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.

Comparative Analysis of Zearalenone Effects on Thyroid Receptor Alpha (TRα) and Beta (TRβ) Expression in Rat Primary Cerebellar Cell Cultures

“…The most specific transporters for THs are organic anion-transporting polypeptides (OATPs), L-type amino acid transporters (LAT1 and LAT2), and monocarboxylate transporters 8 (MCT8) [ 87 , 88 , 89 ] facilitating the uptake of T4 and T3 into neurons and glia in a hormone-specific fashion. These transporters play important roles in the neuron–glia interactions mostly proved in neuron-glia co-cultures [ 90 ] and they are potential targets for different endocrine disruptors [ 91 ]. Modulation of TRs expression by glial cells might be facilitated by a direct neuron–glia metabolic coupling or by direct uptake of ZEN into the glial cells and thus buffering and reducing the applied ZEN concentration.…”

Aging affects sex- and organ-specific trace element profiles in mice

“…For the determination of thyroid I content, equal amounts of thyroid protein (10 μg) were mixed with ammonium persulfate (0.6 M; Sigma-Aldrich/Merck) to a total volume of 50 μL and subsequently heated up to 95°C for 1 h. After cooling, the resulting digest was further diluted (1:20) and 50 μL were transferred to a microtiter plate. Measurement of I was done by the Sandell-Kolhoff reaction in microtiter plate format, as described earlier [ 99 ]. I content was calculated to mg I per g protein.…”

MicroRNA regulation of Transthyretin in trophoblast differentiation and Intra-Uterine Growth Restriction

“…Uptake assay for thyroxin hormone (T4) was performed with microRNA mimic or mimic plus inhibitor transfected BeWo cells by a non-radioactive assay procedure as described in Jayarama-Naidu et al . 62 . In brief, BeWo cells were transfected with miR-141-3p or miR-200a-3p mimic alone or mimic plus inhibitors as described above.…”