(T3) and Thyroxine (T4) in the Human Choriocarcinoma Cell Line
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487 Different transporters for tri-iodothyronine (T3) and thyroxine (T4)in the human choriocarcinoma cell line, JAR K A Powell1, A M Mitchell1, S W Manley2 and R H Mortimer1,3 1Conjoint Endocrine Laboratory, Royal Brisbane Hospital Research Foundation, The Bancroft Centre, 300 Herston Road, Brisbane, Queensland 4029, Australia 2Department of Physiology, The University of Queensland, Queensland 4072, Australia 3Department of Pharmacology, Obstetrics and Gynaecology, The University of Queensland, Queensland 4072, Australia (Requests for offprints should be addressed to K A Powell; Email: [email protected]) Abstract We investigated transport systems for tri-iodothyronine In contrast, the Km for T3 uptake was increased from (T3) and thyroxine (T4) in the human choriocarcinoma a control value of around 300 nM to around 750 nM cell line, JAR, using a range of structurally similar com- (n=4). Diazepam had similar effects. This divergent ffi pounds to determine whether these thyroid hormones shift in a nity for the uptake of T3 and T4 suggested are transported by common or different mechanisms. that separate uptake systems exist for these two thyroid Saturable T3 but not saturable T4 uptake was inhibited hormones. by a wide range of aromatic compounds (nitrendipine, This provides evidence for at least two transporters nifedipine, verapamil, meclofenamic acid, mefenamic mediating uptake of T3 and T4 in JAR cells: a specific T4 acid, diazepam, phenytoin). transporter that does not interact with T3 or structurally Nitrendipine and diazepam were the most similar compounds; and a shared iodothyronine transporter ff e ective inhibitors of saturable thyroid hormone uptake. that interacts with T3,T4, nitrendipine and diazepam. Nitrendipine decreased the Km for T4 uptake from a Journal of Endocrinology (2000) 167, 487–492 control value of around 500 nM to around 300 nM (n=6). Introduction synaptosomes and cardiac myocytes (Kragie 1994 (re- view), Everts et al. 1996) actively transport T3 while T4 is ff Although transfer of thyroxine (T4) from maternal to foetal probably taken up by simple di usion. Separate transport circulation in the perfused placenta is minimal (Mortimer mechanisms for T3,T4 and rT3 have been described in et al. 1996), T4 has been found in cord blood of infants human liver (Kaptein 1997) and separate membrane unable to synthesise their own hormone (Vulsma et al. transporters mediating uptake of T3 and T4 are also 1989). While this provides convincing evidence for the suggested for rat hepatocytes (Krenning et al. 1981). These passage of maternal thyroid hormone across the placenta, at findings taken together suggest that the processes involved least to a foetus lacking thyroid hormone, the mechanisms in uptake and efflux of thyroid hormones may be relatively mediating this transfer are not well understood. specific for individual thyroid hormones and may be We have previously described membrane transporters different depending on tissue type. for thyroid hormones in human trophoblast and the Our previous studies have shown that thyroid hormones human choriocarcinoma cell line, JAR. Transport of are taken up into JAR cells by at least two transporters ff ffi tri-iodothyronine (T3), T4 and reverse tri-iodothyronine with di ering a nities (Mitchell et al. 1999a,b). The aim (rT3) into JAR cells and export of T3 from the cells occurs of this study was to determine if T3 and T4 were ffl by saturable processes. E ux of T4 and rT3, on the other transported by a distinct or shared mechanism in order to hand, occurs by passive diffusion (Mitchell et al. 1992, further characterise iodothyronine transport in JAR cells. 1995, 1999a,b). Membrane transport of thyroid hormone has now been characterised in a wide range of cells, with Materials and Methods reported differences in thyroid hormone uptake and efflux kinetics between and within cell types. In hepatocytes, Reagents for example, uptake of T3 is energy dependent and ffl ff T3 e ux occurs by passive di usion (Hennemann et al. Materials were purchased from the following sources: 125 125 1984). Human and rat lymphocytes and erythrocytes, rat I-T3 (3300 µCi/µg) and I-T4 (1250 µCi/µg) from Journal of Endocrinology (2000) 167, 487–492 Online version via http://www.endocrinology.org 0022–0795/00/0167–487 2000 Society for Endocrinology Printed in Great Britain Downloaded from Bioscientifica.com at 09/26/2021 12:17:42AM via free access 488 K A POWELL and others · Specific thyroxine transporter in JAR cells Du Pont Company, Wilmington, DE, USA; foetal calf reactive red, reactive blue, acid blue and 50 µM iopanoic serum from Commonwealth Serum Laboratories, acid. Drugs and dyes were dissolved in ethanol and diluted Melbourne, Victoria, Australia; bicinchoninic acid Protein in HBSS. The final concentration of ethanol did not ff Reagent from Pierce Chemicals, Rockford, IL, USA; and exceed 0·4%, which had no e ect on cellular uptake of T3 six-well tissue culture plates from Costar, Cambridge, or T4. Results from three to seven determinations were MA, USA. All other chemicals and cell culture media pooled. Further characterisation of the transport system were from Sigma Chemicals, St Louis, MO, USA. was carried out using inhibitor studies to determine the Nitrendipine was a gift from Bayer Pharmaceutical kinetic parameters of inhibition of thyroid hormone Division, West Haven, CO, USA. uptake by various compounds. The cells were incubated 125 for 2 min in the presence of 30 pM I-T3 or 50 pM 125 I-T4 and unlabelled T3 and T4 (0–10 µM) with or Cell culture without 100 µM final concentration of unlabelled The JAR cell line was purchased from American Type nitrendipine and 80–100 µM final concentration of Culture Collection, Rockville, MD, USA. Cells were diazepam. Results from 4–8 determinations were pooled maintained in continuous culture at 37 C in humidified and data fitted to the Michaelis–Menten equation using atmosphere of 95% air and 5% CO2. Growth medium was a non-linear curve-fitting program (GraphPad Prism, RPMI 1640 supplemented with 10% (v/v) foetal calf GraphPad, San Diego, CA, USA). 125 serum. Cells were subcultured three times a week. We sought evidence of metabolism of I-T3 and 5 125 For uptake experiments, 3 10 cells were plated into I-T4 by duplicate cultures of JAR cells during uptake each well of the six-well tissue culture plates. Medium experiments by analysing radioactivity present in the cells was changed 24 h after plating, with cells cultured for and in the medium after incubation with the tracers for 2–3 days. At the end of the uptake experiments, viability 30 min at 37 C. As in our previous studies, we found of the cells was assessed by the trypan blue exclusion test evidence of only minimal metabolism of tracers by the and was always over 90%. cells. Uptake studies Determination of the cellular protein content The procedure for uptake studies and the determination of The protein content of cell lysates was determined with the kinetic parameters (Michaelis constant, Km, and the the bicinchoninic acid reagent (Pierce Chemicals) which maximum velocity, Vmax) of the initial cellular uptake of isamodification of the Biuret reaction using BSA as a 125 125 I-T3 and I-T4 were as previously described (Mitchell standard. et al. 1992, 1995). In brief, prior to all uptake experiments cells were incubated for 1 h in Hanks’ balanced salts solution (HBSS). All incubations were carried out at Statistical analysis 37 C. To terminate uptake, incubation medium was Statistical analysis was performed by Student’s t-test and aspirated, cells were washed twice in ice-cold HBSS and one-way ANOVA followed by multiple comparison of immediately lysed in 1 M NaOH. Cell-associated radio- means against a single control (Bonferonni’s t-test) using activity was determined by counting the radioactivity in a the statistical software package SigmaStat (Jandel Scien- Packard -counter with a counting efficiency of 84%. tific, San Rafael, CA, USA). Results were expressed as 125I-labelled thyroid hormone taken up was expressed as means and standard errors of the mean, and n is the femtomoles per minute per milligram of cellular protein number of independent determinations, each in triplicate. (fmol/min per mg protein). The specificity of the A probability of <0·05 was regarded as significant. uptake process was examined by incubating cells for 125 30 min in the presence of 30 pM I-T3 or 50 pM 125 I-T4, with or without 10 µM excess of unlabelled Results thyroid hormones, 10 mM unlabelled amino acids, 125 125 tryptophan, phenylalanine, leucine, glycine, alanine, Uptake of I-T3 and I-T4 in JAR cells was inhibited glutamine, -(methylamino)-isobutyric acid (Me-AIB) by the L-system amino acids tryptophan, phenylalanine, and 2-amino-2-norbornane-carboxylic acid (BCH). The leucine and the synthetic amino acid analogue 2-amino- effect of various compounds on the initial rate of specific 2-norbornane-carboxylic acid (BCH). Tryptophan was ff 125 uptake of thyroid hormone was determined by incubating the most e ective inhibitor, reducing saturable I-T3 125 125 cells for 2 min in the presence of 30 pM I-T3 or 50 pM and I-T4 uptake by 85·5% (n=3) and 63·6% (n=4) 125 ff I-T4 with or without 10 µM unlabelled thyroid respectively. The e ects of L-system amino acids on hormone and with or without 100 µM nitrendipine, thyroid hormone uptake are shown in Fig. 1. Amino acids nifedipine, verapamil, meclofenamic acid mefenamic acid, transported by other amino acid carriers had no significant ff diazepam, phenytoin, indocyanine green, procion red, e ect on the uptake of T3 and T4 (results not shown). Journal of Endocrinology (2000) 167, 487–492 www.endocrinology.org Downloaded from Bioscientifica.com at 09/26/2021 12:17:42AM via free access Specific thyroxine transporter in JAR cells · K A POWELL and others 489 Figure 1 Effect of amino acids on the saturable uptake of 125 125 (A) I-T3 (n=3–4) and (B) I-T4 (n=4–7) in JAR cells.