaptic receptors and are widely expressed. They are present in the cortex, locus coeruleus, amygdala, septum, hippocampus, and the hypothalamus (Wang et al., 1996). The _2C-adrenoceptors are expressed in the hippocampus, olfactory tubercle, striatum, and the cortex (Wang et al., 1996). _2B-Adrenoceptors have limited distribution and are mainly found in the thalamus (Wang et al., 1996). Clarification of receptor subtypes involved in this response may benefit from studies using ₂-adrenoceptor knockout mice (Scheibner et al., 2001).

Although, the role of specific ₂-adrenoceptor subtypes in modulating SERT activity remains to be defined, pathways involved in ₂-adrenoceptor signaling were investigated as a means of providing insights into the mechanism of UK14304-mediated inhibition of SERT activity. ₂-Adrenoceptors are known to be negatively coupled via G_i/G_o to adenylate cyclase, to inhibit voltage-sensitive Ca²⁺ channels, and to stimulate flux through K⁺ channels (Bylund et al., 1994). Reports of UK14304 inhibiting voltage-dependent Ca²⁺ channels (Chieng and Bekkers 1999) coupled with data on Ca²⁺ activation of SERT (Yura et al., 1996) encouraged us to investigate the role of Ca²⁺ in UK14304-mediated modulation of SERT activity. Our studies corroborate others (Yura et al., 1996) indicating that Ca²⁺ supports basal SERT activity. After Ca²⁺ depletion, rapid recovery of uptake when Ca²⁺ was added to the incubation medium (Fig. 6B) is consistent with previous reports (Yura et al., 1996) indicating that Ca²⁺ from a readily exchangeable pool supports basal SERT activity. There was about a 45-fold difference between the concentrations of verapamil inhibiting 5-HT uptake in synaptosomes and that inhibiting [³H]citalopram binding in membranes prepared from synaptosomes (Fig. 6B). A possible interpretation of the data is that verapamil acts via voltage-dependent Ca²⁺ channels at low concentrations but may act directly on the transporter at high concentrations (Tatsumi et al., 1997; this study). Indeed, we observed that at 1 µM, verapamil-induced inhibition of 5-HT uptake was greater in the presence of Ca²⁺ (27.3%) than in the absence of Ca²⁺ (16.2%), suggesting a Ca²⁺-dependent effect (data not shown). Our results showing that inhibition of 5-HT uptake by UK14304 was abolished in the absence of added Ca²⁺ (Fig. 6C) and that the Ca²⁺ channel blocker verapamil blunts the effects of UK14304 in a concentration-dependent manner (Fig. 7C) provide evidence that Ca²⁺ supports the effects of UK14304. Thus, by blocking Ca²⁺ channels UK14304 limits the availability of Ca²⁺ to support 5-HT transport. Downstream of Ca²⁺ influx, Ca²⁺ may be enhancing SERT function by activating calmodulin (Jayanthi et al., 1994) leading to transporter (or associated protein) phosphorylation by calmodulin-dependent protein kinases (Yura et al., 1996). Although the calmodulin antagonist W7 inhibits 5-HT transport in synaptosomes, we did not find it to blunt the effects of UK14304 (data not shown). We also observed no effect of UK14304 on SERT phosphorylation (data not shown). These findings point to other mechanisms independent of direct SERT modification by protein kinases in the regulated function of the transporter.

The inhibition of 5-HT uptake by UK14304 could be attributed to increased 5-HT release but UK14304 inhibits KClevoked 5-HT release (Numazawa et al., 1995) and fenfluramine-mediated 5-HT efflux (Fig. 4B). Additionally, in vivo chronoamperometry uses exogenously applied 5-HT in excess of what is released, yet UK14304 still decreased clearance of 5-HT (Fig. 5). However, one cannot rule out the possibility that an inhibitor of uptake is released by UK14304.

Typically, activation of ₂-adrenoceptors have been associated with inhibition of 5-HT release (Maura et al., 1992; Gobbi et al., 1993a; Numazawa et al., 1995; Gobert et al., 1998; Scheibner et al., 2001). In that context, our data showing that ₂-adrenoceptor agonists inhibit 5-HT uptake might seem counterproductive to extracellular 5-HT modulation. On the other hand, it could be speculated that the two processes may not occur simultaneously in vivo if different signaling pathways are involved. This intriguing possibility stems from recent studies by Dolmetsch et al. (2001), indicating that specific Ca²⁺-mediated responses may be determined by the mode of Ca²⁺ entry into cells. To date, the signaling pathways mediating ₂-heteroceptor-mediated inhibition of 5-HT release have not been fully delineated. Possibly, uptake inhibition occurs through a pathway that might not impinge on release. Further efforts to target the site of action of Ca²⁺ in both the release and uptake processes are warranted.

	Acknowledgements

 
We acknowledge the expert technical assistance of Qiao Han with aspects of cell culture and transport assays. Dexmedetomidine was kindly provided by Dr. Lee Limbird (Vanderbilt University). Synaptosomal norepinephrine levels were determined by high-performance liquid chromatography in the Neurochemistry Core Facility of the Center for Molecular Neuroscience (Vanderbilt University).

	Footnotes

 
This work was supported by National Institute on Drug Abuse Award DA07390 to R.D.B., National Institute of Mental Health Award MH62612 to S.R., and National Institute of Mental Health Award MH64489 and National Alliance for Research on Schizophrenia and Depression Young Investigator Award to L.C.D.

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

DOI: 10.1124/jpet.102.047134.

ABBREVIATIONS: 5-HT, 5-hydroxytryptamine (serotonin); NE, norepinephrine; CNS, central nervous system; SERT serotonin transporter; DAT, dopamine transporter; NET, norepinephrine transporter; BAPTA-AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester; KRB, Krebs-Ringer bicarbonate; HEK, human embryonic kidney; PBS, phosphate-buffered saline; DMSO, dimethyl sulfoxide; ANOVA, analysis of variance.

Address correspondence to: Dr. Twum A. Ansah, Department of Pharmacology, Meharry Medical College, 1005 D.B. Todd Blvd., Nashville, TN 37208. E-mail: tansah{at}mmc.edu

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