Biomedical subjects
Publications and source records attributed to H H Samuels.
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We have previously demonstrated that L-triiodothyronine (L-T3) induces an increase in growth hormone synthesis and messenger RNA in cultured GH1 cells, a rat pituitary cell line. In addition to regulating the growth hormone response, L-T3 elicits a time- and dose-dependent reduction in the level of its nuclear receptor, which is a direct function of the occupancy of the receptor binding site. In this study we have compared the relative affinity of L-T3, triiodothyroacetic acid, D-triiodothyronine (D-T3), and L-thyroxine (L-T4) for the receptor with the induction of the growth hormone synthesis and the ability of these compounds to elicit a reduction in thyroid hormone nuclear receptor levels. Triiodothyroacetic acid and D-T3 were specifically examined because the biologic effect of these compounds in the intact rat is significantly lower than predicted by their affinity for the receptor using isolated rat liver nuclei in vitro. In intact cells each compound demonstrated an excellent relationship between the relative receptor affinity, the induction of growth hormone production, and the concentration-dependent reduction in nuclear receptor levels. With the exception of D-T3, the relative affinity of iodothyronine was identical for the receptor using intact cells in serum-free media, or isolated GH1 cell nuclei in vitro. The apparent receptor affinity of D-T3 with intact cells was 5.5-fold lower than with isolated nuclei, which suggests a decrease in cell entry of D-T3 relative to the other iodothyronines. Quantitation of the [125I]iodothyronine associated with the receptor in GH1 cells after a 36-h incubation with L-125I-T4 was 90% L-T4 and 10% L-T3, which indicates that the major effect of L-T4 in GH1 cells is a result of intrinsic L-T4 activity. Studies with dispersed rat anterior pituitary cells demonstrated that L-T3 induces growth hormone synthesis and elicits a reduction in nuclear receptor levels in the same fashion as GH1 cells. The observation that thyroid hormone influences dispersed rat pituitary cells in a fashion qualitatively similar to GH1 cells may have implications for the growth hormone response of the somatotroph cell in vivo to different thyroidal states.
GH1 cells are a clonal strain of rat pituitary tumor cells which synthesize GH and PRL. We have previously demonstrated that these cells respond to physiological concentrations of L-T3 and L-T4 when cultured with medium supplemented with thyroidectomized calf serum to achieve a thyroid hormone-depleted state under cell culture conditions. In this study, we describe a method to deplete euthyroid calf serum of L-T3 and L-T4 using an anion exchange resin. We demonstrate that the procedure only minimally alters the low molecular weight anion components of the serum and does not change the total protein content or the electrophoretic pattern of serum proteins. Moreover, we show that euthyroid calf serum depleted of L-T3 and L-T4 by this procedure yields serum which, when used as a medium supplement, results in biological responses identical to those obtained with media supplemented with thyroidectomized calf serum. In addition, resin treatment does not alter the growth-promoting properties of the serum if the thyroid hormone concentration is restored. This procedure should be useful in preparing thyroid hormone-depleted serum for cell culture studies in situations where thyroidectomy is not feasible or would require surgical procedures on a large number of small animals.
The induction of growth hormone synthesis and mRNA by thyroid hormone in cultured GH1 cells is mediated by the thyroid hormone nuclear receptor. In addition, the regulation of the growth hormone response by glucocorticoid is highly dependent on the action of thyroid hormone. To clarify whether thyroid hormone has a general influence on glucocorticoid action in GH1 cells, the glucocorticoid induction of growth hormone and glutamine synthetase was simultaneously examined. In contrast to the growth hormone response, the induction of glutamine synthetase by glucocorticoid was not influenced by thyroid hormone. Both responses appear to be modulated by the glucocorticoid receptor, and thyroid hormone had no influence on nuclear-associated glucocorticoid receptor levels. These results suggest that the thyroid hormone control of glucocorticoid induction of growth hormone may be a selective process, and the nuclear associated receptors for both thyroid and glucocorticoid hormones interrelate to control the growth hormone response.
We have previously demonstrated that thyroid hormone controls growth hormone synthesis in GH1 cells and that the induction of the growth hormone response by glucocorticoid appears to be highly dependent on thyroid hormone action. Thyroid hormone induces growth hormone synthesis approximately 5- to 20-fold and cortisol increases this response 2- to 6-fold further. Long-term kinetics of the growth hormone response show that, without added thyroid hormone, cortisol can induce a small-growth hormone response after 48 hr of incubation. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of proteins synthesized in intact cells demonstrates that the cortisol enhancement of growth hormone synthesis in cells incubated with thyroid hormone is a relatively selective process. Quantitation of growth hormone mRNA levels by cell-free protein synthesis demonstrates that the regulation of growth hormone synthesis by thyroid and glucocorticoid hormones is explained by a synergistic pretranslational control mechanism, presumably at the level of the growth hormone gene.
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We have previously demonstrated with radioimmunoassay techniques that physiologic concentrations of thyroid hormone stimulate an increase in growth hormone production in GH1 cells in culture. In this study, with radiolabel techniques and selective immunoprecipitation, we demonstrate that L-triiodothyronine stimulates growth hormonw production solely by inducing an increase in the de novo synthesis of the polypeptide hormone. L-Triiodothyronine stimulated synthetic rates of growth hormone by 1.5-fold in 1.25 hr, 2-fold in 2.5 hr, to a maximal of 3- to 4-fold after 8.5 hr of incubation. The time interval between significant L-triiodothyronine binding to putative nuclear receptors and a detectable increase in growth hormone synthesis was 45-60 min. Studies on the effect of actinomycin D, 3'-deoxyadenosine, and cycloheximide support the thesis that L-triiodothyronine induces the accumulation of an RNA species which is rate limiting for growth hormone synthesis and lends further support for a primary action of thyroid hormone at the nuclear level.
The relationship between the binding of L-triiodothyronine (T3) to nuclear receptors and the induction of growth hormone synthesis was examined in cultured GH1 cells, a rat pituitary cell line. After 24 hr, T3 induced a maximal 4-fold increase in the rate of growth hormone wynthesis; the T3 concentration that induced a half-maximal increase was 0.22 nM. The biologic dose-response curve was shifted to the left of the receptor occupancy curve (Kd = 0.5 nM) by a factor of approximately 2 when receptor binding was examined for 4 hr but showed a significantly closer agreement when examined for the same 24-hr period as the biologic response. This shift in the fractional occupancy curve is not due to further equilibration but occurs as a result of a time- and dose-dependent depletion of the nuclear receptor by T3.
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Saturable binding activities for triiodothyronine were demonstrated in vitro with isolated nuclei and soluble nuclear extracts of rat liver, kidney, and cultured GH(1) cells. The binding activity can be extracted from nuclei in soluble form with no significant change in hormone affinity and has properties of a nonhistone protein.
High-affinity, limited-capacity nuclear binding activities, putative receptors for triiodothyronine, were detected after incubation of hormone with intact rat pituitary GH1 cells in culture, isolated GH1 cell nuclei, or rat liver nuclei. The total number of triiodothyronine binding sites per nucleus was similar in each case (approximately 8,000). The estimated equilibrium dissociation constants were virtually identical in isolated GH1 cell nuclei and rat liver nuclei, and both values were similar to that determined in intact GH2 cells. These results suggest that mechanisms of thyroid hormone action defined in cell culture could apply to thyroid hormone regulatory effects in vivo.
We previously reported that putative nuclear receptors for thyroid hormone can be demonstrated by incubation of hormone either with intact GH(1) cells, a rat pituitary tumor cell line, or with isolated GH(1) 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 KCl. In vitro binding of [(125)I]T3 and [(125)I]T4 with GH(1) cell and rat liver nuclear extract was examined at 0 degrees C and 37 degrees C. Equilibrium was attained within 5 min at 37 degrees C and 2 h at 0 degrees C. The binding activity from GH(1) cells was stable for at least 1 h at 37 degrees C and 10 days at - 20 degrees C. 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 (K(d)) for hormone binding to the solubilized nuclear binding activity was 1.80 x 10(-10) M (T3) and 1.20 x 10(-9) M (T4) for GH(1) cells and 1.57 x 10(-10) M (T3) and 2.0 x 10(-9) M (T4) for rat liver. These K(d) values for T3 are virtually identical to those which we previously reported with isolated rat liver nuclei and GH(1) 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 GH(1) cells. In addition, the [(125)I]T3 and [(125)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. In contrast, the binding activity for T4 and T3 in GH(1) cell cytosol was markedly different from that observed with nuclear extract (K(d) values were 2.87 x 10(-10) M for T4 and 1.13 x 10(-9) M for T3). Our results indicate that nuclear receptors for T3 and T4 can be isolated in a soluble and stable form with no apparent change in hormonal affinity. This should allow elucidation of the mechanisms of thyroid hormone action at the molecular level.
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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.
Triiodothyronine and thyroxine induce a 3-fold increase in the rate of growth of GH(1) cells in culture. To study further the action of these hormones, we examined the binding of [(125)I]triiodothyronine and purified [(125)I]thyroxine to cellular fractions after incubation with intact cells in serum-free medium. High-affinity, low-capacity binding sites for the hormones were demonstrated in nuclear but not in mitochondrial or cytosol fractions. Chromatographic analysis of the bound nuclear radioactivity from cells incubated with [(125)I]thyroxine demonstrated 97% thyroxine, 1% iodide, and 1% triiodothyronine. Apparent equilibrium dissociation constants, determined by Scatchard analysis, were 29 pM for triidothyronine and 260 pM for thyroxine. The maximal binding capacity was identical for both hormones, with about 5000 sites per cell nucleus. [(125)I]Thyroxine binding was competitively inhibited by triiodothyronine. These data suggest that triiodothyronine and thyroxine interact with identical nuclear receptors, and that conversion of thyroxine to triiodothyronine may not be a prerequisite for biologic activity. Similar high-affinity, low-capacity nuclear binding sites were also demonstrated by incubation of [(125)I]triidothyronine directly with isolated nuclei. Incubation of cells with increasing concentrations of nonradioactive triidothyronine results in a subsequent increase in binding when [(125)I]triiodothyronine is then incubated directly with isolated nuclei. This result suggests that nuclear receptors are not fixed, but increase after exposure of intact cells to hormone. This increase in nuclear receptor content may result from the transfer of an unstable cytosol receptor to the nucleus.
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