QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.105.084459v1 313/3/983    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Miller, G. M. Articles by Madras, B. K. Search for Related Content PubMed PubMed Citation Articles by Miller, G. M. Articles by Madras, B. K.

NEUROPHARMACOLOGY

Primate Trace Amine Receptor 1 Modulation by the Dopamine Transporter

Division of Neurochemistry, New England Primate Research Center, Harvard Medical School, Southborough, Massachusetts

Received for publication February 3, 2005
Accepted March 8, 2005.

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
Recently identified trace amine receptors are potential direct targets for drugs of abuse, including amphetamine and 3,4-methylenedioxymethamphetamine (MDMA). We cloned full-length rhesus monkey trace amine receptor 1 (rhTA₁) that was 96% homologous to human TA₁. The trace amines tyramine and -phenylethylamine (PEA) and the monoamine transporter substrates (±)-amphetamine and (±)-MDMA stimulated cAMP accumulation in rhTA₁-expressing cell lines, as measured by a cAMP response element-luciferase assay. Cocaine did not stimulate cAMP accumulation in rhTA₁ cells, but it blocked [³H]PEA transport mediated by the dopamine transporter. Cotransfection with the human dopamine transporter enhanced PEA-, amphetamine-, and MDMA-mediated rhTA₁ receptor activation, but it diminished tyramine activation of rhTA₁. Because TA₁ (EGFP-rhTA₁ chimera) was largely intracellular, conceivably the dopamine transporter can facilitate access of specific agonists to intracellular TA₁. rhTA₁ mRNA expression was detected in rhesus monkey substantia nigra, implying that TA₁ may be colocalized with the dopamine transporter in dopamine neurons. In summary, primate TA₁ receptors are direct targets of trace amines, amphetamine, and MDMA. These receptors could also be indirect targets of amphetamine, MDMA, and cocaine through modification of monoamine transporter function. Conceivably, rhTA₁ receptors may be located on pre- or postsynaptic membranes. Interference with the carrier function of monoamine transporters with a consequent rise of extracellular levels of trace amines could activate these receptors. The cloning of a highly homologous TA₁ from rhesus monkey and demonstration that rhTA₁ receptors are activated by drugs of abuse, indicate that nonhuman primates may serve to model physiological and pharmacological TA₁-mediated responses in humans.

Octopamine and tyramine function as principal insect neurotransmitters, and cloning of receptors for trace amines was initially accomplished in insects and mollusks (Axelrod and Saavedra, 1977; Arakawa et al., 1990; Saudou et al., 1990; Gerhardt et al., 1997; Han et al., 1998; Blenau et al., 2000). Genes encoding mammalian trace amine receptors, in contrast, were discovered serendipitously in the course of cloning novel serotonin 5-HT₁ receptor subtypes (Borowsky et al., 2001). A human trace amine receptor, designated TA₁, bound trace amines with high affinity and triggered cAMP accumulation. A systematic cloning strategy revealed the existence of 14 rat TAs (rTA₁–rTA₄, rTA₆–rTA₁₅) and four human TAs (hTA₁, hTA₃, hTA₄, and hTA₅). Unexpectedly, a wide range of drugs of abuse, including amphetamine, 3,4-methylenedioxymethamphetamine (MDMA), and d-lysergic acid diethylamide-like hallucinogens, stimulated rat TA₁-mediated cAMP accumulation at a 1 µM concentration (Bunzow et al., 2001). Because this newly discovered receptor family presents a novel target for psychoactive drugs, including drugs of abuse, the physiological and pharmacological role of trace amine receptors warrants systematic investigation and development of appropriate animal models.

Rodents present a challenge for modeling human trace amine receptors and responses to drugs because human and rodent TAs diverge significantly in sequence (76–78%) compared with sequence homology of other G protein-coupled receptors expressed in brain, in subtype number (14 in rat, four in human), in brain distribution of TA₁ (Borowsky et al., 2001), and possibly in pharmacological specificity (Borowsky et al., 2001). Conceivably, these key differences reflect rapid evolution of trace amine receptors and warrant investigation of the trace amine receptor family in a species with evolutionary proximity to humans. Accordingly, we explored whether the rhesus monkey genome encodes TA₁, whether it has a higher degree of homology to human than the rodent TA₁, and whether it responds to psychostimulant drugs of abuse, directly or indirectly.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Genomic DNA Sample Isolation. Venous blood samples (10 ml) were collected from rhesus monkeys in Vacutainer tubes (BD Biosciences, Franklin Lakes, NJ) containing EDTA and stored at –80°C. Genomic DNA was isolated from 400 µl of whole blood using a Generation Capture Column kit (Gentra Systems, Inc., Minneapolis, MN). Eluted DNA was then ultrapurified using the Wizard DNA Cleanup system (Promega, Madison, WI).

PCR Protocol for Cloning rhTA₁ Full-Length Coding Sequences. Oligonucleotide primer sets that anneal outside the coding region were designed based on the human TA₁ subtype sequence embedded within human DNA sequence data from clone RP11-295F4 on chromosome 6 (GenBank AL513524 [GenBank] ). One set of primers that resulted in a PCR product of the expected size from genomic DNA (gDNA) samples was selected. All amplifications were carried out in a total volume of 30 µl containing 1 µl of Elongase enzyme mix (Invitrogen, Carlsbad, CA), 6 µlof5x buffer B [300 mM Tris-SO₄, pH 9.1, 10 mM MgSO₄, 90 mM (NH₄)₂SO₄], 1 µl of 40 µM dNTPs, 10 pmol of each oligonucleotide, and 25–100 ng of gDNA template. Oligonucleotides were designed using Oligo software (Molecular Biology Insights, Inc., Cascade, CO), compared with GenBank sequence libraries to ensure specificity, and custom synthesized (Alph-aDNA, Montreal, QC, Canada). Direct utilization of gDNA as template was possible because the rhTA₁ gene is presumed to be intronless (based on human sequence data). Two primers were used (5' to 3'): TA₁-f, GAT TGA CAG CCC TCA GGA ATG ATG and TA₁-r, CAA AAC AGT AAA ATA TAA TAA TTC TA. Parameters were 94°C for 1 min, 35 cycles of denaturation at 94°C for 12 s, annealing at 54°C for 30 s, and elongation at 72°C for 3 min, with a final elongation at 72°C for 10 min. PCR products were run on 1% agarose gels that were stained with ethidium bromide. A single band was excised from the gel on a DarkReader transilluminator (Clare Chemical Research, Denver, CO), and the DNA was purified using a QIAquick gel extraction kit (QIAGEN, Valencia, CA). Isolated DNA was cloned into pcDNA3.1/V5/His-TOPO (Invitrogen) according to the manufacturer's protocol. After transformation, bacterial colonies were reserved and screened for insert orientation by PCR using a pcDNA3.1 reverse primer (Invitrogen) and a receptor-specific internal forward primer. Colonies with positive PCR results were selected for plasmid DNA isolation with a miniprep (Promega) or maxiprep (QIAGEN) kit according to the manufacturer's protocols. Eight rhTA₁ clones were sequenced from different rhesus monkeys. Occasionally, an individual clone had one or more aberrant nucleotides relative to all other clones. Two of these loci were screened as potential single nucleotide polymorphisms in the rhTA₁ coding sequence, but screening results were negative (data not shown). To ensure that the clone of rhTA₁ selected for study was accurate, it was chosen on the basis of every nucleotide being represented in the majority of the clones.

DNA Sequencing. Sequencing of both strands was performed on selected clones using a CEQ8000 genetic analysis system and CEQ DTSC quick start kit (Beckman Coulter, Fullerton, CA). The system, housed at The New England Primate Research Center (Harvard Medical School, Southborough, MA), uses four-color dye-labeled terminator cycle sequencing reactions that are detected by laser-induced fluorescence in four spectral channels via capillary electrophoresis.

Prediction of Transmembrane Domains and Potential Sites of Phosphorylation. Determination of potential transmembrane regions was performed de novo on all trace amine receptor (TAR) subtype clones using TMpred-prediction of transmembrane regions and orientation algorithm, which is based on a combination of several weight matrices for scoring. Prediction of potential phosphorylation sites was done using NetPhos 2.0 protein phosphorylation prediction server (http://www.cbs.dtu.dk/services/NetPhos/), a neural network-based method for predicting potential phosphorylation sites at serine, threonine, or tyrosine residues in protein sequences. Prediction of potential kinases that may phosphorylate the selected residues was determined using PhosphoBase version 2.0, a prediction algorithm based on substrate consensus sequence logos of some common protein kinases (Kreegipuu et al., 1999).

CRE-Luc and hDAT Vectors. We used a cAMP-response element (CRE)-driven luciferase reporter construct (CRE-Luc; pTLNC121-3; made by Dr. Graeme Bilbe, Novartis, Basel, Switzerland; obtained from Dr. Walter Borne, University of Zurich, Zurich, Switzerland) to promote firefly luciferase expression in response to cAMP. The construct contains a 21X CRE cassette positioned upstream from a TATA box minimal promoter element. This construct is a highly sensitive and quantitative reporter of total cAMP induction over time and thus results in high baseline levels due to constitutive activation of cAMP during cell growth. A human dopamine transporter (hDAT) expression vector was generated from a full-length hDAT cDNA (generous gift of Dr. B. Hoffman, Eli Lilly, Indianapolis, IN) that was isolated from a human substantia nigra cDNA library and ligated into pcDNA3.1 (Invitrogen), resulting in the human dopamine transporter expression vector pcDNA3.1/hDAT.

Cell Culture and Transient Transfections. HEK-293 cells and stably transfected hDAT cells (American Type Culture Collection, Manassas, VA) were grown in 145-mm untreated tissue culture dishes (Greiner America, Inc., Lake Mary, FL) in DMEM supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 µg of streptomycin, and 0.1 mM nonessential amino acids (all reagents from Invitrogen). Cell lines were grown at 5% CO₂ in a 37°C water-jacketed incubator. An Effectine reagent kit (QIAGEN) was used for DNA transfection procedures, according to the manufacturer's protocol. Cells were plated in 24-well plates on the day before transfection. Transient transfection with the CRE-Luc reporter construct, or double transfections with the CRE-Luc reporter construct and an rhTA₁ receptor expression construct in equal molar ratios were performed. The total amount of DNA transfected was held constant, and stable hDAT cells were always transfected simultaneously with wild-type HEK cells, and with the same reagent preparation. In this regard, Cre-Luc only controls were transfected with twice as much reporter as double transfection cells, and basal relative light units (RLUs) were twice as high in these control experiments as in double transfection cells. The transfection media remained on the cells for 5 h and then was replaced with 1 ml of unsupplemented DMEM with no serum. All drugs and trace amines were dissolved in DMEM and were added to the triplicate wells at each dose within 0.5 h after the replacement of the transfection medium. The cell plates were gently swirled, placed back in the incubator for 18 to 20 h, and then assayed for luciferase.

Luciferase Reporter Assays. Stock solutions of test compounds (10 mM) were made and further diluted to various concentrations in unsupplemented DMEM. Drugs were sequentially diluted and added in volumes ranging from 1 to 10 µl, such that final concentrations ranged from 10 nM to as high as 100 µM and were accurate to within 1%. Approximately 18 to 20 h after drug exposure, the wells were washed with phosphate-buffered saline, pH 7.4 (Ca²⁺ and Mg²⁺ free), and reporter lysis buffer (200 µl; Promega) was added to each well. Cell lysates were removed from the wells by vigorous washing. Luciferase assay reagent was made by adding 10 ml of luciferase assay buffer to the lyophilized luciferase assay substrate (Promega). Cell lysates were collected and spun down in Microfuge tubes and lysates (20 µl) from each well were placed in test tubes. Luciferase assay reagent (100 µl) was injected into each sample, and after a 2-s delay, luciferase concentration was measured as RLUs for 10 s.

Time Course and Transport Assays. Initially, a time course for PEA transport into hDAT and untransfected HEK-293 cells was performed to determine an optimal incubation period for transport assays. hDAT cells were incubated for various times (0, 2, 4, 6, 8, 10, 15, 20, 30, 40, and 60 min) with 0.2 ml of cell buffer (5 mM Tris, 8.5 mM HEPES, 120 mM NaCl, 5.4 mM KCl, 1.2 mM CaCl₂, 1.2 mM MgSO₄, 10 mM glucose, and 10 mM tropolone, pH 7.4), 0.2 ml of cell suspension, and 0.2 ml of [³H]PEA [50 nM; 4 nM [³H]PEA, 77.0 Ci/mmol, custom-synthesized by Amersham Biosciences, Inc., Piscataway, NJ, and 46 nM unlabeled PEA]. Nonspecific uptake was determined in the presence of 10 µM mazindol. The linear part of the association time study indicated that 6 min was optimal for [³H]PEA transport experiments.

[³H]PEA transport was determined as a function of temperature and presence of hDAT. To measure energy dependence of [³H]PEA transport, a time-course experiment was conducted at 0–4°C. To determine whether transport was hDAT-dependent, corresponding assays were conducted in hDAT and in untransfected HEK-293 cells at 37°C. The affinity of cocaine for blocking [³H]DA and [³H]PEA transport by hDAT was determined in stably transfected hDAT cells. Cells were washed in cell buffer, pH 7.4, centrifuged, and resuspended in cell buffer, and cell concentration was adjusted to 1.25 x 10⁶ cells/ml using a hemacytometer. Cocaine was dissolved in cell buffer to 1 mM, whereas mazindol was dissolved in cell buffer supplemented with 40 µl of lactic acid and brought to 1 mM, and serial dilutions were prepared. Microfuge tubes, in triplicate, containing 0.2 ml of cells, 0.2 ml of diluted test drug or baseline drug, and 0.2 ml of [³H]DA (20 nM) or [³H]PEA (20 nM) were incubated for 10 or 6 min, respectively, at 37°C, as determined by time course experiments. After incubation, cells were harvested by centrifugation at 16,600g for 15 min at 0°C. The cell supernatant was removed, and the pellet was dissolved in 100 µl of 0.1% SDS. The Microfuge tube was clipped and added to 4 ml of Readysafe scintillation fluid, and radioactivity was measured for 5 min on an LS6000IC Beckman scintillation spectrometer. Nonspecific binding was determined in the presence of 10 µM mazindol. Data analyses were performed using GraphPad Prism software (GraphPad Software Inc., San Diego, CA).

N-Terminal EGFP-rhTA₁ Chimera. The rhTA₁ expression vector was linearized with BamHI and Bsu36I. The BamHI site is native to the MCS of pcDNA3.1-V5/His-TOPO and 5' to the ATG start codon of the rhTA₁ cDNA insert. A Bsu36I site is native to the rhTA₁ sequence, lies downstream from the MCS, 5' to the ATG start codon in the rhTA₁ insert, and is unique with regard to the greater construct. The EGFP coding region was amplified by low-cycle, high-fidelity PCR using pIRES2-EGFP (BD Biosciences Clontech, Palo Alto, CA) as template. Primers for this PCR reaction were designed to incorporate BamHI and Bsu36I sites in the 5' end of the upper and lower primers, respectively, such that the ligated EGFP insert was in frame with the rhTA₁ cDNA and lacked only the STOP codon. Primer design left a neutral tether between EGFP and the rhTA₁ sequence. The following primers were used to generate PCR-amplified and -modified EGFP: EGFP-BamHI-f (5' to 3') TTG AGG ATC CCG ATG ATA ATA TGG CCA CAA CCA and EGFP-Bsu-r (5'-3') GCG GCC TGA GGA CTT GTA CAG CTC GTC CAT GCC. The amplification product was digested with BamHI and Bsu36I, and cloned into the linearized rhTAR1 vector directionally. Sequencing confirmed the integrity of the selected clone. This clone was transfected into HEK-293 cells for localization studies as well as cotransfected into these cells with CRE-Luc for functional assessment.

Confocal Microscopy. HEK-293 cells plated in eight-well tissue culture glass slides were transiently transfected with an EGFP-rhTA1 chimera for 24 to 48 h. Cells were fixed in 3.7% paraformaldehyde for 5 min. Cells were stained with TO-PRO-3 for 2 min for nuclei staining and/or Vibrant CM-DiI for 1.5 min for membrane staining (Molecular Probes, Eugene, OR). Cells were then washed with phosphate-buffered saline, pH 7.4, coverslipped using Vectashield hard set mounting medium for fluorescence (Vector Laboratories, Burlingame, CA), and painted with clear nail polish to seal the coverslip. Visualization was performed using a Leica TCS SP laser scanning microscope equipped with three lasers (Leica Microsystems, Exton, PA). The fluorescence of individual fluorochromes was captured simultaneously, after optimization to reduce bleedthrough channels (photomultiplier tubes) using the Leica software.

Real-Time RT-PCR of rhTA₁ in the Substantia Nigra. Brain tissue was harvested from three rhesus monkeys euthanized for other purposes. The substantia nigra was dissected, placed in RNA-later (Ambion, Austin, TX), and stored at 4°C for less than 1 week. RNA was extracted from homogenized tissue (Dounce tissue grinder, Kontes; VWR Scientific, West Chester, PA) using TRIzol reagent (Invitrogen). RNA was then treated with RQ1 RNase-free DNase (Promega) for 1 h, and 1 µg was reverse transcribed using Superscript II RNase H^– reverse transcriptase (Invitrogen), according to the manufacturer's protocols. Oligonucleotides were designed using Oligo software (Molecular Biology Insights, Inc.), compared with GenBank sequence libraries to ensure specificity, and custom synthesized (AlphaDNA). PCR reactions were set up in a reaction volume of 20 µl using components supplied in the LightCycler RNA Master SYBR Green I kit for one-step RT-PCR using the LightCycler 2 instrument (Roche Diagnostics, Indianapolis, IN), and either 1 µlof water or 1 µl (50 ng) of cDNA. In separate reactions, we amplified TA₁, DAT, and a portion of the dynorphin promoter as a control for genomice DNA contamination with the following primer sets (5' to 3'): TA1–862-f, TTG ATT TGG TTT GGC TAC TTG and TA1-r1, CAA AAC AGT AAA ATA TAA TAA TTC TA; DAT7-f, GGC TTC TTC TAC AAC GTC ATC ATC GC and DAT21-r, GCT CTG GTG GAG GTG CAG CAC G; DynProm-15f, AGA GGT TGA AGT TGG CAG CTT ATC; and DynProm-496r, CCA GGC GGT TAG GTA GAG TTG TCA. All amplifications were carried out in glass capillary tubes with rapid cycling with the following conditions: preincubation at 95°C for 7 min; cycling at 95°C for 10 s, 58°C for 15 s, and 72°C for 10 s (12 s for DAT); melting at 95°C for 0 s, 65°C for 15 s, and 95 for 0 s at ramp of 0.1°C/s.

Materials. Tyramine and -phenylethylamine were purchased from Sigma-Aldrich (St. Louis MO). (+)-Amphetamine, (–)-amphetamine, (+)-MDMA (hydrochloride), (–)-MDMA (hydrochloride), and (–)-cocaine hydrochloride were obtained from the National Institute on Drug Abuse (Bethesda, MD). [³H]DA (20.5 Ci/mmol) was purchased from PerkinElmer Life and Analytical Sciences (Boston, MA), and [³H]PEA (77.0 Ci/mmol) was custom-synthesized by Amersham Biosciences Inc.

GenBank. The complete coding sequence of Macaca mulatta TAR1 gene is entered in GenBank AY135366 [GenBank] .

	Results

Top Abstract Materials and Methods Results Discussion References

 
rhTA₁ Sequence. Borowsky et al. (2001) identified four human trace amine receptor subtype genes (hTA₁, hTA₃, hTA₄, and hTA₅) that are intronless and occur in tandem on chromosome 6. Accordingly, we speculated that it would be feasible to clone rhesus monkey TA₁ coding sequences from genomic DNA. Using a PCR strategy based on the human DNA sequence on chromosome 6 (clone RP11-295F4, GenBank AL513524 [GenBank] ), full-length rhTA₁ coding sequences were obtained. Sequence comparisons between rhTA₁ and hTA₁ revealed that the coding sequences were 96.9% homologous, and the deduced amino acid sequence was 96.4% homologous (Fig. 1). This contrasts with a 78.7% homology with rat and 76.0% homology with mouse amino acid sequences. Hydrophobicity analysis predicted seven transmembrane regions, an extracellular N terminus, and an intracellular C terminus, as expected for GPCRs. Analysis of potential phosphorylation sites using NetPHOS analysis (Blom et al., 1999), revealed a potential casein kinase II phosphorylation site at position 222 unique to rhTA₁. hTA₁ lacks this site but has a unique potential calmodulin-dependent protein kinase II phosphorylation site at position 224. Additionally, both hTA₁ and rhTA₁ have a potential protein kinase C phosphorylation site at position 327 in the C-terminal tail.

View larger version (66K): [in this window] [in a new window]   Fig. 1. Comparison of the amino acid sequences of TA1 protein from rhesus monkey and human. Rhesus monkey and human TA1 have 96.4% similarity to human. Seven predicted transmembrane regions are underlined. NetPHOS analysis predicts a protein kinase C site at S327 and a casein kinase II site at S222 in rhesus monkey TA1. There are two N-glycosylation sites (gly).

Trace Amine and Psychostimulant Effects on rhTA₁-Transfected HEK-293 Cells. The trace amines tyramine and PEA dose-dependently increased cAMP production in HEK-293 cells transiently transfected with rhTA₁ and CRE-Luc (Fig. 2; Table 1). rhTA₁ activation was discernible in transfected HEK-293 cells with PEA and tyramine in a range of concentrations (10–1000 nM), with similar activation observed in C6 glioma cells transfected with rhTA₁ and CRE-Luc. Drugs of abuse, including amphetamines (amphetamine, methamphetamine, and MDMA), reportedly stimulate rat TA₁-mediated cAMP formation at 1 µM concentrations (Bunzow et al., 1999). To determine whether rhTA₁ is activated by drugs of abuse, we tested whether (+)-amphetamine, (–)-amphetamine, (+)-MDMA, and (–)-MDMA elicited rhTA₁-mediated increases in cAMP formation, as monitored by the CRE-Luc assay. Each drug promoted dose-dependent increases in luciferase expression (Fig. 3) in rhTA₁-transfected cells, but failed to increase luciferase activity in CRE-Luc only-transfected HEK-293 cells (data not shown). PEA and tyramine achieved a signal at lower concentrations (10 nM) than amphetamine or MDMA. The stereoisomers of amphetamine apparently were more potent than (+)- or (–)-MDMA, because rhTA₁ activity was detected with 100 nM of each isomer of amphetamine, whereas rhTA₁ activation by (+)-MDMA required 300 nM and (–)-MDMA required 1000 nM. None of the agonists (tyramine, PEA, amphetamine, mescaline, or MDMA) promoted luciferase production in the absence of rhTA₁. All data with these compounds were obtained from experiments that did not include propranolol pretreatments but were performed in the absence of serum.

View larger version (30K): [in this window] [in a new window]   Fig. 2. Effect of tyramine and PEA (0 nM, 10 nM, 100 nM, and 1 µM) on luciferase activity in HEK-293 cells transiently transfected with rhTA1 and CRE-Luc or in C6 glioma cells transiently transfected with rhTA1 and CRE-Luc. In HEK-293 cells transfected with CRE-Luc only, trace amines elicited no response. Data shown are percentage of increase in RLUs compared with 0 nM drug treatment.

View larger version (27K): [in this window] [in a new window]   Fig. 3. Dose-response effects of (+)-amphetamine (A), (–)-amphetamine (B), (+)-MDMA (C), and (–)-MDMA (D) on luciferase activity in HEK-293 cells transiently transfected with rhTA1 and CRE-Luc. Data shown are percentage of increase in RLUs compared with 0 nM drug treatment. EC50 values using a nonlinear single site competition binding analysis equation for these data are estimated to be 682 nM for (+)amphetamine and 1700 nM for (–)-amphetamine. Comparable data for the isomeric forms of MDMA were not estimated because at the highest concentrations of MDMA (30 µM), the dose-response relationship did not asymptote.

Cocaine Effects on rhTA₁ and [³H]PEA Transport by hDAT. To determine whether another potent stimulant cocaine was a direct agonist at rhTA₁, we transfected HEK-293 cells with rhTA₁ and CRE-Luc and stable hDAT cells with CRE-Luc only, and tested a wide range of doses. In neither cell preparation did cocaine affect CRE-Luc expression (Fig. 4). We reasoned that trace amine levels in brain may be altered by blockade of monoamine transporters, since tyramine has been demonstrated to be a substrate for the dopamine transporter. We were, however, unable to find strong evidence in the literature that PEA serves as a substrate for the dopamine transporter. Accordingly, we tested hDAT cells to determine whether [³H]PEA is a substrate and whether cocaine is an effective blocker of PEA transport by the dopamine transporter. A time course of [³H]PEA transport into hDAT cells was assessed to provide an appropriate incubation period for subsequent studies. [³H]PEA (50 nM) was quickly sequestered by hDAT cells with a peak transport occurring at 8 min and stabilizing thereafter (Fig. 5A; n = 4). Nonspecific binding was measured using 10 µM mazindol to inhibit the hDAT. In subsequent studies, an incubation period of 6 min was used as this time period fell within the linear part of the curve. To ensure the uptake observed was due to the presence of hDAT, we conducted similar experiments in native HEK-293 cells. [³H]PEA was bound by untransfected HEK-293 cells, but unlike the hDAT cells, association was not time-dependent and was less than 50% compared with hDAT cells (Fig. 5B; n = 4). Presumably, this represents nonspecific binding of [³H]PEA seen in hDAT cells in the presence of excess mazindol (Fig. 5A). To ascertain whether transport of [³H]PEA was an energy-dependent process, we compared transport at 0 and at 37°C. Transport of [³H]PEA at 0°C was minimal, suggesting that [³H]PEA was a temperature-dependent process (Fig. 5B; n = 2). Cocaine blocked [³H]dopamine transport in a concentration-dependent manner with moderately high affinity (Fig. 5C; IC₅₀ = 167.3 ± 81.8 nM; n = 4), confirming previous data (Yatin et al., 2002). Intriguingly, cocaine was a more potent inhibitor of [³H]PEA transport, mediated by the dopamine transporter (Fig. 5D; IC₅₀ = 19.8 ± 3.5 nM; n = 4).

View larger version (7K): [in this window] [in a new window]   Fig. 4. Effect of cocaine on luciferase activity in HEK-293 cells transiently transfected with rhTA and CRE-Luc (A) and hDAT cells transiently transfected with CRE-Luc only (B). Data shown are percentage of increase in RLUs compared with 0 nM cocaine treatment.

View larger version (24K): [in this window] [in a new window]   Fig. 5. Characterization of HEK-293 cells stably transfected with the hDAT. A, time course of [3H]PEA transport in HEK-293 cells stably transfected with hDAT. hDAT or native HEK cells were incubated at 37°C with 50 nM [3H]PEA for various times (the curves are representative; n = 4). Nonspecific binding was measured using 10 µM mazindol to inhibit the hDAT (Baseline). B, untransfected HEK-293 cells were incubated at 37°C. The curve is the mean of four independent experiments ± S.E.M.; n = 4. The data are compared with hDAT cells incubated at 0°C (this curve is the mean of two independent experiments, ± S.E.M.) with [3H]PEA for various times. C, representative example of cocaine blockade of [3H]DA transport (IC50 = 167.3 ± 81.8 nM; n = 4). D, representative example of cocaine blockade of [3H]PEA transport (IC50 = 19.8 ± 3.5 nM; n = 4). All experiments were conducted in triplicate.

Cellular Location of an EGFP-rhTAR1 Chimera. In view of the low expression of TA₁ binding sites and previous reports demonstrating that a FLAG-tagged rat TA₁ chimera was localized intracellularly in transfected HEK-293 cells (Bunzow et al., 2001), we investigated the cellular localization of TA₁ in transfected HEK-293 cells. To this end, we constructed an N-terminal EGFP-rhTA₁ chimera containing a SER-SER-GLY-MET linker (Fig. 6A). This linker was selected for methodological convenience, but the additional SER residues in this construct did not erroneously create novel potential phosphorylation sites that could be active if intracellular, according to NetPHOS analysis. Cyclase generating activity of the chimera was tested by cotransfecting it with CRE-Luc and exposing the cells to 1 and 10 µM PEA. Although not as robust as the wild-type receptor, the EGFP-rhTA₁ chimera retained the capacity for PEA activation to increase cAMP production (Fig. 6B). The reduced response of the chimera-expressing cells to 10 µM PEA (p < 0.001 by ANOVA/Tukey's) was a result of either diminished function or fewer moles of chimeric molecules than control rhTA₁ molecules being transfected, because we kept constant the amount of DNA transfected. When the transiently transfected chimera-expressing cells were observed on an inverted fluorescence microscope, EGFP-rhTA₁ expression was visible at 24 h and was more intense at 48 h. At 24 h, EGFP-rhTA₁ distribution seemed largely intracellular in normal-looking cells, although in rare instances, EGFP-rhTA₁ was clearly visible in membranes (Fig. 7A). By 48 h, cells with high EGFP-rhTA₁ expression looked vacuolized and round, the green fluorescent signal being predominant in misshaped cells. By confocal microscopy, the EGFP-rhTA₁ chimeric protein showed robust fluorescence intracellularly in normal-looking HEK-293 cells at 24 h (Fig. 7, B–D).

View larger version (22K): [in this window] [in a new window]   Fig. 6. A, schematic illustrating the artificial linker region of EGFP-rhTA1 chimera and sequence verification. B, response to PEA treatments in rhTA1 and CRE-Luc-cotransfected HEK-293 cells (black columns) and in EGFP-rhTA1 and CRE-Luc-cotransfected HEK-293 cells (gray columns). Data shown are percentage of increase in RLUs compared with 0 nM drug treatment. ***, treatment effects of 10 µM were significantly different from each other (p < 0.001; ANOVA/Tukey's).

View larger version (22K): [in this window] [in a new window]   Fig. 7. Photomicrographs of EGFP-rhTA1 chimera transiently expressed in HEK-293 cells. A, photomicrograph obtained on an inverted fluorescence microscope (Olympus IX70). A rare instance of EGFP-rhTA1 expression observed in the cell membrane at 24 h. B and C, confocal micrographs that indicate intracellular distribution of EFP-rhTA1 expression at 24 h. Green, EGFP-rhTA1; red, nuclei. D, confocal micrographs that indicate intracellular distribution and minimal membrane expression of EFP-rhTA1 expression at 24 h. Green, EGFP-rhTA1; red, membrane staining.

Modulation of rhTA₁ Activity by hDAT. Our observations with EGFP-rhTA₁ and those reported with a FLAG-tagged rat TA₁ chimera (Bunzow et al., 2001), suggested that a high proportion of TA₁ may be localized intracellularly. Conceivably, the access of TA₁ agonists tyramine and PEA to intracellular rhTA₁ could be compromised in the transfected HEK-293 cells. To improve trace amine transport to the intracellular milieu, we simultaneously transfected HEK-293 cells and stably expressing hDAT cells with rhTA₁ and CRE-Luc and conducted side-by-side luciferase assays. In HEK-293 cells transiently transfected with rhTA₁ and CRE-Luc, once again PEA dose-dependently increased cAMP production (Fig. 8A, left; n = 3). In simultaneously and identically treated stable hDAT cells, PEA dose-dependently increased cAMP production, but luciferase response was strikingly potentiated at 1 µM PEA (Fig. 8A, right; p < 0.001 versus rhTA₁ at 1 µM). In sharp contrast, the presence of the hDAT significantly diminished tyramine activation of rhTA1-mediated cAMP production (Fig. 8B; p < 0.01 at 100 nM, p < 0.001 at 1 µM).

View larger version (15K): [in this window] [in a new window]   Fig. 8. A, effect of PEA (0 nM, 10 nM, 100 nM, and 1 µM) on luciferase activity in HEK-293 cells and in stable hDAT cells transiently transfected with rhTA1 and CRE-Luc (rhTA1 and rhTA1/hDAT, respectively). ***, p < 0.001 versus 1 µM rhTA1. B, effect of tyramine (0 nM, 10 nM, 100 nM, and 1 µM) on luciferase activity in HEK-293 cells and in stable hDAT cells transiently transfected with rhTA1 and CRE-Luc (rhTA1 and rhTA1/hDAT, respectively). **, p < 0.01 versus 100 nM rhTA1; ***, p < 0.001 versus 1 µM rhTA1. Data shown are percentage of increase in RLUs compared with 0 nM drug treatment.

To determine whether hDAT influence on TA₁ activity extended to amphetamines, parallel experiments were conducted with (±)-amphetamine and (±)-MDMA in simultaneously and identically treated HEK-293 cells and hDAT cells transiently transfected with rhTA₁ and CRE-Luc. As shown previously, both isomeric forms of amphetamine and MDMA activated TA₁ activity, but the presence of the hDAT significantly potentiated rhTA₁ activity (Fig. 9, A and B).

View larger version (14K): [in this window] [in a new window]   Fig. 9. A, effect of (+)-amphetamine (left) and (–)-amphetamine (0 nM, 10 nM, 100 nM, and 1 µM) on luciferase activity in HEK-293 cells and in stable hDAT cells transiently transfected with rhTA1 and CRE-Luc (rhTA1 and rhTA1/hDAT, respectively). ***, p < 0.001 versus 1 µM rhTA1; *, p < 0.05 versus 1 µM rhTA1. B, effect of (+)-MDMA and (–)-MDMA (0 nM, 10 nM, 100 nM, 1 µM, and 10 µM) on luciferase activity in HEK-293 cells and in stable hDAT cells transiently transfected with rhTA1 and CRE-Luc (rhTA1 and rhTA1/hDAT, respectively). **, p < 0.01 versus 1 µM rhTA1. All data are n = 3, with the exception of 10 µM MDMA (n = 1), which was excluded from statistical analysis (ANOVA/Tukey's). Data shown are percentage of increase in RLUs compared with 0 nM drug treatment.

View larger version (46K): [in this window] [in a new window]   Fig. 10. rhTA1 expression in the rhesus monkey substantia nigra. Real-time PCR followed by melting analysis was performed on a LightCycler 2.0 instrument with SYBR Green. A, rhTA1 amplification curve from substantia nigra (SN) compared with no template control (NTC). B, melting analysis of rhTA1 amplification product. C, region of the dynorphin promoter was amplified to assay for trace genomic contamination. D, melting analysis of reaction products to indicate a higher melting temperature for the genomic product and a near complete absence of specific signal generated from SN cDNA. E, DAT amplification curve from SN compared with NTC. F, melting analysis of DAT amplification product.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
The recent discovery of a family of mammalian trace amine receptors with high affinity for trace amines and activation of rat TA₁ by psychostimulant and hallucinogenic drugs of abuse (Borowsky et al., 2001; Bunzow et al., 2001) presents a significant new resource for exploring the physiological, pathological, and pharmacological relevance of trace amines and their receptors in brain. We postulated that nonhuman primates would offer a more effective preclinical model system for exploring trace amines and their receptors in primate species. In support of this premise, rhTA₁ displayed high structural similarity to hTA₁ and was activated by PEA and tyramine, analogous to their activation of hTA₁. We furthermore demonstrated that primate trace amine receptors may contribute to the stimulant and reinforcing properties of psychostimulant drugs, because amphetamine, MDMA, but not cocaine, activated rhTA₁ at pharmacologically relevant concentrations. Even though we ruled out a direct role of cocaine at TA₁, cocaine may potentially function as an indirect agonist at postsynaptic TA₁ receptors because it effectively blocked PEA transport by the dopamine transporter. We also provide evidence that coexpression of hDAT with rhTA₁ amplified TA₁ activity in an agonist-specific manner, indicative of a potential mechanism for trace amine regulation of monoamine release (Berry, 2004).

The rhTA₁ coding sequence is 96.9% homologous to hTA_1. Previous comparisons of hTA₁ and rodent TA₁ receptors revealed striking differences between coding sequences that are reportedly less than 79% homologous with human. The deduced amino acid sequence of rhTA₁ is 96.4% homologous to human, 78.7% homologous to rat, and 76.0% homologous to mouse sequences. The extracellular N-terminal arm of rhTA₁ and hTA₁ are identical, whereas only 10 of 22 amino acids in mouse TA₁ matched hTA₁. We particularly note this divergence because we have previously found that a single amino acid difference in the N-terminal arm of the µ-opiate receptor, another GPCR, alters affinity of an endogenous ligand in rhesus monkey (Miller et al., 2004), as well as human (Bond et al., 1998). Notable is the absence of a dileucine motif in hTA₁ and rhTA₁, which is present only in mouse TA₁. Accordingly, internalization and processing of the mouse TA₁ may be unique.

The report that amphetamine, methamphetamine, and MDMA activated rodent TA₁ activity introduced a potentially novel and additional mechanism for their psychostimulant and therapeutic properties (Bunzow et al., 2001). Amphetamines are established substrates for monoamine transporters and the vesicular amine transporter-2 as well as other targets (Sulzer et al., 1993; Uhl et al., 2000; Krasnova et al., 2001; Yatin et al., 2002; Green et al., 2003) and their capacity to increase extracellular levels of monoamines via transporter exchange is a widely accepted mechanism of action (Rothman et al., 2001). We found that amphetamine and MDMA activated rhTA1, with amphetamine stereoisomers being more potent than the MDMA isomeric forms. Significantly, the concentration range of amphetamine (100 nM–>10 µM) and MDMA (1–30 µM) used fell within the plasma concentration levels achievable by administration of amphetamine to humans (p.o. approximately 300 nM; Asghar et al., 2003) or MDMA to primates (i.m., approximately 6–12 µM; Bowyer et al., 2003).

The discovery that cocaine is a more potent inhibitor of [³H]PEA transport than [³H]dopamine transport by hDAT (Table 2) supports the view that cocaine may indirectly activate rhTA_1. Accordingly, by blocking trace amine transport and increasing extracellular trace amine levels, cocaine could indirectly activate membrane-bound pre- or postsynaptic trace amine receptors. Alternately, if rhTA₁ is localized intracellularly in dopamine neurons, cocaine may block trace amine efflux through the DAT, thereby increasing the availability of intracellular trace amines. In view of these findings, psychostimulant drugs may directly or indirectly activate rhTA₁. Conceivably, the higher potency of cocaine for inhibiting [³H]PEA compared with [³H]dopamine transport may be attributable to high membrane absorption by [³H]PEA, resulting in significant ligand depletion. Alternately, [³H]PEA transport may promote a conformational change different from that of [³H]dopamine and give rise to increased cocaine accessibility to binding site on the DAT.

The potential interaction between the cloned rhTA₁ receptor and monoamine transporters extended beyond overlapping substrate specificity and influence of transport inhibitors. Converging observations led us to investigate a potential role of the DAT in modulating rhTA₁ activity. A previous report that FLAG-tagged rat TA₁ chimera was localized almost exclusively in the intracellular compartment of transfected HEK-293 cells (Bunzow et al., 2001), suggested that access of agonists to the receptor may require translocation to the cell interior. Accordingly, we directly assessed cellular distribution and activation of an EGFP-rhTA₁ chimera transiently expressed in HEK-293 cells. In conformity with the previous report using a different construct, the majority of fluorescently labeled TA₁ sites were located intracellularly. Detectable rhTA₁ activation by trace amines could be mediated by low levels of extracellular membrane-bound TA₁ interacting with extracellular trace amines or by intracellular TA₁, activated by trace amines that diffuse into the cell in the course of the 20 h used for the CRE-Luc assay. Conceivably, TA₁ activity is enhanced by DAT-driven delivery of tyramine and PEA to intracellular TA₁ receptors. In support of this, we demonstrated dopamine transporter-mediated translocation of [³H]PEA as have others with [³H]tyramine (Sitte et al., 1998). Paradoxically, PEA activation of rhTA₁ was strikingly enhanced by coexpression of the hDAT, whereas tyramine activation of cAMP production was significantly attenuated. Several mechanisms may account for the divergent influence of DAT on PEA or tyramine activation of rhTA₁ receptors. In contrast to tyramine, PEA 1) may be sequestered efficiently by hDAT to intracellular compartments accessible to rhTA₁; 2) may facilitate formation of an active hDAT-rhTA₁ dimer; 3) is more stable in this cell line, although less likely as both amines are of similar potencies in the absence of the hDAT; 4) may promote formation of a rhTA₁-hDAT complex that is relatively ineffective in extruding PEA but effective is releasing tyramine; and 5) may promote rhTA₁ trafficking differently from tyramine, thereby enhancing PEA access to rhTA₁ to a greater extent than tyramine. Intriguingly, rhTA₁ activation was also enhanced by amphetamine and MDMA, furnishing evidence that the rhTA₁-hDAT interaction generalized to exogenous substrates. Thus far, the depression of rhTA₁ activity observed in the presence of hDAT is unique to tyramine and permits speculation on whether the physiological effects of the endogenous trace amines PEA and tyramine are distinguishable in brain and whether different transporters will display different influences on rhTA₁ activity in their presence. Clearly, there are notable differences in the relative concentrations of PEA and tyramine in different brain regions and in concentrations required to promote release or inhibit monoamine transport (Berry, 2004), but a side-by-side comparison of their electrophysiological effects in midbrain dopamine neurons failed to reveal distinct differences (Geracitano et al., 2004). The relevance of these findings to DAT-TA₁ interaction in brain and to brain function in general is unknown. Only trace amounts of TA₁ mRNA were detected by RT-PCR in discrete regions of human brain (Borowsky et al., 2001), but these low levels of expression may be attributable to methodologies, tissue quality, or subject variations. Relevant to our findings, mouse brain TA₁ mRNA is detectable within several monoaminergic cell groups, including the dorsal raphe, locus coeruleus, ventral tegmental area, and substantia nigra (Bunzow et al., 2001). Equally germane to the physiological relevance of rhTA₁-hDAT interaction in HEK-293 cells, is our detection of rhTA₁ expression in the dopamine transporter-rich rhesus monkey substantia nigra by real-time RT-PCR.

In primates, a detailed analysis of the pharmacological specificity of TA₁ and other TA subtypes is a necessary next step in deciphering the physiological and pharmacological relevance of this intriguing class of receptors. Notwithstanding this void, the present study provides evidence that TA₁ is a potential contributor to the effects of drugs of abuse in the primate via at least four putative mechanisms. Psychostimulant drugs of abuse (e.g., amphetamine) may 1) directly activate trace amine receptor subtypes; 2) increase endogenous trace amine levels by modulating monoamine transporter function, thereby indirectly activating trace amine receptors and/or possibly other biogenic amine receptors; 3) increase release or block transport of endogenous biogenic amines that activate trace amine receptors; and 4) alter trace amine levels, thereby modulating release of other neurotransmitters (Juorio and Danielson, 1978). These postulates are predicated on the production and release of trace amines at relevant concentrations. Because amphetamines but not cocaine activate rat TA₁ (Bunzow et al., 2001), this receptor system may differentiate the neurochemical targets and adaptive processes mediated by these two classes of psychostimulants. rhTA₁ functional responses to psychostimulants may have profound relevance to mechanisms of psychostimulant drug action and to physiological and behavioral parameters of human drug addiction. The recent discovery of polymorphisms in human trace amine receptor 4 associated with schizophrenia and a null mutation in the human TA₃ (Vanti et al., 2003; Duan et al., 2004) highlights the need to explore the relationship between molecular and functional properties of the trace amine receptors and neuropsychiatric disorders. Accordingly, we postulate that nonhuman primates will provide an appropriate resource for clarifying the physiological, pharmacological, and pathological relevance of TA subtypes relevant to humans.

	Acknowledgements

 
We thank Teresa Branchek for helpful insights.

	Footnotes

 
This study was supported by Grants DA06303 (to B.K.M.), DA016606 (to G.M.M.), DA15305 (to B.K.M.), and RR00168. The research was presented in abstract form at Society for Neuroscience (2002, 2004), the annual meeting of the College on Problems of Drug Dependence (2004), and the American College of Neuropsychopharmacology (2004).

doi:10.1124/jpet.105.084459.

ABBREVIATIONS: DA, dopamine; NE, norepinephrine; 5-HT, 5-hydroxytryptamine (serotonin); PEA, -phenylethylamine; TA, trace amine; rTA, rat trace amine; hTA, human trace amine; MDMA 3,4-methylenedioxymethamphetamine; TAR, trace amine receptor; PCR, polymerase chain reaction; gDNA, genomic DNA; hDAT, human dopamine transporter; CRE, cAMP response element; Luc, luciferase; HEK, human embryonic kidney; DMEM, Dulbecco's modified Eagle's medium; RLU, relative light unit; EGFP, enhanced green fluorescent protein; RT-PCR, reverse transcriptase-polymerase chain reaction; ANOVA, analysis of variance.

Address correspondence to: Dr. Bertha K. Madras, Department of Psychiatry, New England Primate Research Center, Harvard Medical School, One Pine Hill Dr., Southborough, MA 01772. E-mail: bertha_madras{at}hms.harvard.edu

	References

Top Abstract Materials and Methods Results Discussion References

 

Altar CA, Wasley AM, and Martin LL (1986) Autoradiographic localization and pharmacology of unique [3H]tryptamine binding sites in rat brain. Neuroscience 17: 263–273.[CrossRef][Medline]
Arakawa S, Gocayne JD, McCombie WR, Urquhart DA, Hall LM, Fraser CM, and Venter JC (1990) Cloning, localization and permanent expression of a Drosophila octopamine receptor. Neuron 4: 343–354.[CrossRef][Medline]
Asghar SJ, Tanay VA, Baker GB, Greenshaw A, and Silverstone PH (2003) Relationship of plasma amphetamine levels to physiological, subjective, cognitive and biochemical measures in healthy volunteers. Hum Psychopharmacol 18: 291–299.[CrossRef][Medline]
Axelrod J and Saavedra JM (1977) Octopamine. Nature (Lond) 265: 501–504.[CrossRef][Medline]
Berry MD (2004) Mammalian central nervous system trace amines. Pharmacologic amphetamines, physiologic neuromodulators. J Neurochem 90: 257–271.[CrossRef][Medline]
Blenau W, Balfanz S, and Baumann A (2000) Amtyr1: characterization of a gene from honeybee (Apis mellifera) brain encoding a functional tyramine receptor. J Neurochem 74: 900–908.[CrossRef][Medline]
Blom N, Gammeltoft S, and Brunak S (1999) Sequence and structure-based prediction of eukaryotic protein phosphorylation sites. J Mol Biol 294: 1351–1362.[CrossRef][Medline]
Bond C, LaForge KS, Tian M, Melia D, Zhang S, Borg L, Gong J, Schluger J, Strong JA, Leal SM, et al. (1998) Single-nucleotide polymorphism in the human mu opioid receptor gene alters beta-endorphin binding and activity: possible implications for opiate addiction. Proc Natl Acad Sci USA 95: 9608–9613.[Abstract/Free Full Text]
Borowsky B, Adham N, Jones KA, Raddatz R, Artymyshyn R, Ogozalek KL, Durkin MM, Lakhlani PP, Bonini JA, Pathirana S, et al. (2001) Trace amines: identification of a family of mammalian G protein-coupled receptors. Proc Natl Acad Sci USA 98: 8966–8971.[Abstract/Free Full Text]
Boulton AA (1976) Identification, distribution, metabolism and function of meta and para tyramine, phenylethylamine and tryptamine in brain. Adv Biochem Psychopharmacol 15: 57–67.[Medline]
Bowyer JF, Young JF, Slikker W, Itzak Y, Mayorga AJ, Newport GD, Ali SF, Frederick DL, and Paule MG (2003) Plasma levels of parent compound and metabolites after doses of either d-fenfluramine or d-3,4-methylenedioxymethamphetamine (MDMA) that produce long-term serotonergic alterations. Neurotoxicology 24: 379–390.[CrossRef][Medline]
Branchek TA and Blackburn TP (2003) Trace amine receptors as targets for novel therapeutics: legend, myth and fact. Curr Opin Pharmacol 3: 90–97.[CrossRef][Medline]
Bunzow JR, Sonders MS, Arttamangkul S, Harrison LM, Zhang G, Quigley DI, Darland T, Suchland KL, Pasumamula S, Kennedy JL, et al. (2001) Amphetamine, 3,4-methylenedioxymethamphetamine, lysergic acid diethylamide and metabolites of the catecholamine neurotransmitters are agonists of a rat trace amine receptor. Mol Pharmacol 60: 1181–1188.[Abstract/Free Full Text]
Calligaro DO and Eldefrawi ME (1987) Central and peripheral cocaine receptors. J Pharmacol Exp Ther 243: 61–68.[Abstract/Free Full Text]
Debler EA, Hashim A, Lajtha A, and Sershen H (1988) Ascorbic acid and striatal transport of [3H] 1-methyl-4-phenylpyridine (MPP+) and [3H] dopamine. Life Sci 42: 2553–2559.[CrossRef][Medline]
Duan J, Martinez M, Sanders AR, Hou C, Saitou N, Kitano T, Mowry BJ, Crowe RR, Silverman JM, Levinson DF, et al. (2004) Polymorphisms in the trace amine receptor 4 (TRAR4) gene on chromosome 6q23.2 are associated with susceptibility to schizophrenia. Am J Hum Genet 75: 624–638.[CrossRef][Medline]
Durden DA and Philips SR (1980) Kinetic measurements of the turnover rates of phenylethylamine and tryptamine in vivo in the rat brain. J Neurochem 34: 1725–1732.[Medline]
Eshleman AJ, Carmolli M, Cumbay M, Martens CR, Neve KA, and Janowsky A (1999) Characteristics of drug interactions with recombinant biogenic amine transporters expressed in the same cell type. J Pharmacol Exp Ther 289: 877–885.[Abstract/Free Full Text]
Friedman J, Babu B, and Clark RB (2002) Beta (2)-adrenergic receptor lacking the cyclic AMP-dependent protein kinase consensus sites fully activates extracellular signal-regulated kinase 1/2 in human embryonic kidney 293 cells: lack of evidence for G(s)/G(i) switching. Mol Pharmacol 62: 1094–1102.[Abstract/Free Full Text]
Geracitano R, Federici M, Prisco S, Bernardi G, and Mercuri NB (2004) Inhibitory effects of trace amines on rat midbrain dopaminergic neurons. Neuropharmacology 46: 807–814.[CrossRef][Medline]
Gerhardt CC, Lodder HC, Vincent M, Bakker RA, Planta RJ, Vreugdenhil E, Kits KS, and van Heerikhuizen H (1997) Cloning and expression of a complementary DNA encoding a molluscan octopamine receptor that couples to chloride channels in HEK293 cells. J Biol Chem 272: 6201–6207.[Abstract/Free Full Text]
Green AR, Mechan AO, Elliott JM, O'Shea E, and Colado MI (2003) The pharmacology and clinical pharmacology of 3,4-methylenedioxymethamphetamine (MDMA, "ecstasy"). Pharmacol Rev 55: 463–508.[Abstract/Free Full Text]
Han KA, Millar NS, and Davis RL (1998) A novel octopamine receptor with preferential expression in Drosophila mushroom bodies. J Neurosci 18: 3650–3658.[Abstract/Free Full Text]
Hauger RL, Skolnick P, and Paul SM (1982) Specific [3H]beta-phenylethylamine binding sites in rat brain. Eur J Pharmacol 83: 147–148.[CrossRef][Medline]
Hyttel J (1978) Inhibition of [3H]dopamine accumulation in rat striatal synaptosomes by psychotropic drugs. Biochem Pharmacol 27: 1063–1068.[CrossRef][Medline]
Jones RS and Boulton AA (1980) Interactions between p-tyramine, m-tyramine, or beta-phenylethylamine and dopamine on single neurones in the cortex and caudate nucleus of the rat. Can J Physiol Pharmacol 58: 222–227.[Medline]
Juorio AV (1976) Presence and metabolism of beta-phenylethylamine, p-tyramine, m-tyramine and tryptamine in the brain of the domestic fowl. Brain Res 111: 442–445.[CrossRef][Medline]
Juorio AV and Danielson TJ (1978) Effect of haloperidol and d-amphetamine on cerebral tyramine and octopamine levels. Eur J Pharmacol 50: 79–82.[CrossRef][Medline]
Karoum F, Chuang LW, and Wyatt RJ (1981) Presence and distribution of phenylethylamine in the rat spinal cord. Brain Res 225: 442–445.[CrossRef][Medline]
Kellar KJ and Cascio CS (1982) [3H]Tryptamine: high affinity binding sites in rat brain. Eur J Pharmacol 78: 475–478.[CrossRef][Medline]
Krasnova IN, Ladenheim B, Jayanthi S, Oyler J, Moran TH, Huestis MA, and Cadet JL (2001) Amphetamine-induced toxicity in dopamine terminals in CD-1 and C57BL/6J mice: complex roles for oxygen-based species and temperature regulation. Neuroscience 107: 265–274.[CrossRef][Medline]
Kreegipuu A, Blom N, and Brunak S (1999) PhosphoBase, a database of phosphorylation sites: release 2.0. Nucleic Acids Res 27: 237–239.[Abstract/Free Full Text]
Matecka D, Rothman RB, Radesca L, de Costa BR, Dersch CM, Partilla JS, Pert A, Glowa JR, Wojnicki FH, and Rice KC (1996) Development of novel, potent and selective dopamine reuptake inhibitors through alteration of the piperazine ring of 1-[2-(diphenylmethoxy)ethyl]-and 1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-(3-phenylpropyl)piperazines (GBR 12935 and GBR 12909). J Med Chem 39: 4704–4716.[CrossRef][Medline]
Miller GM, Bendor J, Tiefenbacher S, Yang H, Novak MA, and Madras BK (2004) A mu-opioid receptor single nucleotide polymorphism in rhesus monkey: association with stress response and aggression. Mol Psychiatry 9: 99–108.[CrossRef][Medline]
Perry DC (1986) [3H]Tryptamine autoradiography in rat brain and choroid plexus reveals two distinct sites. J Pharmacol Exp Ther 236: 548–559.[Abstract/Free Full Text]
Rothman RB, Baumann MH, Dersch CM, Romero DV, Rice KC, Carroll FI, and Partilla JS (2001) Amphetamine-type central nervous system stimulants release norepinephrine more potently than they release dopamine and serotonin. Synapse 39: 32–41.[CrossRef][Medline]
Saudou F, Amlaiky N, Plassat JL, Borrelli E, and Hen R (1990) Cloning and characterization of a Drosophila tyramine receptor. EMBO (Eur Mol Biol Organ) J 9: 3611–3617.[Medline]
Sitte HH, Huck S, Reither H, Boehm S, Singer EA, and Pifl C (1998) Carrier-mediated release, transport rates, and charge transfer induced by amphetamine, tyramine and dopamine in mammalian cells transfected with the human dopamine transporter. J Neurochem 71: 1289–1297.[Medline]
Sulzer D, Maidment NT, and Rayport S (1993) Amphetamine and other weak bases act to promote reverse transport of dopamine in ventral midbrain neurons. J Neurochem 60: 527–535.[Medline]
Uhl GR, Li S, Takahashi N, Itokawa K, Lin Z, Hazama M, and Sora I (2000) The VMAT2 gene in mice and humans: amphetamine responses, locomotion, cardiac arrhythmias, aging and vulnerability to dopaminergic toxins. FASEB J 14: 2459–2465.[Abstract/Free Full Text]
Ungar F, Mosnaim AD, Ungar B, and Wolf ME (1977) Tyramine-binding by synaptosomes from rat brain: effect of centrally active drugs. Biol Psychiatry 12: 661–668.[Medline]
Vanti WB, Muglia P, Nguyen T, Cheng R, Kennedy JL, George SR, and O'Dowd BF (2003) Discovery of a null mutation in a human trace amine receptor gene. Genomics 82: 531–536.[CrossRef][Medline]
Yatin SM, Miller GM, Norton C, and Madras BK (2002) Dopamine transporter-dependent induction of C-Fos in HEK cells. Synapse 45: 52–65.[CrossRef][Medline]

This article has been cited by other articles:

				T. D. Sotnikova, M. G. Caron, and R. R. Gainetdinov Trace Amine-Associated Receptors as Emerging Therapeutic Targets Mol. Pharmacol., August 1, 2009; 76(2): 229 - 235. [Abstract] [Full Text] [PDF]

				Z. Xie and G. M. Miller A Receptor Mechanism for Methamphetamine Action in Dopamine Transporter Regulation in Brain J. Pharmacol. Exp. Ther., July 1, 2009; 330(1): 316 - 325. [Abstract] [Full Text] [PDF]

				J. J. Maguire, W. A. E. Parker, S. M. Foord, T. I. Bonner, R. R. Neubig, and A. P. Davenport International Union of Pharmacology. LXXII. Recommendations for Trace Amine Receptor Nomenclature Pharmacol. Rev., March 1, 2009; 61(1): 1 - 8. [Abstract] [Full Text] [PDF]

				L. S. Barak, A. Salahpour, X. Zhang, B. Masri, T. D. Sotnikova, A. J. Ramsey, J. D. Violin, R. J. Lefkowitz, M. G. Caron, and R. R. Gainetdinov Pharmacological Characterization of Membrane-Expressed Human Trace Amine-Associated Receptor 1 (TAAR1) by a Bioluminescence Resonance Energy Transfer cAMP Biosensor Mol. Pharmacol., September 1, 2008; 74(3): 585 - 594. [Abstract] [Full Text] [PDF]

				Z. Xie and G. M. Miller {beta}-Phenylethylamine Alters Monoamine Transporter Function via Trace Amine-Associated Receptor 1: Implication for Modulatory Roles of Trace Amines in Brain J. Pharmacol. Exp. Ther., May 1, 2008; 325(2): 617 - 628. [Abstract] [Full Text] [PDF]

				Z. Xie, S. V. Westmoreland, and G. M. Miller Modulation of Monoamine Transporters by Common Biogenic Amines via Trace Amine-Associated Receptor 1 and Monoamine Autoreceptors in Human Embryonic Kidney 293 Cells and Brain Synaptosomes J. Pharmacol. Exp. Ther., May 1, 2008; 325(2): 629 - 640. [Abstract] [Full Text] [PDF]

				C. D Verrico, L. Lynch, M. A Fahey, A.-K. Fryer, G. M Miller, and B. K Madras MDMA-induced impairment in primates: antagonism by a selective norepinephrine or serotonin, but not by a dopamine/norepinephrine transport inhibitor J Psychopharmacol, March 1, 2008; 22(2): 187 - 202. [Abstract] [PDF]

				E. A. Reese, J. R. Bunzow, S. Arttamangkul, M. S. Sonders, and D. K. Grandy Trace Amine-Associated Receptor 1 Displays Species-Dependent Stereoselectivity for Isomers of Methamphetamine, Amphetamine, and Para-Hydroxyamphetamine J. Pharmacol. Exp. Ther., April 1, 2007; 321(1): 178 - 186. [Abstract] [Full Text] [PDF]

				Z. Xie, S. V. Westmoreland, M. E. Bahn, G.-L. Chen, H. Yang, E. J. Vallender, W.-D. Yao, B. K. Madras, and G. M. Miller Rhesus Monkey Trace Amine-Associated Receptor 1 Signaling: Enhancement by Monoamine Transporters and Attenuation by the D2 Autoreceptor in Vitro J. Pharmacol. Exp. Ther., April 1, 2007; 321(1): 116 - 127. [Abstract] [Full Text] [PDF]

				Z. Xie and G. M. Miller Trace Amine-Associated Receptor 1 Is a Modulator of the Dopamine Transporter J. Pharmacol. Exp. Ther., April 1, 2007; 321(1): 128 - 136. [Abstract] [Full Text] [PDF]

				B. K. Madras, Z. Xie, Z. Lin, A. Jassen, H. Panas, L. Lynch, R. Johnson, E. Livni, T. J. Spencer, A. A. Bonab, et al. Modafinil Occupies Dopamine and Norepinephrine Transporters in Vivo and Modulates the Transporters and Trace Amine Activity in Vitro J. Pharmacol. Exp. Ther., November 1, 2006; 319(2): 561 - 569. [Abstract] [Full Text] [PDF]

				H. A. Navarro, B. P. Gilmour, and A. H. Lewin A Rapid Functional Assay for the Human Trace Amine-Associated Receptor 1 Based on the Mobilization of Internal Calcium J Biomol Screen, September 1, 2006; 11(6): 688 - 693. [Abstract] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.105.084459v1 313/3/983    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Miller, G. M. Articles by Madras, B. K. Search for Related Content PubMed PubMed Citation Articles by Miller, G. M. Articles by Madras, B. K.