Thyroid Hormone Action in Cell Culture: Demonstration of Nuclear Receptors in Intact Cells and Isolated Nuclei

Abstract

Triiodothyronine and thyroxine induce a 3-fold increase in the rate of growth of GH, cells in culture.To study further the action of these hormones, we examined the binding of [1211] (Grand Island Biological Co., Grand Island, N.Y.). The hormone concentrations in these sera are physiologic and are about 2 nM for T3 and 100 nM for T4, as determined by gas-liquid chromatography and specific radioimmunoassay (5). For hormone-binding studies, the medium was replaced with hypothyroid medium (Ham's F-10 medium supplemented to 10%o with hypothyroid calf serum obtained from a thyroidectomized calf, Rockland Farms, Pa.) and the cultures were incubated for 24-48 hr to deplete the cells of hormone. The hormone concentrations in the thyroidectomized calf serum are 0.3 nM for T3 and 3 nM for T4 (5, 7).Binding Studies with Intact Cells. For binding studies, the cells were harvested in the late logarithmic phase of growth with a rubber policeman and centrifuged at 500 X g for 5 min. The cell pellet was washed three times with 10 ml of serum-free Ham's F-10 medium by repeated dispersion and centrifugation. The cells were then suspended in serum-free medium with various concentrations of [125IVT3 or [125I]T4 and incubated at 370 for the times indicated. All radioactive analysis was done with a gamma spectrometer. After incubation with hormone, the cell suspensions were centrifuged at 500 X g for 5 min and the medium supernatant was saved for determination of hormone concentration. All further procedures were done at 0°. The cell pellet was homogenized in 10 volumes of STM buffer (0.25 M sucrose-20 mM Trist HCl-1.1 mM MgCl2, at pH 7.85 at 250) by 15 strokes at 5000 rpm with a motorized pestle (Tri R, New York). The homogenate was centrifuged at 800 X g for 10 min, and the resultant supernatant was then centrifuged at 16,000 X g -to obtain crude "mitochondrial" and "cytosol" fractions. The homogenate pellet was used to prepare nuclei either by centrifugation at 40,000 X g through 10 ml of 2.2 M sucrose or by two successive suspensions and centrifugations in 10 ml of STM-Triton buffer (0.25 M sucrose-20 mM Tris * HCl-

Thyroid Hormone Nuclear Receptors in Skeletal Muscle as Influenced by Environmental Temperature and Energy Intake

“…Assay of the number of receptors was carried out according to the method of Samuels & Tsai (1973). Aliquots of the suspension of nuclei were incubated for 30 min in the dithiothreitol solution at 37 'C in the presence of[1251I]T3 and a range of concentrations of unlabelled T3.…”

The Action of Thyroid Hormone

“…This nuclear T3 binding protein is heat labile and has a 4-10 times greater affinity for L-T3 than L-T4. However, D-T3 binds with nearly equal affinity as the naturally occurring L-isomer, and triiodothyroacetic acid (TRIAC) has even greater affinity (Oppenheimer et QZ., 1972a;Samuels & Tsai, 1973;DeCroot & Torresani, 1975;Docter et al, 1976).…”

Novel aspects of T3 actions on GH and TSH synthesis and secretion: physiological implications

“…THRs can also indirectly bind to DNA through proteins such as AP1 (jun/fos) complexes, influencing the gene expression (Flamant et al 2017). The presence of THRA and THRB isoforms in humans, mice, rats, chickens and Xenopus laevis was described and indicates that this mechanism is conserved across a variety of species (Tata & Widnell 1966, Samuels & Tsai 1973, Sap et al 1986, Weinberger et al 1986, Yaoita et al 1990.…”