title: A Nonradioactive Uptake Assay for Rapid Analysis of Thyroid Hormone Transporter Function.
abstract: Thyroid hormones (TH) are actively taken up into target cells via TH-transmembrane transporters (THTT). Their activity and expression patterns define a layer of endocrine regulation that is poorly understood. Therefore, THTT are potential targets for interfering agents (endocrine disruptors) as well as for pharmacological interventions. Inactivating mutations have been identified as the underlying cause of heritable diseases (monocarboxylate transporter 8-associated Allan-Herndon-Dudley syndrome) and might also define a class of subclinical TH insensitivity. As a basic tool to solve questions regarding THTT substrate specificity, activation or inactivation by compounds and functional changes from mutations, uptake assays with radiolabeled tracers are standard. Due to the need for radioactive isotopes, this technique is limited to screening of labelled substrates and disadvantageous regarding handling, setup, and regulatory issues. To overcome these hurdles, we developed an uptake assay protocol using nonradioactive ligands. In brief, uptake of nonradioactive iodine-containing substrate molecules was monitored via Sandell-Kolthoff reaction. The novel assay was designed to the common microtiter plate layout. As a prove-of-principle, we measured TH uptake by monocarboxylate transporter 8-transfected MDCK1 cells. Titrations with bromosulphthalein as an example for inhibitor screening setups and a side-by-side comparison with the radioactive method prove this assay to be reliable, sensitive, and convenient. Furthermore, the method was applicable on primary murine astrocytes, which enables high-throughput screening studies on in vitro model systems with physiological transporter regulation. Due to its design, it is applicable for high-throughput screening of modulatory compounds, but it is also a safe, inexpensive and an easily accessible method for functional testing of THTT in basic science.
authors.affiliation: Institut für Experimentelle Endokrinologie, Charité - Universitätsmedizin Berlin, D-13353 Berlin, Germany.
authors.forename: Roopa
authors.initials: R
authors.lastname: Jayarama-Naidu
authors.affiliation: Institut für Experimentelle Endokrinologie, Charité - Universitätsmedizin Berlin, D-13353 Berlin, Germany.
authors.forename: Jörg
authors.initials: J
authors.lastname: Johannes
authors.affiliation: Institut für Experimentelle Endokrinologie, Charité - Universitätsmedizin Berlin, D-13353 Berlin, Germany.
authors.forename: Franziska
authors.initials: F
authors.lastname: Meyer
authors.affiliation: Institut für Experimentelle Endokrinologie, Charité - Universitätsmedizin Berlin, D-13353 Berlin, Germany.
authors.forename: Eva Katrin
authors.initials: EK
authors.lastname: Wirth
authors.affiliation: Institut für Experimentelle Endokrinologie, Charité - Universitätsmedizin Berlin, D-13353 Berlin, Germany.
authors.forename: Lutz
authors.initials: L
authors.lastname: Schomburg
authors.affiliation: Institut für Experimentelle Endokrinologie, Charité - Universitätsmedizin Berlin, D-13353 Berlin, Germany.
authors.forename: Josef
authors.initials: J
authors.lastname: Köhrle
authors.affiliation: Institut für Experimentelle Endokrinologie, Charité - Universitätsmedizin Berlin, D-13353 Berlin, Germany.
authors.forename: Kostja
authors.initials: K
authors.lastname: Renko
mesh_terms: D000818:Animals; D001253:Astrocytes / Q000378:metabolism*; D004285:Dogs; D055879:Gene Knock-In Techniques; D007457:Iodine Radioisotopes / Q000378:metabolism; D061985:Madin Darby Canine Kidney Cells; D027501:Monocarboxylic Acid Transporters / Q000235:genetics* / Q000378:metabolism; D014284:Triiodothyronine / Q000378:metabolism*
chemical_list: D007457:Iodine Radioisotopes; D027501:Monocarboxylic Acid Transporters; D014284:Triiodothyronine