Triiodothyronine Receptor Research Articles

A monoclonal antibody to the rat nuclear triiodothyronine receptor: production and characterization.

The nuclear T3 receptor (NTR) was affinity-labeled with bromoacetyl-[125I]T3, purified by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and used to immunize BALB/c mice. Spleen cells from one strongly immunoreactive mouse were fused with Sp2 mouse myeloma cells, and 328 hybridomas were screened by a dot-blot immunoassay using as antigen, a preparation of NTR partially purified by diethylaminoethyl-Sephadex chromatography. Four positive cultures were thus found; three of which were confirmed by comparing Western blotting patterns with the electrophoretic mobility of the affinity-labeled NTR. One of these 3 hybridomas was further subcloned by limiting dilution and gave rise to the 2B3 clone, which produces an immunoglobulin of the immunoglobulin G1 subclass. Several lines of evidence indicated that the 2B3 monoclonal antibody was indeed directed against the NTR. The antibody recognized a protein with the same electrophoretic mobility as the affinity-labeled receptor. Thus, Western blotting revealed a predominant protein with a mol wt of 57,000 and a less abundant 45,000 component on sodium dodecyl sulfate gels, and multiple isoelectric variants of the 57,000 protein, with a predominant form at pI 6.2, were detected on two-dimensional gels. Incubation of the 2B3 antibody with the NTR labeled with [125I]T3 resulted in the formation of an antibody-receptor complex, as indicated by a shift of the radioactivity peak upon gel filtration on Sephacryl S-300. In contrast, control ascitic fluid did not change the elution profile of the labeled NTR. The 2B3 antibody is able to remove the T3-binding activity from rat liver nuclear extracts. Finally, in accordance with previous T3-binding experiments, expected amounts of NTR were found in pituitary, liver, brain, kidney, spleen, and testis with the use of the Western blotting technique and immunohistochemistry on frozen tissue sections. This antibody should prove useful in the characterization and purification of the NTR and also in the study of its distribution in different tissues and cell types.

Time course of the change in nuclear 3,5,3'-triiodothyronine receptors of skeletal muscle in relation to energy intake.

Nuclear triiodothyronine receptors have been investigated in skeletal muscle from young pigs living at thermal neutrality (26 degrees C). Animals were fed at a high (H) or low (L) level of energy intake for 3 or 21 days and killed at 6 or 24 h after the last meal. Receptor numbers were significantly greater on the H than the L intake, even after only 3 days of treatment. The difference in receptors between animals on H and L diets was somewhat less at 6 compared with 24 h after feeding. It is postulated that these changes could be mediated through the action of glucagon.

Evidence for the presence of nuclear 3,5,3'-triiodothyronine receptors in secondary cultures of pure rat oligodendrocytes.

It has been reported that oligodendrocytes do not contain nuclear T3 receptors, which is in apparent contradiction with the well-known effects of thyroid hormones on myelination. In this study we have reexamined the presence of receptors in this cell population, using pure rat oligodendrocyte cultures. T3 binding was also studied with the use of pure rat astrocytes as well as in mixed neuronal-glial cultures. The latter are mainly neuronal during the first days in culture and essentially glial thereafter. Binding studies carried out in intact cells demonstrated the presence of high affinity-low capacity binding sites for thyroid hormones in pure cultures of oligodendrocytes. The maximal binding capacity was 50-60 fmol/100 micrograms DNA and the dissociation constant (Kd) 0.13 nM. Pure rat astrocyte cultures also contained high affinity sites for thyroid hormones, although receptor concentrations was 2-3 times lower than in oligodendrocytes or neurons. This was confirmed in pure cultures of chick astrocytes and in neuronal-glial cultures during the astroglial period. The relative affinity of the receptor for thyroid hormone analogs was triiodothyroacetic acid = T3 greater than T4 greater than tetraiodothyroacetic acid in oligodendrocyte and astrocyte nuclei, and the sedimentation coefficient of the receptor was approximately 3.8S in both cell types. These results demonstrate that nuclear T3 receptors similar to those found in neurons and astrocytes are also present in oligodendrocytes. This suggests that the effects of thyroid hormones on myelination could result from a direct action of the hormone in the oligodendrocytes.

Preferential Saturation of Brain 3,5,3'-Triiodothyronine Receptor during Development in Fetal Lambs*

The concentrations of T4 and T3 were measured in brain, liver, and lung of fetal lambs at 100 days gestational age. The highest concentrations of T4 were found in lung (26.8 ng/g). Brain T4 (8.8 ng/g) was only 30% of lung T4. In contrast, higher concentrations of T3 were found in brain (1.8 ng/g) than in lung (0.39 ng/g) or liver (0.36 ng/g). Nuclear T3 was 16% of the total T3 in brain and 44% of that in lung. The degree of saturation of the nuclear T3 receptor was estimated from the concentrations of nuclear T3 and nuclear receptor. Receptor saturation was low in liver and lung (10%) and high in brain (74%). Receptor occupancy was also measured directly in vitro by comparing the binding of [125I]T3 in nuclear extracts at 0 and 20 C. This method is based on the different rates of dissociation of the T3-receptor complex at these temperatures (0.045 and 0.618 h-1, respectively). Therefore, [125I]T3 was bound mainly to unoccupied sites at 0 C, whereas at 20 C it bound to unoccupied sites plus a fraction (70%) of endogenously occupied sites. There was no difference in binding at the two temperatures using lung extract, reflecting a very low occupancy. Data from brain suggested 61% receptor saturation. Total and free T3 were measured in plasma and in lung and brain cytosols, and the figures were compared to the intranuclear free T3 calculated by the law of mass action, from the affinity and saturation of receptor. In lung, the concentrations of cytosolic (5.4 +/- 1.9 pM) and nuclear (8.6 pM) free T3 were similar to that of plasma T3 (3.7 +/- 0.99 pM). In contrast, brain cytosolic (14.9 +/- 1.2 pM) and nuclear (203 pM) free T3 revealed the presence of free T3 gradients from cytosol to plasma (4-fold) and from nucleus to cytosol (13.6-fold). The data suggest that the sheep brain is a major target of thyroid hormone action at the end of the neuroblast proliferation period. Mechanisms are locally present in the brain at this stage of development to ensure a high saturation of the nuclear T3 receptor.

Nuclear triiodothyronine receptor of human adipose tissue

l-Triiodothyronine induces malic enzyme in explants from human adipose tissue. Consequently, we looked for the presence of receptors for l-triiodothyronine in nuclei isolated from human adipose tissue. The binding of 125I-triiodothyronine by the nuclei was time- and temperature-dependent. At 37°C binding reached a steady state after 60 minutes. Dithiothreitol enhanced total binding and suppressed nonspecific binding. Scatchard analysis showed the presence of a single class of binding sites. The apparent association constant, K a, was 0.13 ± 0.03 × 10 10 M −1, the maximal binding capacity 2.20 ± 0.81 pmol/mg DNA (mean ± SD, n = 7) and the number of binding sites 8,000/nucleus. l-Triiodothyronine and d-triiodothyronine had equal affinity to the nuclear receptor; triiodothyroacetic acid had three times higher affinity. l- and d-thyroxine had 8% and 12%, respectively, and tetraiodothyroacetic acid had 19% affinity compared to that of l-triiodothyronine. Reverse triiodothyronine was a weak competitor. Digestion of nuclei with micrococcal nuclease abolished specific binding. These results show that nuclei from human white adipose tissue possess high affinity receptors for l-triiodothyronine, which are associated with nuclear chromatin. It is likely that induction of malic enzyme in human adipose tissue by l-triiodothyronine is mediated by the nuclear receptors.

Expression and regulation of c-erb-A mRNA from lymphocytes in patients with thyroid dysfunction.

To investigate the expression and the regulation of nuclear triiodothyronine (T3) receptor at the gene level, cellular(c)-erb-A mRNA isolated from lymphocytes in patients with thyroid dysfunction was examined by Northern gel analysis and dot blot hybridization using viral (v)-erb-A cDNA probe. Human lymphocytes contained c-erb-A mRNA (approximately 2.0 kilobase (kb) in length), and c-erb-A mRNA, which was determined by dot blot hybridization, was observed to be increased in hypothyroid patients but unaltered in hyperthyroid patients. The high level of c-erb-A mRNA may contribute in part to the increase in nuclear T3 receptor and these results suggest the presence of up-regulation of nuclear T3 receptor at the gene level in the lymphocytes of hypothyroid patients.

Triiodothyronine effects on RNA polymerase activities in isolated neuronal and glial nuclei of the mature rat brain cortex

Our previous study demonstrated that a high level of nuclear triiodothyronine receptors (NT 3R), which are identical to the hepatic NT 3R, exists in neuronal nuclei of the cerebral cortex from an adult rat brain. To investigate whether thyroid hormone acts through binding to nuclear receptors, we measured RNA polymerase activities in isolated neuronal and glial nuclei of cerebral cortices prepared from three groups of rats with different T 3 levels: T 3 ( 20 μ g 100 g BW/d , for three days)-injected hyperthyroid rats, control normal rats, and thyroidectomized rats. The enzyme activities in both nuclear fractions were assayed under the condition of dose-response linearity. When RNA polymerase I activity in neuronal nuclei from control rats was expressed as 100%, the activities from T 3-injected and hypothyroid rats were 112.3 ± 3.4% (n = 5, P < .05) and 86.9 ± 3.5% (n = 5, P < .05), respectively. The increase in the enzyme activities were parallel to the increase in T 3 content in neuronal nuclei among the groups. Glial nuclear RNA polymerase I showed the same tendency in response to T 3, although the enzyme activity was smaller than from neuronal nuclei. RNA polymerase II, however, showed no significant change in response to altered T 3 levels. The existence of numerous receptors and an induction of increased RNA polymerase I activity by T 3 in neuronal nuclei raise the possibility that thyroid hormone through a NT 3R pathway in the cerebral cortex of even the mature rat brain.

Putative thyroid hormone receptors in red blood cells of some reptiles

Putative triiodothyronine (T 3) receptors have been detected in the nuclei of red blood cells (RBC) in a number of reptile species. The binding characteristics of T 3 receptors in vitro were dissociation constant ( K d ) 9.1 to 28.58, 36.8 and 40, and 11.12 and 11.36 p M, and binding capacity ( B max) 0.12 to 0.37, 0.17 and 0.24, and 0.19 and 0.28 fmol per million cells in the rat snake ( Ptyas korros), soft-shelled turtle ( Trionyx sinensis), and tokay gecko ( Gekko gecko), respectively. These data were obtained in all species using in vitro incubation of whole cell according to current receptor studies on living cells. With modified technique in subsequent experiments, these values of the binding characteristics were seemingly low. The discrepancy was ascribed to the assessment of “free” fraction of hormone which would be used in subsequent calculation.

Nuclear 3,5,3'-triiodothyronine receptor occupancy, phosphoenolpyruvate carboxykinase (PEPck) messenger ribonucleic acid levels and PEPck enzyme activity in rat liver.

The expression of the gene coding for phosphoenolpyruvate carboxykinase (PEPck) is under complex multi-hormonal control. A stimulatory effect of T3 on the expression of this gene has been described, but it has not been related to nuclear T3 receptor occupancy. We therefore studied simultaneously nuclear T3 receptor occupancy, PEPck enzyme activities, and PEPck messenger RNA (mRNA) levels in the liver of thyroidectomized, T3-treated rats. A dose-dependent increase in receptor occupancy and in enzyme activities was observed 24 h after a single ip injection of T3. However, no increase in the levels of PEPck mRNA was observed, using a specific complementary DNA (pPCK10). To evaluate this further, time-course experiments were performed over a 24 h period after a single ip injection of a receptor saturating dose of T3. These experiments showed simultaneous increases of PEPck enzyme activities and PEPck mRNA levels. Receptor occupancy had increased to 95% 3 h after injection of T3, whereas maximal levels of enzyme activity and mRNA were obtained 6-12 h after T3 administration. Translational efficiency did not change during induction. Twenty-four hours after T3 administration, mRNA levels and enzyme activity had declined, but receptor occupancy was still maximal. We conclude that T3 influences PEPck gene expression primarily at a pretranslational (transcriptional) level. The transient increase in PEPck mRNA despite persisting full receptor occupancy suggests a modification in time of the interaction between the T3-receptor complex and the regulatory region of the PEPck gene.

Diabetes decreases liver and kidney nuclear 3,5,3'-triiodothyronine receptors in rats.

The liver and kidney nuclear T3 content and the maximal nuclear T3-binding capacity (MBC) were measured 1 month after streptozotocin administration and compared with values in controls either fed ad libitum (C) or offered a restricted diet (FR). A group of insulin-treated diabetic (D+I) rats was also included. Plasma T4 and T3 concentrations decreased to low levels in diabetic (D) rats. Plasma T3 levels were decreased in FR rats, whereas circulating T4 was in the normal range for C animals. The MBC (nanograms of T3 per mg DNA) for liver and kidney nuclear T3 was determined by an in vivo saturation technique. The respective results for all groups were as follows (asterisks denote values differing from C with P values less than 0.05): C, 0.601 and 0.414; FR, 0.583 and 0.369; D, 0.310 and 0.220; D+I, 0.630 and 0.394. Nuclear T4 and T3 concentrations were determined by an isotopic equilibrium technique. Nuclear T3 (nanograms per mg DNA) for liver and kidney were, respectively, 0.298 and 0.176 for C, 0.208 and 0.135 for FR, 0.109 and 0.070 for D, and 0.270 and 0.168 for D+I rats. The decreased liver and kidney nuclear T3 content in D rats appears to be due to a marked reduction of their available intracellular T4 pool, from which T3 could be generated, but most likely represents a decreased T3 uptake into liver and kidney nuclei, as the nuclear to plasma ratios of labeled T3 were decreased in D rats. The low levels of T3 in nuclei of FR rats could be attributed to an inhibition of T4 to T3 conversion, since the intracellular pool of T4 appears to be normal. The possibility that diabetes and food restriction might affect the thyroid activity was examined by measurement of the activities of alpha-glycerophosphate dehydrogenase and cytosol malic enzyme, two liver and kidney enzymes regulated by thyroid hormone. Furthermore, although the measurements made in FR rats excluded the possibility that the alterations in MBC found in D animals were nutrition dependent, the reduced nuclear T3 content concomitant with food restriction may account for some of the quantitative changes in the alpha-glycerophosphate dehydrogenase and cytosol malic enzyme activity found in D rat tissues. In conclusion, the present findings suggest that the observed changes in indices of thyroid hormone action in liver and kidney of D rats could be related to alterations in nuclear T3 receptor concentrations and the concentration of T3 bound to the receptor.

The effects of postoperative factors on serum thyroid hormones and rat liver nuclear 3,5,3'-triiodothyronine receptor concentrations in surgical models of uremia and regenerating liver.

We have determined the individual effects of postoperative fasting, surgical/anesthetic factors, acute uremia (AU), and regenerating liver (RL) on nucleoplasmic (NP; 0.15 M KCl-extractable) and chromatin-bound (CB; 0.4 M KCl-extractable) rat liver T3 receptors. AU and RL rats were studied 24 h after bilateral nephrectomy (blood urea nitrogen, 128 +/- 13 mg/dl) or two thirds hepatectomy, respectively. The effects of postoperative fasting were assessed by comparison of normal rats (N) with control rats (N6) pair-fed to match the caloric intake of the AU and RL rats. Surgical/anesthetic effects were determined by comparison of N6 rats with sham-operated pair-fed rats (S6). The effects of AU or RL were obtained by comparison with S6 controls. Changes in mean body weight attributable to fasting (N6-N), surgical/anesthetic effects (S6-N6), acute uremia (AU-S6), and regenerating liver (RL-S6) were: -17.3 (P less than 0.001), -4.0 (P = NS), -4.5 (P less than 0.05), and -1.0 g/24 h (P = NS), respectively. Changes in mean serum T4 (N, 5.3 +/- 1.3 micrograms/dl) were: -1.0 (P = NS), -0.6 (P = NS), -0.9 (P less than 0.05), and -1.0 micrograms/dl (P less than 0.05), respectively. Changes in mean serum T3 (N, 53 +/- 23 ng/dl) were: -8 (P = NS), -18 (P less than 0.05), -10 (P = NS), and -14 ng/dl (P less than 0.05), respectively. The NP and CB receptor pools of the AU and RL rats were not significantly different from those of age-matched N rats (NP, 25 +/- 5 fmol/mg DNA; CB, 405 +/- 134 fmol/mg DNA). Chronically uremic (CU) rats 2 weeks after five sixths nephrectomy (blood urea nitrogen, 36 +/- 2 mg/dl) did not exhibit significant change in their extractable receptor pools. Complete starvation for 24 h (NO) or 72 h (NOO) generally resulted in marked reductions in receptor concentrations compared to those in age-matched N rats fed ad libitum: NP pool (N, 31 +/- 17 fmol/mg DNA): NO-N, -40% (P = NS); NOO-N, -59% (P less than 0.01); CB pool (N, 303 +/- 105 fmol/mg DNA): NO-N, -19% (P less than 0.05); NOO-N, -41% (P less than 0.001). These studies indicate that severe AU, moderate CU, and LR have relatively little effect on solubilized rat liver nuclear receptor concentrations. In contrast, complete starvation is a potent depressant of both nuclear receptor pools. In the surgical models of AU and LR, postoperative fasting was the primary cause of weight loss.(ABSTRACT TRUNCATED AT 400 WORDS)

Nuclear triiodothyronine receptor binding characteristics and occupancy in obese (ob/ob) mice.

Obese (ob/ob) mice exhibit reduced adaptive thermogenesis associated with an impairment of thyroid hormone action. The mechanism underlying the latter defect was investigated by comparing the binding characteristics and occupancy of solubilized nuclear 3,5,3'-triiodothyronine (T3) receptors from livers of lean and obese mice. Scatchard analysis showed minimal differences in Bmax and Kd between phenotypes at both 4 and 8-10 wk of age, indicating that reduced hepatic thyroid hormone expression in obese mice is not caused by alterations in nuclear receptor concentration or affinity. In contrast, nuclear T3 receptor occupancy (endogenous T3 associated with the specific receptor divided by Bmax) was 14 and 23% lower in 4- and 8- to 10-wk-old obese mice, respectively. Together with reported changes in hepatic thyroid hormone-sensitive enzymes, these data are consistent with a diminished nuclear T3 signal initiating thyroid hormone action in obese mice. Decreased nuclear T3 receptor occupancy may be secondary to a low transport of plasma T3 to the nuclear pool. In conclusion, impaired hepatic thyroid hormone action in obese mice is mediated in part at least by a reduction in nuclear T3 receptor occupancy.

Triiodothyronine receptors in porcine granulosa cells

Triiodothyronine has been found to enhance gonadotropin- and insulin-stimulated morphologic luteinization and progesterone production by porcine granulosa cells in culture. The role of triiodothyronine in the above events is not well defined. One possibility is that triiodothyronine effects are direct and are receptor mediated. To test this concept, we undertook this study to investigate the presence of triiodothyronine receptors in granulosa cells aspirated from large (6 to 12 mm), medium (3 to 5 mm), and small (1 to 2 mm) porcine ovarian follicles. Crude nuclei were isolated and tested for iodine 125-triiodothyronine specific binding. Binding was time and temperature dependent and maximal in cells from small follicles at pH 8. Scatchard analysis yielded a mean apparent dissociation constant of 5.5 × 10−9mol/L and a mean apparent total number of binding sites of 1.0 pmol/mg of deoxyribonucleic acid. Competition experiments revealed the following relative binding affinities: triiodothyronine >l-thyroxine > reverse triiodothyronine. Gonadotropins, prostaglandins, epidermal growth factor, and insulin did not compete with 125I-triiodothyronine for binding. We conclude that there are nuclear binding sites in porcine granulosa cells with characteristics expected of a triiodothyronine receptor which might mediate a direct action of triiodothyronine on granulosa cells.

Identification and characterization of L-triiodothyronine receptors in cells of glial and neuronal origin.

High affinity-low capacity binding sites for thyroid hormone have been identified in the nuclei of glial (C6) and neuronal (Neuro 2A) cultured cells. Equilibrium dissociation constants, determined by Scatchard analysis, were very similar in both types of cells (0.2-0.3 nM). The relative affinity of hormonal analogs was also similar: the affinity for T3 was lower than for triiodothyroacetic acid and higher than for T4 or tetraiodothyroacetic acid. The sedimentation coefficients obtained by gradient centrifugation of nuclear receptor extracted with 0.4 M KCl or excised by micrococcal nuclease digestion were 3.5 S and 6.5 S, respectively. These results suggest that the thyroid hormone receptor is not restricted to neuronal cells, but also appears in cells of glial origin.

Effects of an Acute Bacterial Infection on Serum Thyroid Hormones and Nuclear Triiodothyronine Receptors in Mice*

Serum T3, T4, and rT3 levels as well as liver nuclear T3 receptors (NT3R) were measured in mice with a bacterial infection. Pseudomonas aeruginosa were injected into one thigh of ICR mice, resulting in a severe infection at sacrifice 15 h later. Since food intake, which influences serum thyroid hormone levels and NT3R, was 75% lower in infected than in control mice, infected mice were either fed and compared with pair-fed controls or fasted and compared with fasted and fed controls. Fasting induced a fall in serum T3 and T4 levels, which was even more pronounced in infected fasted animals. However, while fasting caused an approximately 80% increase in serum rT3 concentrations, serum rT3 levels in infected fasted animals were not different from those in fed controls. The combination of infection and fasting thus prevented the rise in serum rT3 otherwise invariably associated with fasting. NT3R measurements on isolated nuclei revealed the presence of NT3R in mouse liver similar to those reported in rat liver. The NT3R Kd (approximately 2 X 10(-10) M) was not affected by decreased food intake, infection, or a combination thereof. The NT3R maximum binding capacity (MBC) was decreased in fasted animals (460 vs. 306 pg/mg DNA). However, the MBC of infected fasted mice was not different from that of fasted mice. Similarly, no difference in MBC was found between infected fed and pair-fed control mice. In mice injected with heat-killed P. aeruginosa to evaluate potential effects of endotoxins, neither serum thyroid hormone levels nor hepatic NT3R were different from those of controls. These data show that in mice, a severe bacterial infection with P. aeruginosa has effects on serum hormone levels not explained by the disease-associated diminished food intake, whereas it has no effects on liver NT3R beyond those due to the disease-related decreased food intake.

Ontogenesis of triiodothyronine nuclear receptors in three skeletal muscles in male and female chicks

The ontogenesis of nuclear triiodothyronine receptors was determined in the pectoralis (αW fibers: fast contracting, glycolytic metabolism), adductor brevis (αR fibers: fast contracting, oxido-glycolytic metabolism) and adductor magnus (βR fibers: slow contracting, oxidative metabolism), muscles of male and female chickens at 18 days in ovo and 0, 6, and 30 days ex ovo. In the fast muscles (adductor brevis and pectoralis major), the T 3 receptor number decreases from the 18th day of incubation to hatching or the 6th day after hatching, respectively, and then increases. In the slow muscle (adductor magnus), the T 3 receptor number increases from 18 days in ovo to hatching and then decreases. At 30 days after hatching, the number of T 3 receptors was higher in the fast muscles than in the slow one. A comparison of the two fast muscles reveals that the number of T 3 receptors was more elevated in the pectoralis (glycolytic) fibers than in the adductor brevis (oxido-glycolytic) fibers. An overall test of significance showed a higher number of T 3 nuclear receptors in muscles from females than from males.

Thyroxine increases neonatal mouse submandibular gland mRNA-directed synthesis of epidermal growth factor

Thyroid hormones are known to modulate the concentrations of epidermal growth factor (EGF) in the mouse submandibular gland (SMG); this action is presumably mediated by the nuclear triiodothyronine receptor. To test the hypothesis that thyroid hormones act to increase SMG EGF concentrations by increasing the number of poly(A)+ -specific mRNA, poly(A)+ RNA was isolated from SMGs of neonatal mice which had been treated daily from birth through to 21 days of age with thyroxine (T4,0.4 microgram/g body weight). Poly(A)+ RNA also was extracted from SMGs of intact 21-day-old mice which had received vehicle alone. No significant differences in total nucleic acid, total RNA, or poly(A)+ RNA yields were noted between the two groups of animals. The isolated poly(A)+ RNAs from T4-treated and control mice were translated in an in vitro wheat germ system. Although no significant differences in efficiency of [35S]cysteine incorporation into trichloracetic acid precipitable material were noted between the two poly(A)+ RNA preparations, a significantly greater proportion of radioactivity was immunoprecipitable by anti-EGF antiserum in the translation medium derived from T4-treated mice (17.2 +/- 0.9%, mean +/- SEM) than in that of control mice (7.3 +/- 0.5%, P less than 0.001). Polyacrylamide gel electrophoresis of the immunoprecipitates (IMMP) revealed the presence of three radioactive bands with apparent relative masses (MrS) of 12,000, 9000, and 6000. The latter species comigrated with purified EGF, [125I]EGF, and an IMMP of a SMG extract. The translation product IMMPs following polyacrylamide gel electrophoresis were iodinated and digested with alpha-chymotrypsin.(ABSTRACT TRUNCATED AT 250 WORDS)

Triiodothyronine receptors in developing mouse neuronal and glial cell cultures and in chick-cultured neurones and astrocytes.

The evolution of L-triiodothyronine (T3) receptors was studied in developing cultures of cells dissociated from cerebral hemispheres of 14-day-old mouse embryos, which present successive distinct periods of cell proliferation and/or maturation. These periods are characterized essentially as neuronal from 1 to 12 days in vitro (DIV) and glial between 12 and 60 DIV. Furthermore myelin-related membranes are produced in this culture system. Binding capacities of the T3 nuclear receptors increased from 1 to 6 DIV, when it reached a maximum (16 fmol/100 micrograms DNA). A similar increase of the DNA content of the cell was observed until 8 DIV. Thereafter a sharp fall of receptor concentration leading to a 5-fold decrease in the binding capacity occurred until day 15, a period at which neurones disappeared from the cultures. From 25 to 50 DIV (coinciding with the glial period), the concentration of receptor remained more or less constant (1-2 fmol/100 micrograms DNA). In parallel, the DNA content did not vary greatly between 30 and 50 DIV. Scatchard analysis revealed the presence of a single class of receptors at 6 and 20 DIV, representative of 'neuronal' and 'glial' periods, respectively. The equilibrium dissociation constant (Kd) of the nuclear receptor from cells at 6 DIV (2 X 10(-10) M) was similar to that found at 20 DIV. These results were confirmed using pure cultured neurones and astrocytes prepared from embryonic chick brain. The effect of T3 on the cellular gangliosides used as an index of neuronal cell maturation, and on cerebroside sulfotransferase (CST), an enzyme involved in the production of myelin sulfatides, was studied to determine a possible correlation between the binding capacity of the T3 nuclear receptor and the response of the cultured cells to thyroid hormone. Our data demonstrate that T3 had no significant effect either on the content of gangliosides or on their developmental pattern, while it increased the level of CST activity by 75% between 18 and 25 DIV. These results show that, although the concentration of T3 receptors per 100 micrograms DNA in glial cells was lower than that in neurones, it was nevertheless sufficient to elicit a response in oligodendrocytes.

Dissociation of the triiodothyronine receptor from the nucleus induced by cations: evidence for divalent cation sensitive and insensitive nuclear sites.

Aliquots of purified rat liver nuclei were diluted at 0 degrees C in isotonic buffers containing monovalent (Na+) or divalent (Ca2, Mg2+) cations. At different times following dilution the nuclear suspensions were centrifuged and the T3 receptor was measured in KCl extracts of the nuclear pellets. The rate of receptor loss after dilution in EDTA was 0.0025 min-1. Dilution in the presence of cations caused a fast release of receptor during the first 10 min. This phase, which was not observed when the nuclei were diluted in EDTA without salt, was followed by a second phase where the receptor was released at the same rate as in EDTA. Receptor release was only dependent on the presence of cations in the dilution buffer during the first 10 min after dilution. The amounts of receptor remaining in the nuclei after the first 10 min of dilution were 51.8 +/- 9.2%, in the presence of Ca2+ and Mg2+, 38.6 +/- 8.9% in 0.15 M NaCl, and 18.0 +/- 4.8% in 0.15 M NaCl in the presence of Ca2+ and Mg2+. The release of receptor was not influenced by the integrity of the nuclear membrane. These results suggest the presence of divalent cation sensitive and insensitive nuclear sites for the T3 receptor, in amounts which could be estimated to be about 48 and 52%, respectively. Other interpretations are also possible, such as the presence of a high proportion of free receptors in the nucleosol, which could be released during the first phase of dilution if the negative charges in chromatin are blocked by cations to avoid redistribution of receptors immediately after dilution.

BINDING SITES IN FETAL AND GROWTH PLATE CARTILAGE

In addition to genetic and nutritional factors, linear growth during the prenatal and postnatal periods is controlled by peptide, steroid, and thyroid hormones interacting with the receptors present on the membrane or in the cytosol and nuclei of growth plate cartilage. Using standard procedures, insulin and nonsuppressible insulin-like activity (a somatomedin) showed significant binding in 600, 15,000, and 105,000 g membrane fractions of epiphyseal cartilage of immature animals. The binding of growth hormone and prolactin was small and probably not significant. Specific uptake of glucocorticoid was demonstrated in viable canine chondrocytes, but not of androgen, estrogen, or vitamin D3 metabolite. A triiodothyronine receptor was present in nuclei from dog epiphyseal cartilage. Hormones that lack binding may affect cartilage only indirectly. Hormone receptors were studied in those portions of fetal growth cartilage that will later evolve into an ossification center, articular cartilage, and epiphyseal cartilage. Cytosol fractions contained a receptor for glucocorticoid but not for androgen or estrogen. Zonal analysis showed a higher level in the peripheral and central sections than in the palisade section. Triiodothyronine binding was also detected in nuclei prepared from whole fetal cartilage. Heterogeneity of cell function was obvious in fetal cartilage. Cell division was high in the central and peripheral zones as well as the upper half of the palisade zone, but low in the lower palisade section. Proline and sulfate incorporation predominated in the palisade section compared with the central and peripheral sections. Disease states with changes of metabolic activities in the cartilage may perhaps be better understood with a clearer knowledge of receptor levels and interactions.