β-Phenylethylamine Alters Monoamine Transporter Function via Trace Amine-Associated Receptor 1: Implication for Modulatory Roles of Trace Amines in Brain

Trace amines, including β-phenylethylamine (β-PEA), tyramine, tryptamine, and octopamine, have been known to be heterogeneously distributed in mammalian brain tissues for decades, and their distribution spatially parallels the origins and terminal projection areas of the monoaminergic neurons (Durden et al., 1973; Philips et al., 1978). In monoaminergic neurotransmission, common biogenic amines, including dopamine, norepinephrine, and serotonin, are well established as neurotransmitters, which are synthesized from their precursor amino acids in neurons, stored in vesicles of neuronal terminals and released into the synaptic clefts to interact with both presynaptic and postsynaptic receptors. Although trace amines share similarities with common biogenic amines in structure, metabolism, and distribution, their roles and functional profiles in the brain are far from being as clear as those for common biogenic amines.

With high turnover rates, trace amines are dynamically regulated by enzymes and are present in the brain at generally low levels (Durden and Philips 1980; Durden and Davis, 1993). No dedicated neurons to date have been found to use any of the trace amines exclusively, and therefore, for a long period, the trace amines were thought of as byproducts or false neurotransmitters (Parker and Cubeddu, 1988; Janssen et al., 1999). However, the rate in synthesis of trace amines is equivalent to that of dopamine and norepinephrine (Durden and Philips 1980; Paterson et al., 1990), and altered levels of the trace amines are associated with various neuropsychiatric disorders (Boulton, 1980; Davis and Boulton, 1994; McClung and Hirsh, 1999; D'Andrea et al., 2003), which are hallmarked by changes in monoaminergic activity. These properties and amphetamine-like effects of the trace amines (Parker and Cubeddu, 1988; Paterson, 1993; Barroso and Rodriguez, 1996) suggest that trace amines may serve as neuromodulators in brain. Over the years, several studies have reported specific binding sites for [³H]β-PEA, [³H]tryptamine, and [³H]tyramine in brain (Hauger et al., 1982; Kellar and Cascio, 1982; Vaccari, 1986), which displayed a high correlation in their density with levels of endogenous trace amines (Karoum et al., 1981; Hauger et al., 1982).

In 2001, a family of related G protein-coupled receptors, now referred to as trace amine-associated receptors (TAARs), were identified, and the first solid evidence of TAARs in mammalian brain was reported (Borowsky et al., 2001). Among TAARs, TAAR1 has been well established to be functionally related with trace amines (Borowsky et al., 2001; Bunzow et al., 2001), and TAAR1 signaling has been demonstrated to be associated with the cAMP signaling pathway (Borowsky et al., 2001; Bunzow et al., 2001; Miller et al., 2005; Xie et al., 2007b). We previously revealed that TAAR1 is widely expressed in rhesus monkey brain, notably in monoaminergic nuclei (Miller et al., 2005; Xie et al., 2007b), and that rhesus monkey TAAR1 responds to a wide spectrum of chemicals, including trace amines (Miller et al., 2005, Xie et al., 2007b). However, the functional interaction of TAAR1 with trace amines and the role that TAAR1 plays in the brain remain unclear. Our previous data showed that TAAR1 signaling is enhanced by monoamine transporters and that TAAR1 activation can modulate DAT kinetics in vitro (Miller et al., 2005; Xie et al., 2007b; Xie and Miller, 2007). Our data also showed colocalization of TAAR1 and DAT in a subset of dopamine neurons in the rhesus monkey and mouse substantia nigra (Xie et al., 2007b; Xie and Miller, 2007). We have also observed TAAR1 expression in norepinephrine transporter (NET)-positive neurons in the locus coeruleus of rhesus monkey (unpublished data). Lindemann et al. (2008) recently reported TAAR1 expression in the dorsal raphe nucleus. These findings suggest that TAAR1 may serve as a presynaptic regulator in functional modulation of monoamine transporters. With respect to monoamine autoreceptors, it has been reported that their activation by monoamine neurotransmitters provides feedback regulation of monoamine neurotransmitter release (Hjorth et al., 2000; Garcia et al., 2004), but it is unknown whether trace amines exert any effects via monoamine autoreceptors.

In this study, we evaluated the interaction of trace amines with TAAR1 and monoamine autoreceptors and investigated whether monoamine autoreceptors influence TAAR1 signaling and monoamine transporter function in response to trace amines. We used transfected HEK293 cells and brain synaptosomes derived from nonhuman primates, wild-type mice, and TAAR1 knockout mice to investigate the effects of β-PEA activation of TAAR1 on the uptake and efflux function of DAT, NET, and serotonin transporter (SERT). The data confirm that all the trace amines tested recognize and excite TAAR1 and reveal that trace amines do not interact with monoamine autoreceptors to alter TAAR1 activity. The data also reveal that β-PEA alters monoamine transporter function via interacting with TAAR1 but not monoamine autoreceptors, which may suggest a common mechanism by which trace amines exert a modulatory role on monoamine transporters in brain.

Materials and Methods

Materials. β-Phenylethylamine, indatraline, methylphenidate, desipramine, citalopram, raclopride, SKF86466, and methiothepin were purchased from Sigma-Aldrich (St. Louis, MO). Dulbecco's modified Eagle's medium (DMEM), 100× nonessential amino acids, fetal bovine serum, 100× penicillin/streptomycin, Geneticin (G418), and trypsin/EDTA were purchased from Invitrogen (Carlsbad, CA). Dual-Luciferase Reporter Assay System kit, pGL4.73, and Passive Lysis Buffer (5×) were obtained from Promega (Madison, WI). [³H]Dopamine (60 Ci/mmol), [³H]norepinephrine (49.5 Ci/mmol), and [³H]serotonin (20.3 Ci/mmol) were purchased from PerkinElmer Life and Analytical Sciences (Boston, MA). ReadySafe scintillation cocktail was obtained from Beckman Coulter (Fullerton, CA). Rabbit anti-human trace amine receptor 1 antibody was obtained from ABR Affinity BioReagents (Golden, CO). Goat anti-rabbit IgG (H+L) was purchased from Millipore Bioscience Research Reagents (Temecula, CA). SuperSignal West Pico Chemiluminescent Substrate was from Pierce Chemical (Rockford, IL). The expression constructs of human α_2A, α_2B, 5-HT_1A and 5-HT_1B were purchased from UMR cDNA Resource Center (Rolla, MO). Rhesus monkey dopamine D2 receptor short isoform (D2s) was constructed in pcDNA3.1(+) in our laboratory.

Cell Culture and Transfection. Cells were grown in DMEM supplemented with fetal bovine serum (10%), penicillin (100 U/ml), streptomycin (100 μg/ml), and nonessential amino acids (0.1 mM) at 5% CO₂ and 37°C, and G418 was used for selection or maintenance of selection of stable cell lines. Calcium phosphate transfection was performed as described elsewhere (Xie et al., 2007a,b) to introduce the necessary receptor and transporter into different cells for assays. For cotransfection, the ratio of constructs and the amount of total DNA was held constant with pcDNA3.1(+).

Generation of Cell Lines for Assays. All cell lines in this study were generated originally from HEK293 cells (American Type Culture Collection, Manassas, VA). TAAR1 are HEK293 cells transiently transfected with rhesus monkey TAAR1, CRE-Luc, and pGL4.73; HEK are HEK293 cells transiently transfected with pcDNA 3.1, CRE-Luc, and pGL4.73; D2s-TAAR1 are stable D2s cells transiently transfected with rhesus monkey TAAR1, CRE-Luc, and pGL4.73; α_2A-TAAR1 are stable α_2A cells transiently transfected with rhesus monkey TAAR1, CRE-Luc, and pGL4.73; α_2B-TAAR1 are stable α_2B cells transiently transfected with rhesus monkey TAAR1, CRE-Luc, and pGL4.73; 5HT_1A-TAAR1 are stable 5HT_1A cells transiently transfected with rhesus monkey TAAR1, CRE-Luc, and pGL4.73; 5HT_1B-TAAR1 are stable 5HT_1B cells transiently transfected with rhesus monkey TAAR1, CRE-Luc, and pGL4.73; DAT are HEK293 cells transiently transfected with human DAT; NET are HEK293 cells transiently transfected with human NET; SERT are HEK293 cells transiently transfected with human SERT; TAAR1-DAT are stable TAAR1 cells transiently transfected with human DAT; TAAR1-NET are stable TAAR1 cells transiently transfected with human NET; TAAR1-SERT are stable TAAR1 cells transiently transfected with human SERT; D2s-DAT are stable D2s cells transiently transfected with human DAT; α_2A-NET are stable α_2A cells transiently transfected with human NET; and 5HT_1B-SERT are stable 5HT_1B cells transiently transfected with human SERT.

SDS-Polyacrylamide Gel Electrophoresis and Western Blotting. Cells were grown to more than 90% confluence and collected through treatment with ice-cold 0.05% trypsin/EDTA and centrifugation at 1000g for 5 min at 4°C. The cell pellets were rinsed once with ice-cold phosphate-buffered saline and centrifuged again. Cell lysates were prepared by the addition of ice-cold radioimmunoprecipitation assay buffer (150 mM NaCl, 1.0% Igepal CA-630, 0.5% sodium deoxycholate, 0.1% SDS, and 50 mM Tris, pH 8.0) in a ratio of 10⁷ cells/ml to break cells for 30 min at 4°C, which was supplemented with 1× protease inhibitor cocktail. The cell lysates were centrifuged at 12,000g for 10 min at 4°C, and the supernatants were analyzed for protein concentration using Quick Start Bradford Protein Assay (Bio-Rad, Hercules, CA). The supernatant was mixed with Bio-Rad Laemmli sample buffer at 1:2 (v/v), heated for 5 min at 95°C, and centrifuged at 12,000g for 5 min at 4°C. Proteins (10 μg) were separated by SDS-polyacrylamide gel electrophoresis (10% acrylamide running gel and 4% acrylamide stacking gel) and electrotranslocated onto polyvinylidene difluoride membrane (0.45 μm). The membrane was rinsed three times with TTBS (25 mM Tris, 150 mM NaCl, and 0.1% Tween 20, pH 7.5), blocked for 12 h at 4°C in TTBS with 5% nonfat dry milk, and then incubated with rabbit anti-human trace amine receptor 1 antibody at 1:1000 overnight at 4°C and goat anti-rabbit IgG (H+L) at 1:5000 for 2 h at room temperature in blocking buffer. SuperSignal West Pico chemiluminescent substrate was used to visualize the blots under a luminescent image analyzer (LAS-1000; Fujifilm, Tokyo, Japan).

Animals and Synaptosome Preparation. Monkey brains were obtained from juvenile rhesus (Macaca mulatta) and tamarin (Sanguinus Oedipus) monkeys that were sacrificed for other purposes or euthanized for necropsy, and the striatum (only putamen) and thalamus were collected. TAAR1 knockout and wild-type mouse colonies were established in New England Primate Research Center (Southborough, MA) from six pairs of heterozygous mice given to us as a gift by Lundbeck Research USA, Inc. (Paramus, NJ). The mice were sacrificed at 8 to 10 weeks old to collect the striatum and thalamus. The operations were conducted in accordance with the Animal Experimentation Protocol approved by the Harvard Medical Area Standing Committee on Animals. Fresh tissues were homogenized in 1.5-ml Eppendorf centrifuge tubes with 10 volumes of ice-cold un-buffered 0.32 M sucrose solution (pH 7.0), using a motor-driven pellet pestle purchased from Sigma-Aldrich for 30 up-and-down strokes. The homogenate was centrifuged (1000g, 10 min at 4°C) to yield crude nuclear pellet and low-speed supernatant. The low-speed supernatant fraction was carefully transferred into another fresh tube and centrifuged at 10,000g and 4°C for 20 min to yield synaptosome-containing pellet. The pellet was resuspended in an appropriate volume of ice-cold uptake buffer (a modified Krebs buffer: 25 mM HEPES, 120 mM NaCl, 5 mM KCl, 2.5 mM CaCl₂, 1.2 mM MgSO₄, 1 μM pargyline, 2 mg/ml glucose, and 0.2 mg/ml ascorbic acid, pH 7.5) for further assays.

Dual Luciferase Reporter Assay. Cells were placed in wells of 48-well plates (75,000 cells/well in 0.5 ml DMEM). At 60 to 70% cell confluence, a luciferase reporter construct CRE-Luc (cAMP-sensitive) and a reporter control construct pGL4.73 (cAMP-irresponsive) were introduced into the cells along with rhesus monkey TAAR1. After 12 h of incubation under transfection condition, the cells were exposed to vehicle or different concentrations (10^–9–10^–4 M) or 1 μM of trace amines in serum-free DMEM for 18 h. Passive lysis buffer (PLB) and luciferase assay substrate reagents were prepared according to manufacturer's protocol. Cell lysates were prepared by adding 100 μl of 1× PLB into each well to break the cells and shaking the plate on a platform at 25°C for 30 min. The lysate (20 μl) from each well was transferred into wells of opaque 96-well microplates (PerkinElmer Life and Analytical Sciences). Luciferase substrate reagents (50 μl) were injected into each well, and after a 2-s delay, luciferase levels were measured as RLUs for 12 s on a Wallac 1420 multilabel counter, Victor 3V (PerkinElmer Life and Analytical Sciences).

Binding Assays. The binding assays were performed according to procedures described elsewhere (Xie et al., 2005). In brief, stable D2s, α_2A, α_2B, 5HT_1A, and 5HT_1B cells were placed in 145-mm cell culture dishes (1.5 × 10⁵ cells/ml) and grown to approximately 90% confluence, dispersed with ice-cold trypsin/EDTA, and collected via centrifugation at 1000g for 5 min. The cell pellets were rinsed once with ice-cold phosphate-buffered saline (pH 7.4) and resuspended in serum-free DMEM at a density of 5 × 10⁶ cells/ml. The cell suspension (100 μl) was added into binding buffer (25 mM HEPES, 120 mM NaCl, 1.5 mM CaCl₂, 5 mM KCl, and 1.5 mM MgCl₂, pH 7.4) to perform competition binding assay in which whole cells were incubated at 25°C for 1 h with [³H]dopamine (20 nM), [³H]norepinephrine (10 nM), or [³H]serotonin (10 nM), along with different concentrations (10^–10∼10^–4 M) of each of the trace amines or common biogenic amines in a final volume of 500 μl. Nonspecific binding was determined in the presence of 10 μM raclopride (D2s), SKF86466 (α_2A and α_2B), or methiothepin (5-HT_1A and 5-HT_1B). The bound radiant was separated from the free by rapid filtration through a Brandel 24-well harvester (Brandel Inc., Gaithersburg, MD) using Whatman GF/C glass fiber filter paper (Whatman, Clifton, NJ), presoaked in a 0.25% solution of polyethyleneimine in water. The filter patches with trapped cells were rinsed three times with 25 mM HEPES buffer (pH 7.4, precooled to 4°C) and placed in scintillation vials with 4.0 ml of Beckman ReadySafe scintillation cocktail and counted on Beckman LS6000IC scintillation spectrophotometer for 1 min/sample.

[³H]Monoamine Uptake Assays. Cells were placed in 145-mm cell culture dishes (1.5 × 10⁵ cells/ml) and grown to 60 to 70% confluence for transfection. After transfection (incubation under transfection condition for 24 h and then in fresh growth medium for 12 h), the cells were harvested and prepared into suspension at a density of 5 × 10⁶ cells/ml. Brain synaptosomes were prepared from fresh striatal and thalamic tissues of monkey and mouse brains. Fifty microliters of the cell suspension or synaptosome preparation was added into 1.5-ml Eppendorf centrifuge tubes and pretreated with vehicle or 1 μM β-PEA in serum-free DMEM (for cells) or in uptake buffer (for synaptosomes) at 25°C for 10 min. After the drug pretreatment, the samples were centrifuged at 1000g (for cells) or at 10,000g (for synaptosomes) at 4°C for 3 min and washed twice with 1 ml of ice-cold serum-free DMEM (for cells) or ice-cold uptake buffer (for synaptosomes). [³H]Dopamine (10 nM), [³H]norepinephrine (20 nM), or [³H]serotonin (20 nM) diluted with serum-free DMEM or uptake buffer were then used to load the cells or synaptosomes at 25°C for 5 min. The samples pretreated with serum-free DMEM (for cells) or uptake buffer (for synaptosomes) only were taken as baseline (100% uptake). For time course uptake assays, synaptosome samples were exposed to [³H]dopamine (10 nM), [³H]norepinephrine (20 nM), or [³H]serotonin (20 nM) only or their mixture with 100 nM β-PEA at 25°C in uptake buffer for 1, 2, 3, 4, 5, 10, 20, and 30 min. The uptake of the synaptosomes at 30 min in [³H]dopamine, [³H]norepinephrine, or [³H]serotonin only was taken as maximal uptake (100%). Nonspecific uptake was defined in the presence of 10 μM indatraline for cells or 10 μM methylphenidate, desipramine, or citalopram for synaptosomes. Uptake reactions were terminated by addition of 1 ml of ice-cold serum-free DMEM (for cells) or uptake buffer (for synaptosomes) into the tubes and immediate centrifugation at 1000 (for cells) or 10,000g (for synaptosomes) at 4°C for 3 min. The resulted pellets were rinsed twice with 1 ml of ice-cold serum-free DMEM (for cells) or uptake buffer (for synaptosomes) through centrifugation as above. The washed pellets were incubated in 1× PLB buffer for 30 min on a shaking platform at 200 rpm and then transferred into scintillation vials containing 4 ml of Beckman ReadySafe scintillation cocktail and counted on Beckman LS6000IC scintillation spectrophotometer for 1 min/sample.

[³H]Monoamine Efflux Assays. Cells were placed in wells of 48-well plates (75,000 cells/well in 0.5 ml medium) and grown to 60 to 70% confluence for transfection (incubation under transfection condition for 24 h and then in fresh growth medium for 12 h). Brain synaptosomes were prepared from fresh striatal and thalamic tissues of monkey and mouse brains. The cells and synaptosomes were loaded with [³H]dopamine (10 nM), [³H]norepinephrine (20 nM), or [³H]serotonin (20 nM) at 25°C for 20 min in serum-free DMEM (for cells) or uptake buffer (for synaptosomes), and the assays were conducted in plate wells for cells and in 1.5-ml Eppendorf tubes for synaptosomes. Uploading of [³H]monoamine was terminated by addition of 1 ml of serum-free DMEM into the wells or uptake buffer into the tubes and immediate aspiration of the DMEM or centrifugation at 10,000g at 4°C for 3 min (for synaptosomes). Nonspecific loading was defined in the presence of 10 μM indatraline for cells or 10 μM methylphenidate, desipramine, or citalopram for synaptosomes. After preloading, the samples were washed once and exposed to vehicle, 1 μM β-PEA or 1 μM β-PEA plus 10 μM methylphenidate, desipramine, or citalopram in serum-free DMEM (for cells), or efflux buffer (25 mM HEPES, 120 mM NaCl, 5 mM KCl, 1.2 mM MgSO₄, 1μM pargyline, 2 mg/ml glucose, 0.2 mg/ml ascorbic acid, pH 7.5) (for synaptosomes) at 25°C for 30 min. Efflux process was terminated by addition of 1 ml of ice-cold serum-free DMEM into the wells or efflux buffer into the tubes. After aspiration of the DMEM (for cells) or centrifugation at 10,000g at 4°C for 3 min (for synaptosome), the samples were then washed twice and incubated in 1× PLB buffer for 30 min on a shaking platform at 200 rpm. The lysates were transferred into scintillation vials containing 4 ml of Beckman ReadySafe scintillation cocktail and counted on Beckman LS6000IC scintillation spectrophotometer for 1 min/sample. The samples treated with efflux buffer after uploading of [³H]monoamine were taken as baseline (100% retention).

Data Analysis. RLUs represent the luciferase level (firefly and Renilla). The average ratio of firefly luciferase RLU to Renilla luciferase RLU was calculated for each triplicate and then divided by the value at baseline (vehicle treatment), and the results were converted into a percentage value. The RLU increase, which is the percentage value above baseline, represents cAMP accumulation in response to the drug challenge. Specific [³H]monoamine uptake and retention were compared to the baseline or the maximal level and expressed as percentage values in each set of assays. Results were finalized as mean ± S.E.M. of the indicated number of observations. Student's t test was used to judge the difference between two samples, and the results were considered significantly different at p < 0.05.

Results

TAAR1 Is Activated by Trace Amines. We and others have previously reported that TAAR1 is expressed in brain monoaminergic nuclei and activated by trace amines in vitro. To assess the influence of their interaction on monoamine transporter function, we re-evaluated herein TAAR1 activation by trace amines to confirm that TAAR1 is recognized and excited by trace amines using dual luciferase assays. Rhesus monkey TAAR1 was transiently transfected into HEK293 cells along with CRE-Luc and pGL4.73, and CRE-Luc expression was determined as a measurement of cAMP accumulation to assess TAAR1 activity in the cells following exposure to trace amines. The data were normalized to vehicle treatment and presented as RLU increase (percentage). β-PEA increased CRE-Luc expression in a dose-dependent manner in the presence of TAAR1, but the CRE-Luc expression did not vary with the dose increase of β-PEA in the absence of TAAR1. Likewise, tyramine, tryptamine, and octopamine also increased CRE-Luc expression in a TAAR1-dependent manner (Fig. 1). These results confirm our previous findings that trace amine are agonists at rhesus monkey TAAR1 (Miller et al., 2005; Xie et al., 2007b) and give further support for interaction of trace amines with TAAR1 through the cAMP signaling pathway.

TAAR1 Activation by β-PEA Modulates Monoamine Transporter Function in Transfected Cells. To determine whether trace amines modulate monoamine transporter function via activating TAAR1, herein we investigated the effect of TAAR1 signaling in response to β-PEA on DAT, NET, and SERT function. Human DAT, NET, or SERT was transiently transfected into HEK293, stable TAAR1 and stable D2s, and α_2A and 5HT_1B cells to generate different cell lines: DAT, NET, SERT, TAAR1-DAT, TAAR1-NET, TAAR1-SERT, D2s-DAT, α_2A-NET, and 5HT_1B-SERT, respectively. DAT, NET, and SERT cells were used as control.

We first evaluated the influence of TAAR1 activation by β-PEA on the uptake function of monoamine transporters in the cells. The cells were pretreated with vehicle or 1 μM β-PEA for 10 min and then washed twice and uploaded with 10 nM [³H]dopamine, 20 nM [³H]norepinephrine, or 20 nM [³H]serotonin for 5 min. The uptake values were reported as percentage of baseline (uptake in cells pretreated with DMEM for 10 min). β-PEA reduced [³H]dopamine uptake in TAAR1-DAT cells by 29.4 ± 5.2%, relative to vehicle treatment (p < 0.01). However, β-PEA had no such effect on [³H]dopamine uptake in DAT and D2s-DAT cells (Fig. 2A). Likewise, β-PEA reduced [³H]norepinephrine uptake in TAAR1-NET cells by 29.6 ± 4.3%, relative to vehicle treatment (p < 0.01). However, β-PEA had no such effect on [³H]norepinephrine uptake in NET and α_2A-NET cells (Fig. 2B). Likewise, β-PEA reduced [³H]serotonin uptake in TAAR1-SERT cells by 34.6 ± 6.6%, relative to vehicle treatment (p < 0.01). However, β-PEA had no such effect on [³H]serotonin uptake in SERT and 5HT_1B-SERT cells (Fig. 2C).

Next, we evaluated the influence of TAAR1 activation by β-PEA on the efflux function of monoamine transporters in the cells. The cells were preloaded with 10 nM [³H]dopamine, 20 nM [³H]norepinephrine, or 20 nM [³H]serotonin for 20 min and then washed twice and treated with vehicle, 1 μM β-PEA, or 1 μM β-PEA plus 10 μM indatraline for 30 min. The [³H]monoamine retention values were reported as percentage of baseline (retention in cells treated with DMEM for 30 min following preloading of [³H]monoamine). β-PEA reduced [³H]dopamine retention by 22.5 ± 3.6% in TAAR1-DAT cells, in comparison to vehicle treatment (p < 0.01), but it had no such effect in DAT cells. Indatraline attenuated the effect of β-PEA in TAAR1-DAT cells (Fig. 3A). Likewise, β-PEA reduced [³H]norepinephrine retention by 20.6 ± 4.4% in TAAR1-NET cells, in comparison to vehicle treatment (p < 0.01), but it had no such effect in NET cells. Indatraline attenuated the effect of β-PEA in TAAR1-NET cells (Fig. 3B). Likewise, β-PEA reduced [³H]serotonin retention by 22.2 ± 4.8% in TAAR1-SERT cells, in comparison to vehicle treatment (p < 0.01), but it had no such effect in SERT cells. Indatraline attenuated the effect of β-PEA in TAAR1-SERT cells (Fig. 3C).

TAAR1 Activation by β-PEA Modulates DAT Function in Brain Striatal Synaptosomes. To investigate whether TAAR1 activation by β-PEA can modulate DAT function in brain, we prepared synaptosomes from monkey and mouse striatal tissues to evaluate DAT modulation by β-PEA, as there is a high density of DAT expressed in brain striatum (Ciliax et al., 1995; Miller et al., 2001). To distinguish effects at DAT from other monoamine transporters also expressed in the striatum, we used the DAT-selective inhibitor methylphenidate for determination of nonspecific uptake of [³H]dopamine. We first evaluated the influence of β-PEA pretreatment on uptake of [³H]dopamine. The synaptosomes were pretreated with vehicle or 1 μM β-PEA for 10 min and then washed twice and uploaded with 10 nM [³H]dopamine for 5 min. The uptake values were reported as percentage of baseline (uptake in synaptosomes pretreated with uptake buffer for 10 min). β-PEA reduced [³H]dopamine uptake in the synaptosomes of wild-type mice by 17.4 ± 3.2%, relative to vehicle treatment (p < 0.01), but it had no such effect on [³H]dopamine uptake in the synaptosomes of TAAR1 knockout mice (Fig. 4A). We further performed time course studies to observe the effect of β-PEA on [³H]dopamine uptake. The synaptosomes were exposed to 10 nM [³H]dopamine or 10 nM [³H]dopamine plus 100 nM β-PEA for different times. The uptake values were reported as percentage of the maximal uptake (uptake of 10 nM [³H]dopamine alone at 30 min). Under this condition, [³H]dopamine uptake was apparently inhibited—the uptake curves shifted right and down. It is noteworthy that [³H]dopamine uptake temporarily halted after 3 min in synaptosomes of rhesus monkeys and wild-type mice, but no such uptake halt occurred in the synaptosomes of TAAR1 knockout mice (Fig. 4B). Because of such a halt in the uptake, the reduction in the percentage value of [³H]dopamine uptake in striatal synaptosomes of rhesus monkeys (42.8 ± 7.1% at 10 min) and wild-type mice (45.6 ± 6.8% at 10 min) was significantly greater than the reduction in striatal synaptosomes of TAAR1 knockout mice (26.5 ± 4.2% at 10 min) (p < 0.01). We then investigated the influence of β-PEA on efflux of [³H]dopamine. The synaptosomes were preloaded with 10 nM [³H]dopamine for 20 min and then washed twice and exposed to vehicle, 1 μM β-PEA, or 1 μM β-PEA plus 10 μM methylphenidate for 30 min. The [³H]dopamine retention was reported as percentage of baseline (retention in synaptosomes treated with efflux buffer for 30 min following uploading of [³H]dopamine), and the loss of the [³H]dopamine retention was taken as the efflux values. β-PEA promoted [³H]dopamine efflux (retention is reduced) in the synaptosomes of rhesus monkeys and wild-type mice by 20.1 ± 3.5 and 27.6 ± 4.2%, respectively, but it had no such effect on [³H]dopamine efflux in the synaptosomes of TAAR1 knockout mice. Methylphenidate blocked the β-PEA effect in the synaptosomes of wild-type mice (Fig. 4C).

TAAR1 Activation by β-PEA Modulates NET Function in Brain Thalamic Synaptosomes. NET has been found to be expressed in brain thalamus at a relatively high level (Ding et al., 2003; Madras et al., 2006). Therefore, we collected monkey and mouse thalamic tissues to prepare synaptosomes for evaluation of NET modulation by β-PEA. We used the NET-selective inhibitor desipramine for determination of nonspecific uptake of [³H]norepinephrine. We first evaluated the influence of β-PEA pretreatment on uptake of [³H]norepinephrine. The synaptosomes were pretreated with vehicle or 1 μM β-PEA for 10 min and then washed twice and uploaded with 20 nM [³H]norepinephrine for 5 min. The uptake values were reported as percentage of baseline (uptake in synaptosomes pretreated with uptake buffer for 10 min). β-PEA reduced [³H]norepinephrine uptake in the synaptosomes of wild-type mice by 20.0 ± 3.9%, relative to vehicle treatment (p < 0.01), but it had no such effect on [³H]norepinephrine uptake in the synaptosomes of TAAR1 knockout mice (Fig. 5A). We further performed time course studies to observe the effect of β-PEA on [³H]norepinephrine uptake. The synaptosomes were exposed to 20 nM [³H]norepinephrine or 20 nM [³H]norepinephrine plus 100 nM β-PEA for different times. The uptake values were reported as percentage of the maximal uptake (uptake of 20 nM [³H]norepinephrine alone at 30 min). Under this condition, [³H]norepinephrine uptake was apparently inhibited—the uptake curves shifted right and down. [³H]Norepinephrine uptake temporarily halted after 3 min in synaptosomes of rhesus monkeys and wild-type mice, but no such uptake halt occurred in the synaptosomes of TAAR1 knockout mice (Fig. 5B). Because of such a halt in the uptake, the reduction in the percentage value of [³H]norepinephrine uptake in thalamic synaptosomes of rhesus monkeys (41.0 ± 5.6% at 10 min) and wild-type mice (39.3 ± 4.5% at 10 min) was significantly greater than the reduction in thalamic synaptosomes of TAAR1 knockout mice (26.6 ± 4.9% at 10 min) (p < 0.01). We then investigated the influence of β-PEA on the efflux of [³H]norepinephrine. The synaptosomes were preloaded with 20 nM [³H]norepinephrine for 20 min and then washed twice and exposed to vehicle, 1 μM β-PEA, or 1 μM β-PEA plus 10 μM desipramine for 30 min. The [³H]norepinephrine retention was reported as percentage of baseline (retention in synaptosomes treated with efflux buffer for 30 min following uploading of [³H]norepinephrine), and the loss of the [³H]norepinephrine retention was taken as the efflux values. β-PEA promoted [³H]norepinephrine efflux (retention is reduced) in the synaptosomes of rhesus monkeys and wild-type mice by 27.2 ± 5.5 and 26.8 ± 5.1%, respectively, but it had no such effect on [³H]norepinephrine efflux in the synaptosomes of TAAR1 knockout mice. Desipramine blocked β-PEA effect in the synaptosomes of wild-type mice (Fig. 5C).

TAAR1 Activation by β-PEA Modulates SERT Function in Brain Striatal Synaptosomes. SERT has been also found to be expressed in brain striatum at a high level (Sur et al., 1996; Kish et al., 2005). Therefore, we collected monkey and mouse striatal tissues to prepare synaptosomes for evaluation of SERT modulation by β-PEA. We used the SERT-selective inhibitor citalopram for determination of nonspecific uptake of [³H]serotonin. We first evaluated the influence of β-PEA pretreatment on uptake of [³H]serotonin. The synaptosomes were pretreated with vehicle or 1 μM β-PEA for 10 min and then washed twice and uploaded with 20 nM [³H]serotonin for 5 min. The uptake values were reported as percentage of baseline (uptake in synaptosomes pretreated with uptake buffer for 10 min). β-PEA reduced [³H]serotonin uptake in the synaptosomes of wild-type mice by 22.0 ± 5.1%, relative to vehicle treatment (p < 0.01), but it had no such effect on [³H]serotonin uptake in the synaptosomes of TAAR1 knockout mice (Fig. 6A). We further performed time course studies to observe the effect of β-PEA on [³H]serotonin uptake. The synaptosomes were exposed to 20 nM [³H]serotonin or 20 nM [³H]serotonin plus 100 nM β-PEA for different times. The uptake values were reported as percentage of the maximal uptake (uptake of 20 nM [³H]serotonin alone at 30 min). Under this condition, [³H]serotonin uptake was apparently inhibited—the uptake curves shifted right and down. [³H]Serotonin uptake temporarily halted after 4 min in synaptosomes of rhesus monkeys and wild-type mice, but no such uptake halt occurred in the synaptosomes of TAAR1 knockout mice (Fig. 6B). Because of such a halt in the uptake, the reduction in the percentage value of [³H]serotonin uptake in striatal synaptosomes of rhesus monkeys (45.8 ± 6.6% at 10 min) and wild-type mice (44.1 ± 5.8% at 10 min) was significantly greater than the reduction in striatal synaptosomes of TAAR1 knockout mice (30.0 ± 5.1% at 10 min) (p < 0.01). We then investigated the influence of β-PEA on efflux of [³H]serotonin. The synaptosomes were preloaded with 20 nM [³H]serotonin for 20 min and then washed twice and exposed to vehicle, 1 μM β-PEA or 1 μM β-PEA plus 10 μM citalopram, for 30 min. The [³H]serotonin retention was reported as percentage of baseline (retention in cells treated with DMEM or synaptosomes treated with efflux buffer for 30 min following uploading of [³H]serotonin), and the loss of the [³H]serotonin retention was taken as the efflux values. β-PEA promoted [³H]serotonin efflux (retention is reduced) in the synaptosomes of rhesus monkeys and wild-type mice by 20.0 ± 3.6 and 16.8 ± 3.3%, respectively, but it had no such effect on [³H]serotonin efflux in the synaptosomes of TAAR1 knockout mice. Citalopram blocked the β-PEA effect in the synaptosomes of wild-type mice (Fig. 6C).

TAAR1 Signaling Is Not Altered by Monoamine Autoreceptors at Exposure to Trace Amines. Monoamine autoreceptors, including dopamine D2, adrenergic α₂, and serotonin 5HT₁ receptors, are presynaptic feedback regulators for the respective monoamine neurotransmitter release. To determine whether the trace amines interact with these receptors to alter TAAR1 signaling, we assessed the influence of autoreceptors, D2s, α_2A, α_2B, 5HT_1A, and 5HT_1B coexpressed with TAAR1, respectively, on TAAR1 activation in the cells at exposure to trace amines. We transiently transfected rhesus monkey TAAR1 along with CRE-Luc and pGL4.73 into HEK293 and stable D2s, α_2A, α_2B, 5HT_1A, and 5HT_1B cells to generate different cell lines (TAAR1, D2s-TAAR1, α_2A-TAAR1, α_2B-TAAR1, 5HT_1A-TAAR1, and 5HT_1B-TAAR1) and then determined CRE-Luc expression as a measurement of cAMP accumulation to assess TAAR1 activity. The data were normalized to vehicle treatment and are presented as RLU increase (percentage).

We first evaluated TAAR1 activation by β-PEA in the cells. When the cells were exposed to 1 μM β-PEA, the monoamine autoreceptors D2s, α_2A, α_2B, 5HT_1A, or 5HT_1B had no significant effect on TAAR1 signaling; TAAR1 activation in the presence of each type of autoreceptor was similar in amplitude to that in the absence of the autoreceptor (Fig. 7A). In contrast, parallel to β-PEA, we also tested dopamine, norepinephrine, and serotonin and found that TAAR1 signaling was largely attenuated by D2s at exposure to 1 μM dopamine, by α_2A or α_2B at exposure to 1 μM norepinephrine, or by 5HT_1A or 5HT_1B at exposure to 1 μM serotonin (Xie et al., 2008), suggesting that the autoreceptors were expressed well and indicating that β-PEA differs from dopamine, norepinephrine, or serotonin in that β-PEA at 1 μM did not interact with the monoamine autoreceptors. We then further evaluated TAAR1 activation by other trace amines in the cells. Likewise, TAAR1 signaling in response to 1 μM tyramine (Fig. 7B), 1 μM tryptamine (Fig. 7C), or 1 μM octopamine (Fig. 7D) was not affected by any of the monoamine autoreceptors tested. To clarify whether there is a significant difference in TAAR1 expression in the cells tested, we performed Western blotting to determine the TAAR1 level. We found that D2s, α_2A, α_2B, 5HT_1A, or 5HT_1B did not significantly alter TAAR1 expression (Fig. 7E).

Trace Amines Have Extremely Low Binding Affinity for Monoamine Autoreceptors. To further determine whether trace amines interact with monoamine autoreceptors, we assessed the binding affinity of trace amines to the monoamine autoreceptors in comparison to dopamine (for D2s), norepinephrine (for α_2A and α_2B), and serotonin (for 5HT_1A and 5HT_1B). Stable D2s, α_2A, α_2B, 5HT_1A, and 5HT_1B cells were used to perform competition binding assays to determine IC₅₀ values of the trace amines. We found that β-PEA, octopamine, tryptamine, and tyramine had poor binding affinity for D2s relative to dopamine (Fig. 8A), for α_2A and α_2B relative to norepinephrine (Fig. 8B), and for 5HT_1A and 5HT_1B relative to serotonin (Fig. 8C). The IC₅₀ of dopamine for D2s was 0.19 ± 0.06 μM, the IC₅₀ of norepinephrine for α_2A and α_2B was 0.025 ± 0.008 and 0.058 ± 0.018 μM, respectively, and the IC₅₀ of serotonin for 5HT_1A and 5HT_1B was 0.048 ± 0.013 and 0.032 ± 0.011 μM, respectively. However, in each case, the IC₅₀ of β-PEA, octopamine, tryptamine, or tyramine was greater than 10 μM.

Discussion

The data presented in this study confirm that trace amines are potent agonists at TAAR1 and demonstrate that TAAR1 signaling in response to trace amines is not affected by monoamine autoreceptors coexpressed with TAAR1 in transfected cells. Trace amines lack interaction with monoamine autoreceptors, whereas they recognize and excite TAAR1. β-PEA activation of TAAR1 modulates monoamine transporter function in the transfected cells and in brain striatal and thalamic synaptosomes, which suggests a modulatory role for trace amines in the functional regulation of monoamine transporters in brain.

β-PEA has been thought of as an endogenous amphetamine due to its similarity to amphetamine in structure and effects. Numerous studies have reported the influence of β-PEA in the central nervous system, especially on catecholaminergic activity in the mammalian brain. It has been reported that β-PEA increases the release of catecholamines and inhibits catecholamine uptake (Paterson et al., 1990). The present study provides evidence that β-PEA alters the uptake and efflux functions of DAT, NET, and SERT via interaction with TAAR1. In time course assays in which brain synaptosomes were exposed to [³H]monoamines in combination with β-PEA, we observed that β-PEA induced a halting in uptake of [³H]monoamines after 3 to 4 min in monkey and wild-type mouse synaptosomes. Because such uptake halting did not occur in TAAR1 knockout mouse synaptosomes, it can be deduced that TAAR1 is mediating this effect. In our pretreatment assays in which cells and synaptosomes were exposed to β-PEA and then washed and exposed to [³H]monoamine, β-PEA did not alter [³H]monoamine uptake in transfected cells lacking TAAR1 or in the TAAR1 knockout mouse synaptosomes but did significantly inhibit [³H]monoamine uptake in cells expressing TAAR1 and in monkey and wild-type mouse synaptosomes, providing further evidence that TAAR1 is a mediator of β-PEA effects on the monoamine transporters. In efflux assays, we also observed that β-PEA promoted [³H]monoamine efflux in a TAAR1-dependent manner. Although TAAR1 is not specific for β-PEA, TAAR1 may be essential for β-PEA exerting non-competitive effects on monoamine transporters in brain.

In addition to β-PEA, brain trace amines also include tyramine, tryptamine, and octopamine. These trace amines are spatially and closely associated with the dopamine, norepinephrine, and serotonin neurotransmitter systems (Philips et al., 1974; Axelrod and Saavedra, 1977; Durden and Philips 1980; Paterson et al. 1990), and their specific binding sites in brain have been known for decades (Hauger et al., 1982; Kellar and Cascio, 1982; Vaccari, 1986). In this study, we confirmed that the trace amines are all agonists at TAAR1. We focused on β-PEA for functional analysis, as β-PEA has been independently reported to be associated with various neuropsychiatric disorders/diseases. It is likely that the functional profile demonstrated for β-PEA in modulation of monoamine transporters is indicative of a common mechanism for trace amines. Although trace amines are generally present in brain at a very low level (Durden and Philips 1980; Durden and Davis, 1993), this does not exclude their interaction with TAAR1 in brain. It is possible that trace amines reach levels suitable for TAAR1 activation at very discrete locations in brain, particularly under certain pathological or pharmacological conditions (Bergman et al., 2001). It is noteworthy that aberrant trace amine levels occur in depression and other neuropsychiatric disorders (Boulton, 1980; Davis and Boulton, 1994; McClung and Hirsh, 1999; D'Andrea et al., 2003). Accordingly, our finding that β-PEA acts via TAAR1 to modulate monoamine transporter function may reveal a principal mechanism for trace amines exerting their effects on brain monoaminergic systems.

Monoamine autoreceptors colocalize with monoamine transporters on presynaptic membranes of monoaminergic neurons and provide feedback regulation of monoamine neurotransmitter release. It has been previously recognized that D2 autoreceptor activation can modulate DAT function and expression (Mayfield and Zahniser, 2001; Bolan et al., 2007). Recently, Wolinsky et al. (2007) reported that the knockout of TAAR1 elicited a large increase of striatal high-affinity D2 receptors, suggesting that TAAR1 function may be related to monoamine autoreceptors. In this regard, we evaluated whether trace amines interact with monoamine autoreceptors, including dopamine D2s, adrenergic α_2A and α_2B, and serotonin 5HT_1A and 5HT_1B receptors, to alter TAAR1 signaling. Our data show that these monoamine autoreceptors are unresponsive to trace amines. TAAR1 signaling in response to trace amines is not affected by these autoreceptors concurrently exposed to trace amines. We previously reported that dopamine, norepinephrine, and serotonin, which are agonists for the respective autoreceptors, are also rhesus monkey TAAR1 agonists in vitro (Xie et al., 2007b). Indeed, we have also demonstrated that TAAR1 signaling was largely attenuated by D2s at exposure to 1 μM dopamine, by α_2A or α_2B at exposure to 1 μM norepinephrine, or by 5HT_1A or 5HT_1B at exposure to 1 μM serotonin (Xie et al., 2008). Accordingly, trace amines may differ from common biogenic amines with regard to modulation of monoamine transporters.

Monoamine transporters are important regulators of brain monoaminergic neurotransmission in clearance of released monoamine neurotransmitters from synaptic clefts and termination of neural signaling (Uhl and Johnson, 1994; Giros et al., 1996; Masson et al., 1999), although diffusion and enzymatic degradation also contribute to reducing the synaptic concentration of the neurotransmitters. Trace amines are substrates for DAT, NET, and SERT and, therefore, can compete with dopamine, norepinephrine, and serotonin for transport through their respective monoamine transporters. Indeed, in time course uptake assays, we observed an uptake inhibition in TAAR1 knockout mouse synaptosomes; the uptake curves in the presence of β-PEA shifted right and down, which also occurred in monkey and wild-type mouse synaptosomes. Such conserved inhibition is most likely due to substrate competition. However, the extracellular concentration of the trace amines in brain is much lower than that for common biogenic amines (Durden and Philips, 1980; Durden and Davis, 1993), suggesting that the competition effects of the trace amines may not significantly affect the transport of the common biogenic amines in vivo. In contrast, TAAR1-mediated effects of the trace amines may play a key role.

That β-PEA activation of TAAR1 modulates monoamine transporter function provides a novel insight into understanding the modulatory roles of brain trace amines. Full elucidation of the influence of TAAR1 interaction with trace amines in the brain is directly dependent on clarification of the cellular distribution of the trace amines and trace amine-associated receptors. Unlike many other G protein-coupled receptors, TAAR1 apparently remains largely intracellularly sequestered in transfected cells (Bunzow et al., 2001; Miller et al., 2005; Xie et al., 2007b). It is predictable that the localization of TAAR1 in the neurons will play an important role determining its signaling in response to trace amines and other activators for which further studies are needed. In addition, although the present study provides initial evidence that TAAR1 may serve as a presynaptic regulator of monoamine transporters, we lack direct evidence for TAAR1 distribution on presynaptic membranes. It is likely that TAAR1 is located on/in neurons both presynaptically and postsynaptically and plays a wider role in monoaminergic regulation than we present here. Nevertheless, this study demonstrates that TAAR1 is a modulator of monoamine transporter function in brain and reveals a mechanism by which β-PEA, and possibly other trace amines, can affect monoamine transporter function.

Monoamine transporter regulators are among the most important drugs used to treat a spectrum of neuropsychiatric disorders, and most of the drugs, such as the selective serotonin reuptake inhibitors, directly interact with monoamine transporters. However, these drugs display a dramatically delayed onset in their clinical effects and have potentially serious adverse effects, particularly in children and adolescents with depression, which stimulates efforts for new drug development. Our data reveal a receptor-mediated mechanism in the functional regulation of monoamine transporters and suggest that novel drugs that activate TAAR1 but not monoamine autoreceptors could be able to improve the medication of neuropsychiatric disorders/diseases. Accordingly, the present study may provide an impetus for drug development. Furthermore, our findings also suggest that the clinical efficacy of monoamine autoreceptor inhibitors may in part result from changes in receptor signaling in which the TAAR1 response to endogenous monoamine neurotransmitters is reinforced due to deprivation of autoreceptor activation. Because of the similarity of β-PEA to amphetamine, our data also suggest that amphetamines may regulate monoamine transporter function similarly to β-PEA, and in this regard, drugs that can antagonize TAAR1 may have clinical efficacy as therapeutics for drug abuse and addiction to amphetamines.