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Normal Thermoregulatory Responses to 3-Iodothyronamine, Trace Amines and Amphetamine-Like Psychostimulants in Trace Amine Associated Receptor 1 Knockout Mice

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Normal Thermoregulatory Responses to 3-Iodothyronamine, Trace Amines and Amphetamine-Like Psychostimulants in Trace Amine Associated Receptor 1 Knockout Mice Journal of Neuroscience Research 88:1962–1969 (2010) Normal Thermoregulatory Responses to 3-iodothyronamine, Trace Amines and Amphetamine-like Psychostimulants in Trace Amine Associated Receptor 1 Knockout Mice Helen N. Panas,1 Laurie J. Lynch,1 Eric J. Vallender,1 Zhihua Xie,1 Guo-lin Chen,1 Spencer K. Lynn,2 Thomas S. Scanlan,3 and Gregory M. Miller1* 1Division of Neuroscience, Harvard Medical School/NEPRC, Southborough, Massachusetts 2Molecular Pharmacology Laboratory, McLean Hospital, Harvard Medical School, Belmont, Massachusetts and Interdisciplinary Affective Science Laboratory, Department of Psychology, Boston College, Chestnut Hill, Massachusetts 3Department of Physiology & Pharmacology, Oregon Health and Science University, Portland, Oregon 3-Iodothyronamine (T1AM) is a metabolite of thyroid hor- 3-Iodothyronamine (T1AM) is an endogenous de- mone. It is an agonist at trace amine-associated receptor rivative of thyroid hormone and itself a possible chemi- 1 (TAAR1), a recently identified receptor involved in mono- cal messenger (Scanlan et al., 2004). In various studies in aminergic regulation and a potential novel therapeutic tar- which the biological response to T1AM has been meas- get. Here, T1AM was studied using rhesus monkey ured, changes in temperature, heart rate and cardiac out- TAAR1 and/or human dopamine transporter (DAT) co- put have been observed. Most strikingly, T1AM pro- transfected cells, and wild-type (WT) and TAAR1 knock- duces a profound hypothermic response when adminis- out (KO) mice. The IC50 of T1AM competition for binding tered in vivo (Scanlan et al., 2004). The robust of the DAT-specific radio-ligand [3H]CFT was highly similar hypothermic action of T1AM suggests that the thyron- in DAT cells, WT striatal synaptosomes and KO striatal amine could serve as a neuroprotectant, and that its re- synaptosomes (0.72–0.81 lM). T1AM inhibition of 10 nM ceptor could be a potential therapeutic target for cryo- 3 [ H]dopamine uptake (IC50:WT,1.46 0.5 lM; KO, 1.2 6 genic agents. In this regard, T1AM has been assessed for 3 0.4 lM) or 50 nM [ H]serotonin uptake (IC50:WT,4.56 its potential neuroprotective action in a stroke injury 0.6 lM; KO, 4.7 6 1.1 lM) in WT and KO synaptosomes model. Treatment with T1AM was found to reduce was also highly similar. Unlike other TAAR1 agonists that infarct volume by about one third when tested in a mid- are DAT substrates, TAAR1 signaling in response to T1AM dle cerebral artery occlusion stroke model in mice was not enhanced in the presence of DAT as determined (Doyle et al., 2007). The study by Doyle et al. (2007) by CRE-luciferase assay. In vivo, T1AM induced robust was the first to use endogenous chemicals including hypothermia in WT and KO mice equivalently and dose dependently (maximum change degrees Celsius: 50 mg/ Additional Supporting Information may be found in the online version kg at 60 min: WT 26.0 6 0.4, KO 25.6 6 1.0; and 25 of this article. mg/kg at 30 min: WT 22.7 6 0.4, KO 23.0 6 0.2). Other The first two authors contributed equally to this work. TAAR1 agonists including beta–phenylethylamine (b-PEA), Contract grant sponsor: National Institute on Drug Abuse; Contract MDMA (3,4-methylenedioxymethamphetamine) and meth- grant sponsor: National Institute of Diabetes and Digestive Kidney Dis- amphetamine also induced significant, time-dependent eases; Contract grant sponsor: National Center for Research Resources; thermoregulatory responses that were alike in WT and KO Contract grant number: RR00168; Contract grant number: DA022323 mice. Therefore, TAAR1 co-expression does not alter (to G.M.M.); Contract grant number: DA016606 (to G.M.M.); Contract T1AM binding to DAT in vitro nor T1AM inhibition of grant number: DA025802 (to G.M.M.); Contract grant number: DA025697 (to G.M.M.); Contract grant number: DK52798 (to T.S.S.). [3H]monoamine uptake ex vivo, and TAAR1 agonist- induced thermoregulatory responses are TAAR1-inde- *Correspondence to: Gregory M. Miller, New England Primate pendent. Accordingly, TAAR1-directed compounds will Research Center, Harvard Medical School, One Pine Hill Drive, South- likely not affect thermoregulation nor are they likely to be borough, MA. E-mail: [email protected] cryogens. VC 2010 Wiley-Liss, Inc. Received 22 October 2009; Revised 30 November 2009; Accepted 2 December 2009 Key words: TAAR1; thyronamine; psychostimulant; Published online 12 February 2010 in Wiley InterScience (www. thermoregulation; monoamine interscience.wiley.com). DOI: 10.1002/jnr.22367 ' 2010 Wiley-Liss, Inc. TAAR1 and Thermoregulation 1963 T1AM to induce prophylactic hypothermia to treat MATERIALS AND METHODS stroke, and to suggest a therapeutic use of T1AM as a Animals cryogen. The body’s varied and rapid response to T1AM, as well as the potential use of thyronamine as a Animals were approximately 3 months old at the time cryogenic agent has made determining the mechanism of of the studies and only males were tested. TAAR1 KO and action of T1AM as well as its biological role a relevant WT mouse colonies were established at the New England Pri- goal of recent experimental work. mate Research Center (Southborough, MA) from six pairs of Central to recent studies of T1AM is the specula- heterozygous mice received as a gift from Lundbeck Research USA, Inc. (Paramus, NJ). All animals were maintained on a tion of the involvement of trace amine associated recep- 6 8 tor 1 (TAAR1) in its mechanism of action. TAAR1 is a 12-hr light/dark schedule at a room temperature of 22 1 C G protein-coupled receptor that is activated by a wide with free access to food and water. Animal care was in ac- spectrum of compounds, including common biogenic cordance with the Guide for the Care and Use of Laboratory amines, trace amines, and amphetamine-like psychosti- Animals (National Research Council, National Academy mulant drugs, and plays an important role in modulating Press, 1996) and all procedures were conducted in accordance brain monoamine systems (reviewed in Xie and Miller, with the Animal Experimentation Protocol approved by the 2009a). T1AM has been found to be a potent agonist of Harvard Medical Area Standing Committee on Animals. rat and mouse TAAR1 (EC50: 14 nM and 112 nM, Synaptosome Preparation respectively) and to stimulate cAMP production in rat and mouse TAAR1-expressing cells in a dose dependent Synaptosomes were prepared from adult male TAAR1 manner (Scanlan et al., 2004). It is also recognized that KO and WT mice as described previously (Xie and Miller, 2008). Briefly, freshly dissected striatal tissue was homogenized T1AM is a monoamine transporter inhibitor, making 3 this TAAR1 agonist different from other TAAR1 ago- in cold 0.32 M sucrose (10 volume) using a Kontes Pellet Pestle with 15 up and down strokes. The mixture was centri- nists such as endogenous monoamines (e.g., trace amines 8 and common biogenic amines) and exogenous ampheta- fuged at low speed (1,000g, 10 min, 4 C) and the supernatant carefully removed and centrifuged at high speed (10,000g, mine-like psychostimulants (e.g., amphetamine and 8 methamphetamine) that are monoamine transporter sub- 20 min, 4 C) to yield the pelleted synaptosomes. The pellet strates (Snead et al., 2007; Xie et al., 2007). Accordingly, was resuspended in ice-cold uptake buffer (25 mM HEPES, 120 mM NaCl, 5 mM KCl, 2.5 mM CaCl2, 1.2 mM the demonstrated effects of T1AM could be mediated by l TAAR1 or could be a result of altered monoamine con- MgSO4,1 M pargyline, 2 mg/ml glucose, 0.2 mg/mL ascor- centration via blockade of monoamine transporters, or bic acid, pH 7.5) and used immediately in assays. All reagents some other mechanism. Also, TAAR1 is under current and the pestles used in the preparation of synaptosomes were investigation as a potential target for novel therapeutics purchased from Sigma-Aldrich (St. Louis, MO). which may regulate monoaminergic function, and so determining whether TAAR1 mediates thermoregula- Cell Culture tory responses is important with regard to medications Cell lines were generated as described previously (Xie development. et al., 2008). Cell lines were cultured in untreated tissue cul- Accordingly, we took advantage of a TAAR1 ture dishes (Greiner America, Inc., Lake Mary, FL) in knockout mouse model to directly address the question DMEM supplemented with 10% fetal bovine serum, 100 of whether TAAR1 mediates the thermoregulatory units/ml penicillin, 100 lg/ml streptomycin and 0.1 mM response of T1AM in vivo, and compared responses of nonessential amino acids. Cell transfections were performed T1AM to two trace amines, tyramine and beta-phenyle- using calcium phosphate (250 mM calcium chloride and 23 thylamine (b-PEA), and two amphetamine-like psychos- HBS buffer [50 mM HEPES, 1.5 mM Na2HPO4 (Fisher Sci- timulants, methamphetamine and MDMA, which also entific, Pittsburgh, PA), 280 mM NaCl, 10 mM KCl, 12 mM affect thermoregulation. We verified the interaction of glucose, pH 7.05]). With the exception of reagents noted T1AM with TAAR1 and monoamine transporters in above, all reagents were obtained from Invitrogen Corpora- transfected cells and striatal synaptosomes prepared from tion, Carlsbad, CA. wild-type (WT) and TAAR1 knockout mice (KO). We measured cAMP production using a CRE-Luciferase Dual Luciferase Reporter Assays (CRE-Luc) reporter assay in TAAR1 and TAAR1/ Cells were plated into 48-well plates (75,000 cells/well DAT cells after exposure to T1AM and assessed whether in 0.5 mL DMEM) 24 hr prior to transfection. On the fol- DAT could potentiate TAAR1 signaling in response to lowing day the cells were transfected with CRE-Luc, a re- T1AM, as is the case for other TAAR1 agonists that are porter control construct pGL4.73 (cAMP irresponsive) and ei- DAT substrates (Miller et al., 2005; Xie et al., 2007).
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