No 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-

A B S T R A C T We previously reported that putative nuclear receptors for thyroid hormone can be demonstrated by incubation of hormone either with intact GH1 cells, a rat pituitary tumor cell line, or with isolated GH1 cell nuclei and rat liver nuclei in vitro.We characterized further the kinetics of triiodothyronine (T3) and thyroxine (T4) binding and the biochemical properties of the nuclear receptor after extraction to a soluble form with 0.4 M KCI. In vitro binding of [lI]T3 and ['I]T4 with GH1 cell and rat liver nuclear extract was examined at 0C and 370C. Equilibrium was attained within 5 min at 370C and 2 h at 0C.The binding activity from GH1 cells was stable for at least 1 h at 370C and 10 days at -200C. Chromatography on a weak carboxylic acid column and inactivation by trypsin and Pronase, but not by DNase or RNase, suggested that the putative receptor was a nonhistone protein. The estimated equilibrium dissociation constants (Kd) for hormone binding to the solubilized nuclear binding activity was 1.80 x 10-10 M (T3) and 1.20 X 10" M (T4) for GH1 cells and 1.57 X 10`" M (T3) and 2.0 X 10' M (T4) for rat liver. These Kd values for T3 are virtually identical to those which we previously reported with isolated rat liver nuclei and GH1 cell nuclei in vitro.The 10-fold greater affinity for T3 compared to T4 in the nuclear extract is also identical to that observed with intact GH1 cells. In addition, the ['I]T3 and ['I]T4 high-affinity binding in the nuclear extract were inhibited by either nonradioactive T3 or T4, which suggests that the binding activity in nuclear extract was identical for T3 and T4.Dr. Samuels is the recipient of a PHS Research Career Development Award AM 46546.

Cells from a rat pituitary tumor cell line will respond in vitro to physiological concentrations of L-thyroxine and L-triiodothyronine. The cells are grown in a cultutre medium that contains serum from a hypothyroid calf. Dose-response relationships of a vacriety of thyronine derivatives indicate that this system has a specificity of response which is similar to that observed in vitro.