Effect of follicle-stimulating hormone on insulin-like growth factor-I-stimulated rat granulosa cell deoxyribonucleic acid synthesis.

Granulosa cells from diethylstilbestrol-treated immature rats were cultured in a defined medium on collagen-coated plates. Thymidine incorporation was significantly increased by insulin (ED50, 656 +/- 110 ng/ml) and insulin-like growth factor (IGF-I; ED50, 95 +/- 10 ng/ml). Insulin and IGF-I stimulations were amplified by methylisobutylxanthine an inhibitor of phosphodiesterase activity. The effect of both peptides were also enhanced by low doses of (Bu)2cAMP (0.2-1 mM). In contrast, higher concentrations were inhibitory. Similarly, FSH produced a biphasic enhancement of the insulin- and IGF-I-stimulated DNA synthesis. Maximal effects (2- to 6-fold increases) were observed with the lower doses (2-20 ng/ml) of the gonadotropin. FSH enhancement of IGF-I-stimulated DNA synthesis was dependent on cell density. Plating densities of 3-5 x 10(5) cells/cm2 were required for a maximal interaction. It is concluded that FSH, acting through a cAMP-mediated pathway, may regulate granulosa cell proliferation by modulating the mitogenic effects of insulin and/or IGF-I.     

Organic iodine inhibits deoxyribonucleic acid synthesis and growth in FRTL-5 thyroid cells

FRTL-5 thyroid epithelial cells in culture were used to study the possible inhibitory effects of iodine on thyroid growth. NaI exerted a dose-dependent, thyroid epithelial cell-specific inhibitory effect on [methyl-3H]thymidine incorporation into DNA, reduced the DNA content in the cell layer, and limited the increase in cell number mediated by TSH. The inhibitory effects of sodium iodide applied to growth stimulated by TSH-, cAMP-, and non-cAMP-dependent mechanisms and were prevented by 1-methylimidazole-2-thiol (methimazole) and 2-ethylthioisonicotinamide (ethionamide). The latter findings indicate that the inhibitory effects of NaI are mediated by some iodine-containing organic compound. The inhibitory effects of organic iodine on growth subsided 24-48 h after removal of excess NaI from the culture medium. In contrast, NaI had no effect on normal rat kidney fibroblast or thyroid fibroblast [methyl-3H]thymidine incorporation stimulated by epidermal growth factor or serum. These data demonstrate a specific inhibitory effect of organic iodine on thyroid epithelial cell growth.     

Existence of nuclear thyroid hormone receptors in the porcine thyroid gland and the rat thyroid cell line FRTL-5

We have investigated whether nuclear T3 receptors exist in the thyroid cell. Nuclear proteins extracted from porcine thyroid nuclei with 0.4 mol/l KCl were incubated with [125I]T3. The mixture was then analysed by sucrose density gradient ultracentrifugation which revealed that the T3-binding proteins migrated at the same position of 3.6 S as rat liver nuclear T3 receptors. Fractionation by high performance liquid chromatography using a size exclusion column and an ion exchanger column also demonstrated elution patterns of T3-binding similar to those of the rat liver receptor. Scatchard plots of crude nuclear extracts from porcine thyroid represented a curvilinear pattern. However, when the nuclear proteins partially purified by a DEAE column chromatography were analysed, a single binding component was found; the association constant was 4.1 x 10(10) l/mol and the maximal binding capacity was 602 fmolT3/mg protein. Displacement study with several T3 analogues showed a highly selective affinity for L-T3. Cultured rat thyroid cells of the FRTL-5 line also contained a single class of saturable, high affinity T3-binding site. Subconfluent cells in 100-mm dishes were incubated with increasing amounts of [125I]T3 at 37 degrees C for 3 h and radioactive T3 in isolated nuclei was counted. Scatchard analysis of data showed that the association constant and the maximal binding capacity were 3.44 +/- 0.63 x 10(10) l/mol and 63.7 +/- 17.8 fmolT3/mg protein, respectively. These results strongly suggest that there are nuclear T3 receptors, indistinguishable from the hepatic T3 receptors, in the porcine thyroid and rat FRTL-5 cells.     

Expression of glucocorticoid, retinoid, and thyroid hormone receptors during human lung development

CONTEXT: Although glucocorticoid hormone, thyroid hormone, and retinoic acid play important roles in fetal development, the expression of their receptors in human lung is still unknown. OBJECTIVE: The aim of this study was to investigate the ontogeny of glucocorticoid receptor (GR)alpha, thyroid hormone receptors (TRs), retinoic acid receptors (RARs), and retinoid X receptors (RXRs) mRNA expression in human lungs. DESIGN: Lungs from human fetuses and neonates (13.5-41 wk gestation; n = 20) as well as adults (n = 5) were analyzed by real-time PCR to monitor the ontogeny of mRNA expression for each receptor. In addition, immunohistochemistry was performed to show the cellular distribution of the different receptors. RESULTS: The expression of GRalpha, TRs, RARs, and RXRs was already detected in the earliest developmental stages analyzed. There was no significant difference in mRNA expression between developmental groups for any of the genes studied. However, for fetal and neonatal samples, there were positive correlations between gestational age and mRNA expression for RARalpha (r = 0.665; P = 0.001), RXRalpha (r = 0.444; P = 0.050), and RXRgamma (r = 0.464; P = 0.039). Immunohistochemical studies showed the presence of GRalpha, TRs, RARs, and RXRs in the nuclei of both epithelial and mesenchymal cells, albeit more pronounced in epithelium of larger airways. CONCLUSIONS: The detection of GRalpha, TRs, RARs, and RXRs expression in human lung as early as 13.5 wk gestation implies an early potential for therapeutic or toxic effects by exogenous analogs or by excess of endogenous ligands.     

Differences in nuclear thyroid hormone receptors among species

Hepatic nuclear thyroid hormone receptors from rat, dog, chicken, and rainbow trout were compared. Receptor affinities for 3,5,3'-triiodo-L-thyronine (T3) were similar in preparations from rat, dog, and chicken, using isolated nuclei and nuclear extracts. Rainbow trout nuclear receptor showed a lower affinity for T3. Almost half of the receptors were released into the medium with rat and chicken nuclei, and 79.7 +/- 1.1% of the receptors were released with rainbow trout nuclei, when isolated nuclei were incubated with T3 at 22 degrees for 2 hr. The affinity constant of rat liver receptor for calf thymus DNA-cellulose at 0.17 M KCl, pH 7.4, was 3.98 +/- 1.47 x 10(5) M-1, when determined using DNA-cellulose columns. The number of salt bridges involved in DNA binding of the rat receptor was 5.73 +/- 0.38. When receptor-DNA interactions were compared among species, significant differences were found, but the receptors from dog and rainbow trout liver were similar. Sephacryl S-200 column chromatography showed that chicken receptor had a Stokes radius significantly smaller than that of rat receptor. Partial proteolysis of T3-receptor complex using trypsin alpha-chymotrypsin, elastase, and papain produced distinct T3-binding fragments in different species. Our data provide evidence that nuclear thyroid hormone receptors from different species have significant structural dissimilarities.     

Uterine deoxyribonucleic acid synthesis during preimplantation in precursors of stromal cell differentiation during decidualization

During early pseudopregnancy, DNA synthesis and mitosis in the uterine endometrial stroma precede the development of uterine sensitivity to deciduogenic stimuli. Progesterone redirects the effects of estradiol on endometrial DNA synthesis from the luminal epithelium to the stroma. To determine the time and hormonal control of preimplantation endometrial DNA synthesis, uterine cells were labeled with [3H] thymidine at specific times during early pseudopregnancy or after progestin and estrogen treatment of ovariectomized rats. The fate of these labeled cells after their decidualization was examined by separation of prelabeled deciduomal cells by velocity sedimentation at unit gravity, which separates cells by size. Stromal cells that synthesized DNA during early pseudopregnancy or in response to hormone treatment later differentiated into deciduomal cells. Rates of DNA synthesis increased on days 4 and 5 of pseudopregnancy, with greater incorporation occurring on day 4 in cells that differentiated into polyploid deciduomal cells. In ovariectomized rats, medroxyprogresterone acetate treatment for 15 h increased DNA synthesis in stromal cells that differentiated into diploid and tetraploid deciduomal cells. DNA synthesis increased further at 30 h before returning to basal levels at 48 h. After progestin pretreatment, estradiol treatment increased stromal DNA synthesis again with greater incorporation in cells differentiating into polyploid deciduomal cells. These data indicate that during early pseudopregnancy, both progesterone and estradiol control the DNA synthesis of endometrial stromal cells as a means of reprogramming these cells for the later growth and differentiation of decidualization.     

Purification and characterization of rat liver nuclear thyroid hormone receptors

Nuclear thyroid hormone receptor was purified to 904 pmol of L-3,5,3'-triiodothyronine (T3) binding capacity per mg of protein with 2.5-5.2% recovery by sequentially using hydroxylapatite column chromatography, ammonium sulfate precipitation, Sephadex G-150 gel filtration, DNA-cellulose column chromatography, DEAE-Sephadex column chromatography, and heparin-Sepharose column chromatography. Assuming that one T3 molecule binds to the 49,000-Da unit of the receptor, we reproducibly obtained 6.4-14.7 micrograms of receptor protein with 4.2-4.9% purity from 4-5 kg of rat liver. Elution of receptor from the heparin-Sepharose column was performed using 10 mM pyridoxal 5'-phosphate, which was observed to diminish binding of receptor to heparin-Sepharose or DNA-cellulose. This effect was specific for pyridoxal 5'-phosphate, since related compounds were not effective. Purified receptor bound T3 with high affinity (6.0 X 10(9) liter/mol), and the order of affinity of iodothyronine analogues to purified receptor was identical to that observed with crude receptor preparations [3,5,3'-triiodothyroacetic acid greater than L-T3 greater than D-3,5,3'-triiodothyronine (D-T3) greater than L-thyroxine greater than D-thyroxine]. Purified receptor had a sedimentation coefficient of 3.4 S, Stokes radius of 34 A, and calculated molecular mass of 49,000. Among several bands identified by silver staining after electrophoresis in NaDodSO4/polyacrylamide gels, one 49,000-Da protein showed photoaffinity labeling with [125I]thyroxine that was displaceable with excess unlabeled T3. The tryptic fragment and endogenous proteinase-digested fragment of the affinity-labeled receptor showed saturable binding in 27,000-Da and 36,000-Da peptides, respectively. These molecular masses are in agreement with estimates from gel filtration and gradient sedimentation, indicating that affinity labeling occurred at the hormone binding domain of nuclear thyroid hormone receptor. This procedure reproducibly provides classical native rat liver T3 nuclear receptor in useful quantity and purity and of the highest specific activity so far reported.     

Stimulation of human fibroblast protein, ribonucleic acid, and deoxyribonucleic acid synthesis by bovine growth hormone fragments

Bovine GH and two fragments which were obtained by dissociation of a limited tryptic digest of the hormone stimulated protein, RNA, and DNA synthesis of contact-inhibited human fibroblasts. The stimulation of protein, RNA, and DNA synthesis was similar for the test substances. Maximal stimulation was noted at 10 nM. At that concentration, protein, RNA, and DNA synthesis were respectively increased 1.80, 1.42, and 1.37 times by GH; 1.93, 1.27, and 1.46 times by A-I (the larger fragment); and 1.99, 1.26, and 1.33 times by A-II (the smaller fragment). The action of GH, A-I, and A-II was similar to that of fetal calf serum, but was distinguished by the time course of stimulation and by the magnitude of the response. For example, GH, A-I, and A-II produced their earliest detectable effect at 10 h for protein synthesis, 22 h for RNA synthesis, and 26 h for DNA synthesis. On the other hand, serum stimulated protein, RNA, and DNA synthesis at 6, 10, and 16 h, respectively. These data show that human fibroblasts respond equally to GH, A-I, and A-II and suggest that there may be more than one "active site" in the GH molecule. Lastly, human fibroblasts may represent a useful system to study the actions of GH in vitro.     

Mediation of follicle-stimulating hormone action on follicular deoxyribonucleic acid synthesis by epidermal growth factor.

FSH stimulates DNA synthesis by hamster preantral follicles both in vivo and in vitro, and the in vitro mitogenic effect of FSH is effectively reproduced by epidermal growth factor (EGF). To determine whether follicular EGF is the intracellular transducer of FSH action on hamster preantral follicles, intact follicles at stages 1 to 7 (stages 1-4 = preantral follicles 1-4 layers of granulosa cells, respectively, and no theca; stages 5-6 = 5-6 and 7-8 layers granulosa cells, respectively, and developing theca; and stage 7 = follicles with incipient antrum) were cultured for 24 h in a serum-free culture medium in the absence or presence of 100 ng FSH, 50 ng EGF, 50 ng transforming growth factor-alpha (TGF alpha), or 1 mumol 8-Br-cAMP and challenged with 50 microliters polyclonal antimurine EGF antiserum; the rate of DNA synthesis was determined by [3H]thymidine incorporation. FSH, EGF, and TGF alpha significantly (P less than 0.05) stimulated follicular DNA synthesis; TGF alpha per se was less effective than either FSH or EGF. However, both FSH- and EGF-induced DNA synthesis was drastically attenuated by EGF antiserum; TGF alpha effect remained undisturbed. Interestingly antibody inhibition of FSH-induced DNA synthesis was totally reversed by coexposure to TGF alpha. Follicular DNA synthesis for most stages was stimulated by Br-cAMP, but the effect was significantly (P less than 0.05) inhibited by EGF antibody. Moreover, follicles at different stages responded to EGF with different latency. These results strongly suggest that FSH-induced follicular DNA synthesis in the hamster is mediated by follicular EGF and the pathway of events is FSH action----cAMP production----EGF synthesis----cell proliferation.     

Solubilized nuclear thyroid hormone receptors in circulating human mononuclear cells

In order to assess iodothyronine receptor interactions in man, we have developed a receptor assay for T3 and T4 in solubilized nuclear extracts from circulating mononuclear cells. This assay utilizes the technique of salt solubilization to isolate nuclear receptors and employs standard saturation analysis for T3 and T4 to determine maximal binding capacity (MBC) and equilibrium dissociation constants (Kd). We have determined that 11 normal subjects had a MBC for T3 of 1.20 +/- 0.20 pmol/mg DNA (+/- SE) and a Kd of 3.4 +/- 0.2 X 10(-10) M; the T4 MBC was 8.44 +/- 1.22 pmol/mg DNA and the Kd was 2.7 +/- 0.3 X 10(-10) M. Hypothyroid patients had a mean T3 MBC of 7.32 +/- 2.28 pmol/mg DNA and a mean T4 MBC of 40.04 +/- 21.36 pmol/mg DNA (P less than 0.05 compared to normal). Obese subjects (n = 12) had a basal fed MBC that was 0.66 +/- 0.13 pmol/mg DNA for T3 (P less than 0.05 compared to normal) and was 3.58 +/- 0.56 pmol/mg DNA for T4 (P less than 0.01 compared to normal). During fasting, the average T3 MBC increased to 1.43 +/- 0.31 pmol/mg DNA and the average T4 MBC increased to 9.63 +/- 2.46 pmol/mg DNA, values that are both significantly higher than those in the fed period; the dissociation constants were unaltered in obese subjects (compared to normals) in fed and fasting states. Gel filtration with 0.5 M agarose was employed to ascertain if the physicochemical properties of the solubilized mononuclear human cell receptor were similar to those previously observed in rat and human liver and kidney receptors. The elution profile obtained was similar to that reported earlier. The major binding activity has an estimated Stokes radius of 35 A and a molecular weight ratio of approximately 50,000 daltons. These studies indicate that: 1) high affinity T3 and T4 receptors exist in human mononuclear cells and have properties similar to those for T3 and T4 described previously in rat liver; 2) T3 and T4 receptor number tends to increase in hypothyroid subjects and tend to be lower in obese patients than in normal weight control subjects; 3) fasting is associated with an increase in both T3 and T4 MBC; and 4) despite their apparent physicochemical similarity, T3 receptors in rat liver and human mononuclear cells may be regulated differently, at least during fasting since hepatic T3 receptors decrease in the fasted rat. Collectively, these observations support the concept that human white cell T3 nuclear receptor binding is capable of rapid fluctuations, suggesting a mechanism for homeostatic regulation of T3 action.     

Pituitary expression of type I and type II glucocorticoid receptors during chicken embryonic development and their involvement in growth hormone cell differentiation

Glucocorticoids can induce somatotroph differentiation in vitro and in vivo during chick embryonic and rat fetal development. In the present study, we identified the nuclear receptors involved in somatotroph differentiation and examined their ontogeny and cellular distribution during pituitary development in the chicken embryo. Several steroids were tested for their ability to induce GH cell differentiation. Only glucocorticoids and aldosterone were effective at low nanomolar concentrations, suggesting involvement of both type I (mineralocorticoid) and type II (glucocorticoid) receptors (MR and GR, respectively). ZK98299 and spironolactone (GR and MR antagonists, respectively) when used alone were unable to block corticosterone or aldosterone (2 nm)-induced somatotroph differentiation. However, ZK98299 and spironolactone in combination abolished corticosterone or aldosterone (2 nm)-induced somatotroph differentiation. When used separately, both antagonists attenuated induction of GH mRNA by corticosterone. Spironolactone alone blocked somatotroph differentiation induced by 0.2 nm corticosterone or aldosterone, indicating that corticosteroids at subnanomolar concentrations act only through the MR. GR protein was detected in pituitary extracts as early as embryonic d 8, whereas MR protein was readily detectable only around d 12. GR were expressed in greater than 95% of all pituitary cells, whereas MR were expressed in about 40% of all pituitary cells. Dual-label immunofluorescence revealed that the majority of somatotrophs on d 12 expressed MR. Given the high affinity of corticosteroids for MR and that corticosteroid concentrations during embryonic development are in the subnanomolar range, expression of MR may constitute a significant developmental event during somatotroph differentiation.     

Increase in the number of type II insulin-like growth factor receptors during propylthiouracil-induced hyperplasia in the rat thyroid

We have observed that membranes isolated from rat thyroids contain receptors for the insulin-like growth factors (IGF). As IGFs are known to be important mediators of tissue growth, we conducted this study to determine whether modulation of thyroid IGF receptors might be involved in TSH-stimulated hyperplasia. A substantial increase in both the weight of the thyroid and its DNA content was observed within 2 days of exposing adult male rats to 0.1% propylthiouracil (PTU) in their drinking water. Serum T4 reached unmeasurable levels and serum TSH rose 3-fold over control by the tenth day of treatment. [125I]Iodo-human(h)IGF-II binding to membranes isolated from hyperplastic glands was significantly higher than control beginning at 2 days. A maximum was reached after 5 days (13.3 +/- 0.8%/25 micrograms protein vs. a control level of 6.7 +/- 0.7%, mean +/- SEM). The increase had disappeared by 15 days of PTU exposure, paralleling the drastic fall in the growth rate of the glands. This increase in binding was specific for the thyroid, as it was not seen in other organs. In both treated and control animals, the receptor involved was shown to be type II by preferential binding to IGF-II, lack of interaction with insulin, and molecular sizing. The observed increase in binding could be accounted for by an increase in receptor site number, the affinity remaining essentially the same. We conclude that the TSH-stimulated hyperplasia of the rat thyroid, induced by PTU, is associated with an increase in the binding sites of the type II IGF receptor. This observation raises the possibility that modulation of this receptor may play a role in the mediation of the mitogenic effect of TSH on the thyroid gland.     

Interaction of amiodarone and desethylamiodarone with solubilized nuclear thyroid hormone receptors

The mechanisms of action of the potent antiarrhythmic drug amiodarone are unknown. However, amiodarone and its abundant metabolite, desethylamiodarone, bear a striking structural resemblance to thyroid hormones. In addition, certain cardiac electrophysiologic effects of amiodarone treatment are similar to those of hypothyroidism. These facts suggest that amiodarone or desethylamiodarone could be acting, in part, by blocking thyroid hormone action. Because thyroid hormones are known to act through nuclear receptor proteins, the binding of amiodarone and desethylamiodarone was measured to nuclear extracts derived from human lymphocytes, bovine atrium and ventricle and rat liver. The capacity of increasing concentrations of amiodarone and desethylamiodarone nuclear extracts to block receptor binding of radiolabeled triiodothyronine (T3) in a standard in vitro competition assay was tested. Nuclear extracts demonstrated only minimal binding to amiodarone. However, all receptor preparations had substantial affinities (KD) for the desethyl analog: lymphocyte, 8.6 microM; atrium, 35.0 microM; ventricle, 26.9 microM and liver, 8.6 microM. Desethylamiodarone accumulates in very large quantities in parenchymatous organs during long-term amiodarone treatment. Taking its usual therapeutic serum level (about 4 microM or 2.7 micrograms/ml) as an estimate of intranuclear concentration, desethylamiodarone would partially saturate nuclear thyroid hormone receptors in several different tissues, including the heart. Thus, amiodarone treatment may exert some of its electrophysiologic effects by metabolic conversion to desethylamiodarone. This metabolite may then exclude thyroid hormone from nuclear receptor sites within the myocardium.     

Altered expression of receptors for thyroid hormone and insulin-like growth factor-I during reconstitution after allogeneic hematopoietic stem cell transplantation

Treatment with neuroendocrine hormones has been suggested to promote reconstitution of the immune system after hematopoietic stem cell transplantation (HSCT). We investigated the expression of genes encoding receptors for growth hormone (GH), insulin-like growth factor-I (IGF-I) and triiodothyronine (T3), at various time points after HSCT in 16 patients and 15 healthy controls. Peripheral blood mononuclear cells were isolated and RNA for GH receptor (GHR), IGF-I receptor (IGF-IR) and thyroid hormone receptor (TRalpha1) was amplified by RT-PCR. The expression of the genes was compared with the expression of beta-actin. We demonstrate increased expression of TRalpha1 RNA in patients at 1.5 months post HSCT, compared to a group of healthy controls, and decreased expression of IGF-IR RNA at 2 and 3 months post HSCT, compared to the controls. Serum from three of the patients was also analyzed for levels of T3, T4, TSH and IGF-I at several time points after HSCT. Serum levels for T3, thyroxine (T4), thyroid stimulating hormone (TSH) and IGF-I were within the normal range in all samples. Our results on the molecular level indicate a role for thyroid hormones and IGF-I in immune reconstitution after HSCT, even though the serum levels of T3, T4, TSH and IGF-I are normal.     

Regulation of growth hormone messenger RNA by thyroid and glucocorticoid hormones.

Thyroid and glucocorticoid hormones stimulate growth hormone synthesis in cultured rat pituitary cells (GC). We have compared changes in growth hormone production and mRNA in these cells. Triiodothyronine (10 nM) and dexamethasone (1 micron) stimulated increases in growth hormone production by 2.5- and 3.8-fold, respectively. There were corresponding increases in the capacity of RNA from hormone-treated cells to direct synthesis of pregrowth hormone in a wheat germ cell-free translation system, suggesting hormone-regulated increases in growth hormone mRNA. Hormone-induced changes in mRNA were also demonstrated by determining the kinetics of hybridization of a cDNA probe prepared from RNA enriched (about 70%) for growth hormone translational activity with RNA from control and hormone-treated cells. These results suggest that thyroid and glucocorticoid hormones can regulate growth hormone production by influencing the levels of its mRNA.     

Developmental regulation and function of thyroid hormone receptors and 9-cis retinoic acid receptors during Xenopus tropicalis metamorphosis

Amphibian metamorphosis serves as an excellent model to study T3 function during postembryonic development in vertebrate due to its total dependence on T3. Earlier molecular studies in the model species Xenopus laevis have led to a number of important in vivo findings on the function and mechanisms of T3 receptor (TR) action during vertebrate development. However, the lack of genomic sequence information, its tetraploid genome, and lengthy developmental cycle hinder further analyses on TR functions. In this regard, the highly related species, Xenopus tropicalis, is much more advantageous. Toward developing X. tropicalis for genome-wide and genetic studies of TR function, we analyzed the expression profiles of TRs and their heterodimerization partners, retinoid X receptors (RXRs) or 9-cis retinoic acid receptors. We show that their expression correlates with transformations in different organs and that TR/RXR heterodimers are capable of repressing and activating gene expression in vivo in the absence and presence of T3, respectively. We further demonstrate that TRs are bound to endogenous target genes in X. tropicalis tadpoles. Our results thus support a role of TRs in mediating the metamorphic effects of T3 in X. tropicalis. More importantly, the similarities in the expression and function between X. tropicalis and X. laevis TRs and RXRs as demonstrated by our study also pave the way to take advantages of existing morphological, molecular, and cellular knowledge of X. laevis development and the genetic and sequence superiority of X. tropicalis to dissect the molecular pathways governing tissue/organ-specific transformations during vertebrate postembryonic development.     

Characterization of nuclear thyroid hormone receptors of cultured skin fibroblasts from patients with resistance to thyroid hormone

Nuclear thyroid hormone receptors of patients with the syndrome of resistance to thyroid hormone were investigated in cell lines from seven patients in four affected families and compared to results from six normals. Fibroblasts cultured from skin biopsies were used. When binding affinity and capacity for L-triiodothyronine (T3) were examined by incubating whole cells or isolated nuclei, no significant differences were found. The amount of receptor released during the incubation of nuclei (9.3% to 19.0% of total nuclear receptors) was also within the normal range in these patients. When T3 binding assays were performed on 0.3 mol/L KCl extracted receptor, a significant decrease in binding capacity (MBC) without a difference in binding affinity (Ka) was observed in four patients and a lower Ka with normal MBC was found in two patients. Recovery of receptors in saline extracts, from patients' fibroblasts showing a low MBC, was low in comparison to normals. Lability of salt extracted receptors at 38 degrees C was normal and salt extractability of T3 occupied receptors, examined by incubation of [125I]-T3 labeled nuclei with various concentrations of KCl, was only slightly decreased. This lower salt extractability of receptors was insufficient to account for the low MBC obtained by Scatchard analysis of T3 binding to nuclear extracts. Gel filtration and density gradient sedimentation of salt-extracted receptors showed Stokes radius of 34 A, and sedimentation coefficient of 3.4 S in all patients and normals. From these values, molecular weight of 49,000 and total frictional ratio (f/fo) of 1.4 were calculated for nuclear receptors from patients and normals, suggesting a somewhat asymmetrical shape of receptors.(ABSTRACT TRUNCATED AT 250 WORDS)