Normal thermoregulatory responses to 3-iodothyronamine, trace amines and amphetamine-like psychostimulants in trace amine associated receptor 1 knockout mice

Abstract

3-Iodothyronamine (T1AM) is a metabolite of thyroid hormone. It is an agonist at trace amine-associated receptor 1 (TAAR1), a recently identified receptor involved in monoaminergic regulation and a potential novel therapeutic target. Here, T1AM was studied using rhesus monkey TAAR1 and/or human dopamine transporter (DAT) co-transfected cells, and wild-type (WT) and TAAR1 knock-out (KO) mice. The IC(50) of T1AM competition for binding of the DAT-specific radio-ligand [(3)H]CFT was highly similar in DAT cells, WT striatal synaptosomes and KO striatal synaptosomes (0.72-0.81 microM). T1AM inhibition of 10 nM [(3)H]dopamine uptake (IC(50): WT, 1.4 + or - 0.5 microM; KO, 1.2 + or - 0.4 microM) or 50 nM [(3)H]serotonin uptake (IC(50): WT, 4.5 + or - 0.6 microM; KO, 4.7 + or - 1.1 microM) in WT and KO synaptosomes was also highly similar. Unlike other TAAR1 agonists that are DAT substrates, TAAR1 signaling in response to T1AM was not enhanced in the presence of DAT as determined by CRE-luciferase assay. In vivo, T1AM induced robust hypothermia in WT and KO mice equivalently and dose dependently (maximum change degrees Celsius: 50 mg/kg at 60 min: WT -6.0 + or - 0.4, KO -5.6 + or - 1.0; and 25 mg/kg at 30 min: WT -2.7 + or - 0.4, KO -3.0 + or - 0.2). Other TAAR1 agonists including beta-phenylethylamine (beta-PEA), MDMA (3,4-methylenedioxymethamphetamine) and methamphetamine also induced significant, time-dependent thermoregulatory responses that were alike in WT and KO mice. Therefore, TAAR1 co-expression does not alter T1AM binding to DAT in vitro nor T1AM inhibition of [(3)H]monoamine uptake ex vivo, and TAAR1 agonist-induced thermoregulatory responses are TAAR1-independent. Accordingly, TAAR1-directed compounds will likely not affect thermoregulation nor are they likely to be cryogens.

Figure 1. Activation of TAAR1 by T1AM and comparison to β-PEA

T1AM and β-PEA activation of TAAR1 and TAAR1-DAT cells was determined by a CRE-Luc reporter assay and are reported as percent change in relative light units (RLU). A. T1AM (10 μM) resulted in a 680% increase in RLU in TAAR1 cells versus a 760% RLU increase in TAAR1-DAT cells, whereas β-PEA (10 μM) resulted in a 531% increase in RLU in TAAR1 cells and 1750% increase in RLU response in TAAR1-DAT cells (n=3). B and C. TAAR1-DAT cells were exposed to various DAT blockers at (10 μM) or to vehicle (no DAT blocker), and were then treated with T1AM (10μM) or β-PEA (10μM). Pretreatment with DAT blockers had no significant effect on the response to 10 μM T1AM in TAAR1-DAT cells, whereas all DAT blockers significantly attenuated the enhanced response to 10 μM β-PEA in the TAAR1-DAT cells (n=3). All data shown are values of mean ± SEM. **: p < 0.01; ***: p < 0.001.

Figure 2. T1AM binding and uptake in cells and synaptosomes

A. T1AM binding to the dopamine transporter was measured by competitive binding versus [³H]CFT in DAT/HEK cells. The IC₅₀ was 8.0±1.2 × 10^-7 M (n=2). B. The IC₅₀ of T1AM versus [³H]dopamine was 1.4±0.5 × 10^-6 M for WT and 1.2±0.4 × 10^-6 M for KO striatal synaptosomes (n=2). C. The IC₅₀ of T1AM versus [³H]serotonin was 4.5±0.6 × 10^-6 M for WT and 4.6±1.1 × 10^-6 M for KO striatal synaptosomes (n=3). All data shown are values of mean ± SEM.

Figure 3. Thermoregulatory responses of T1AM, β-PEA, tyramine, MDMA, methamphetamine and saline in WT and KO mice

Wild type (WT) and TAAR1 knockout (KO) male mice were treated with 25 mg/kg or 50 mg/kg T1AM (A), 50 mg/kg β-PEA (B), 50 mg/kg tyramine (C), 25 mg/kg MDMA (D), 3 mg/kg methamphetamine (E) or saline (F). The number of mice in each group is indicated. Body temperature readings were captured using a wireless infra-red thermometer held over the shaved back area of each mouse at 5 minutes prior to the drug injection (-5) and at 15, 30, 45, 60, 75, 90, 120, 150, 180, 240, and 320 minutes post injection. Data was normalized to the saline controls (shown in F). A two-way ANOVA with repeated measures was used to determine if there was a significant difference in the thermoregulatory responses over all time points between WT and KO mice and across different drug treatments. There were no significant main or interactive effects. For each drug treatment, a one way ANOVA was used to determine statistical significance of individual time points versus the baseline (-5) temperature (detailed in supplemental Table S1). All data shown are values of mean ± SEM. *, p < 0.05 or lower versus the -5 time point for both WT and KO analyses except as indicated: in A, ns refers to KO data; in D and E, ns refers to WT data. See supplemental Table S1 for more detail.