Uptake of 3,5,3′-Triiodothyronine by Cultured Rat Hepatoma Cells Is Inhibitable by Nonbile Acid Cholephils, Diphenylhydantoin, and Nonsteroidal Antiinflammatory Drugs*

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Abstract
Cellular uptake of T3 was examined using rat H4 hepatoma cells. Uptake of [125I]T3 (10-11 M) from serum-free medium was measured as the cell-associated counts retained by washed cells (2 .times. 106 per well). Displaceable uptake was 84% of total uptake at 2 min (2.9% of total counts). T4, tetraiodothyroacetic acid, triiodothyroacetic acid, rT3, and D-T3 was 2-5% as effective as T3 in displacing uptake. Nonequilibrium kinetics indicated a half-maximal uptake at 680 nM T3 with approximately 7 million sites per cell. Displaceable uptake was time and temperature dependent and was 73% inhibited by 2 mM KCN and 52% by 10 mM bacitracin but not by 2 mM ouabain or 10 .mu.M cytochalasin B. Phloretin, 100 .mu.M, inhibited uptake by 66%. T3 uptake was directly related to the free T3 concentration over the range of albumin concentrations, 0-10 g/liter. The nonbile acid cholephil compounds, bromosulfophthalein, iopanoic acid, and indocyanine green (all 100 .mu.M) inhibited t3 uptake to 62%, 17%, and 5% of control, respectively. Taurocholate, methylaminoisobutyric acid, and oleic acid were noninhibitory. The half-inhibitory concentrations of reactive nonsteroidal antiinflammatory drugs were: meclofenamic acid (25 .mu.M), mefenamic acid (45 .mu.M), fenclofenac (69 .mu.M), flufenamic acid (100 .mu.M), and diclofenac (230 .mu.m). Aspirin, ibuprofen, oxyphenbutazone, and phenylbutazone (all 100 .mu.M) were noninhibitory. Diphenylhydantoin inhibited uptake to 50% at 75 .mu.M. These findings suggest that T3 uptke by cultured rat hepatocytes is by an energy-dependent, saturable, stereo-selective mechanism that is dependent on cell membrane proteins. This mechanism appears to be shared by a number of other ligands, including nonbile acid cholephils and several nonsteroidal antiinflammatory drugs of the anthranilic and phenylacetic acid classes, as well as diphenylhydantoin. The bile acid taurocholate, oleic acid, and a probe for type A amino acid uptake were inactive. The extent to which these effects may modify expression of thyroid hormone action remains to be established.