Membrane reception of thyroid hormones]

Comparative and competitive analyses of thyroxine (T4) and triiodothyronine (T3) binding to highly purified rat liver, brain and lung cell plasma membranes were carried out. The dependence of hormone binding on the time, temperature and concentration was studied. The effects of trypsin and partial delipidation on the binding parameters of thyroid hormones were investigated. Two thyroid hormone-binding sites were detected in cell plasma membranes of all tissues under study. The maximal binding of T4 to rat liver membranes and the maximal binding of T3 to rat brain membranes was observed in all experiments, the affinity for T3 being higher than that for T4. An important role of both protein and lipid components of plasma membranes in the membrane reception of thyroid hormones is proposed.     

Binding properties of thyroxine to nuclear extract from sea urchin larvae

We previously reported that thyroid hormones are involved in the formation of the adult rudiment and adult-type skeleton in sea urchin larvae, as well as in the resorption of larval tissues. In the present study, to search for the presence of thyroid hormone receptor in sea urchin larvae, we performed a ligand-binding assay between radiolabeled thyroid hormones and nuclear extracts from the larvae of the sea urchin Hemicentrotus pulcherrimus. The presence of binding sites with a high affinity to thyroxine (T4) was detected in the nuclear extract, but not in the cytoplasmic fraction. The dissociation constants for the T4 binding to the nuclear extracts were estimated to be about 18 pM from the mesenchyme-blastula stage to the four-armed pluteus stage. The quantity of T4 binding sites in the nuclear extracts increased during larval development. These results suggest that the binding affinity to T4 in the nuclear extracts was caused by a putative nuclear thyroid hormone receptor in sea urchin larvae.     

Benzodiazepine Receptor and Thyroid Hormones: In Vivo and In Vitro Modulation

In rats rendered hyperthyroid by chronic treatment with L-triiodothyronine (T3) hormone there was a 21 and 27% decrease, respectively, in the number of binding sites for [3H]flunitrazepam ([3H]FNZ) and [3H]ethyl-beta-carboline-3-carboxylate ([3H]beta-CCE) without changes in affinity for the two ligands. Two weeks after thyroidectomy there was a 44% increase in [3H]FNZ sites and a 17% increase in [3H]beta-CCE binding sites. In vitro we found that T3 produces a decrease in Bmax and an increase in KD, both changes being characteristic of a mixed type of inhibition. Thyroid status dramatically affected the Ki of T3 in displacing [3H]FNZ from sites on isolated membranes of the cerebral cortex: in hypothyroid rats the Ki value was 0.9 microM, whereas in hyperthyroid rats, it was 83 microM, a 92-fold difference. In control rats, the Ki was 11 microM. These findings are discussed in relation to a possible modulation of benzodiazepine receptors by thyroid hormones.     

Hormone binding by protein disulfide isomerase, a high capacity hormone reservoir of the endoplasmic reticulum

Protein disulfide isomerase (PDI) is a folding assistant of the eukaryotic endoplasmic reticulum, but it also binds the hormones, estradiol, and 3,3',5-triiodo-l-thyronine (T(3)). Hormone binding could be at discrete hormone binding sites, or it could be a nonphysiological consequence of binding site(s) that are involved in the interaction PDI with its peptide and protein substrates. Equilibrium dialysis, fluorescent hydrophobic probe binding (4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS)), competition binding, and enzyme activity assays reveal that the hormone binding sites are distinct from the peptide/protein binding sites. PDI has one estradiol binding site with modest affinity (2.1 +/- 0.5 microm). There are two binding sites with comparable affinity for T(3) (4.3 +/- 1.4 microm). One of these overlaps the estradiol site, whereas the other binds the hydrophobic probe, bis-ANS. Neither estradiol nor T(3) inhibit the catalytic or chaperone activity of PDI. Although the affinity of PDI for the hormones estradiol and T(3) is modest, the high local concentration of PDI in the endoplasmic reticulum (>200 microm) would drive hormone binding and result in the association of a substantial fraction (>90%) of the hormones in the cell with PDI. High capacity, low affinity hormone sites may function to buffer hormone concentration in the cell and allow tight, specific binding to the true receptor while preserving a reasonable number of hormone molecules in the very small volume of the cellular environment.     

Vitamin A and thyroxine carrier proteins in chicken plasma: steady-state control of the plasma level of free retinol-binding protein and free thyroxine.

1. The binding parameters of prealbumin-2 with retinol-binding protein and thyroxine (T4) revealed the existence of distinct and multiple sites for both retinol-binding protein and T4. 2. From the analysis of binding parameters of retinol-binding protein with prealbumin-2 it is clear that under steady-state conditions about 99% of the holo-retinol-binding protein remains bound to prealbumin-2. 3. Equilibrium dialysis studies on binding properties of thyroid hormones with prealbumin-2 revealed that it has a single high affinity site and three low affinity sites. 4. The occurrence of three carrier proteins for thyroid hormones, thyroxine-binding globulin, prealbumin-2 and albumin has been demonstrated. However, the chicken thyroxine-binding globulin differs from human thyroxine-binding globulin by being relatively less acidic and occurring at a two-fold lower concentration. But the thyroid hormone binding parameters are comparable. 5. Highly sensitive methods were developed for determination of T4 binding capacities of the various proteins and plasma level of total T4 by fractionation of carrier proteins and further quantitatively employing in electrophoresis and equilibrium dialysis. 6. The thyroxine-binding proteins were found to be of two types, one (viz., thyroxine-binding globulin) of great affinity but of low binding capacity, which mainly acts as reservoir of T4, and another (viz., prealbumin-2) of low affinity but of high binding capacity, which can participate predominantly in the control of the free T4 pool.     

Development of support matrices for affinity chromatography of thyroid hormone receptors

Certain cellular responses to thyroid hormones appear to be mediated by non-histone chromatin protein receptors. Purification of these proteins is important for an investigation of the detailed mechanisms of their regulatory role. In the present studies, we report the development of an affinity chromatographic system that can be used to purify thyroid hormone receptors solubilized from nuclei. Amine-substituted hormone analogs were prepared with D and L isomers of T3; these bind to the receptor. This finding supports the hypothesis that thyroid hormones fit into the receptor with the amino groups accessible from outside the binding site. Although L-triiodothyronine (LT3) (the naturally occurring isomer) binds more tightly (relative Kd = 1.0 nM) to the nuclear receptor than D-triiodothyronine (DT3) (relative Kd = 2.0 nM), the amine-substituted analog of DT3 binds more tightly than the LT3 analog (DT3 analog, relative Kd = 40 nM; LT3 analog, relative Kd = 1500 nM). Agarose-based gels containing DT3 and LT3 covalently coupled by their amino groups were also prepared. Binding of receptor to these gels was biospecific in that it could be inhibited by prior incubation of the receptors with LT3. In addition, as predicted by the analog studies, the DT3 affinity gels were more effective than LT3 gels in adsorbing receptor. Elution of receptor from the LT3-derived gels was achieved in a predicted volume and concentration of counter-ligand in elution buffer. These results suggest that affinity chromatography can be applied to the purification of thyroid hormone receptors.     

Multiple thyroid hormone binding sites on male rat liver nuclear matrices

Equilibrium binding of T3 to nuclear matrices isolated from male rat liver occurred after incubation for 3h at 20 degrees C. Two binding sites, having KD's of 6 and 95 nM, were revealed by Scatchard analysis. T3 and Triac competed for the binding of [125I]T3 to the high affinity site whereas only T3 competed for binding to the lower affinity site. Reverse T3 (rT3) did not compete for the binding of T3 to either class of binding sites. The binding sites were highly DNAse-sensitive, and less sensitive to protease treatment. The effect of binding of T3 to nuclear matrices by ATP, DTT and EDTA indicated that the sites are dissimilar to previously identified cytosolic binding sites. The higher affinity site resembles the T3 receptor in affinity and thyroid hormone specificity. The second site represents a new class of thyroid hormone binding sites. Its role in the regulation of thyroid hormone action warrants further investigation.     

In vitro demonstration of putative nuclear 3,5,3'-triiodothyronine receptors in isolated liver nuclei of Singi fish, Heteropneustes fossilis (Bloch)

Putative thyroid hormone (TH) receptors have been demonstrated in the isolated liver nuclei of Singi fish, Heteropneustes fossilis (Bloch), and their binding characteristics have been examined. Nuclear T3 saturation analyses were carried out in vitro at 27 degrees C in a sucrose-Tris-HCl buffer (pH 7.5) containing calcium (2 mM), magnesium (3 mM) and 2-mercaptoethanol (5 mM). After incubation the bound and free hormones were separated by centrifugation and the nuclei were treated with Triton X-100 (final concentration 0.25%) to reduce the non-specific binding. The binding was saturable and reached equilibrium by 20 minutes of incubation and was also stable for 2 hours. The binding was reversible and the rate of dissociation was more or less equal to the rate of association. The binding was linearly increased with the increased concentrations of the DNA (nuclei). Scatchard analyses of the equilibrium binding data revealed that only one class of binding sites for T3 did exist in the hepatic nuclei of Singi fish. The affinity of these sites or the mean dissociation constant (Kd = 0.20 +/- 0.07 x 10(-10) M) and the mean maximum binding capacity (MBC = 0.17 +/- 0.04 pmol/mg DNA) were in reasonable agreement with the values reported for other teleost fishes.     

Decrease in triiodothyronine binding sites in chick embryo erythrocytes during early development

Specific thyroid hormone (TH) binding sites have been detected in nuclei of erythrocytes obtained from developing chick embryos. The binding characteristics and relative affinities for TH analogs were those expected of TH receptors. Nuclear triiodothyronine (T3) saturation analysis was carried out in vitro by incubating intact erythrocytes in M199 medium with 3-200 pM [125I]T3 for 1 hr at 37 degrees C or 20-24 hr at 21 degrees C. Nuclei were obtained by centrifugation after lysing the erythrocytes in a stabilizing buffer containing 0.3% saponin, followed by addition of Triton X-100 (final concentration 0.2%) to minimize the nonspecific binding. Scatchard analysis of equilibrium binding data suggested that the nuclei possess a single class of binding sites. The binding is reversible and the rate of dissociation is temperature dependent. T3 and T4 appear to bind to the same sites, but the affinity of T3 was 16 times greater. Among TH analogs tested, Triac had the highest affinity followed by L-T3, D-T3, Tetrac, L-T4, D-T4, T2, and rT3. Serial studies performed on different days of chick embryogenesis demonstrated a rapid and significant decrease of the erythrocyte nuclear T3 receptor. On Day 5, the number of T3 binding sites was maximal at 1600 +/- 100 per nucleus. The number declined steadily until, by Day 20, it had reached about 60 +/- 10 sites/nucleus. RBC from adult and baby chickens had less than 1% as many binding sites as those from Day 5 embryos. There was no significant change in the affinity of the sites (Kd approximately equal to 20 pM at 37 degrees C). The reason for the loss of T3 binding sites during embryogenesis is not known. Since the plasma level of the TH increases during embryogenesis, this may reflect down regulation. Another possibility is that the change in erythrocyte population which occurs during this period involves production of erythrocytes which contain fewer T3 binding sites.     

Fatty acyl-CoAs are potent inhibitors of the nuclear thyroid hormone receptor in vitro

We report that long-chain fatty acyl-CoAs are potent inhibitors of the thyroid hormone (T3) receptor isolated from rat liver nuclei. Both saturated and unsaturated fatty acyl-CoAs were similarly potent. Fifty per cent inhibition of T3 binding by the receptor was observed at an oleoyl-CoA concentration as low as 1.3 microM, and the affinity of oleoyl-CoA for the receptor (Ki) was estimated to be 0.45 microM. Fatty acyl-CoAs also promoted dissociation of the hormone bound to the receptor. The action of fatty acyl-CoAs was competitive for the hormone binding site, resulting in a reduction in the receptor's affinity for T3. These observations suggest that fatty acyl-CoAs modulate the binding of the thyroid hormone to its nuclear receptor, in vitro. Whether or not such events occur in vivo remains to be determined.     

Modulatory Effects of Thyroxine Treatment on Central and Peripheral Benzodiazepine Receptors in the Rat

The effects of 10 days of D-thyroxine (T4) treatment on central benzodiazepine (BZ) receptors in the brain and on peripheral-type BZ binding sites in the heart, kidney, and testis of rats were studied. The experimental hyperthyroidism resulted in an increase in the density of cortical central BZ receptors, without any alteration of the affinity of the receptors to [3H]flunitrazepam. The increase in cortical central BZ receptors was also accompanied by the up-regulation of peripheral BZ binding sites in the heart, kidney, and testis. The affinity of the peripheral BZ binding sites for the ligand [3H]PK 11195 was not affected by T4 treatment in any of these three organs. The increase in the density of brain cortical central BZ receptors was less prominent than the increase in the peripheral BZ binding sites. The modulatory effect of T4 treatment on central and peripheral BZ receptors might be attributed to the direct interaction of the thyroid hormone at these sites or might reflect a physiological compensatory adaptation mechanism to thyrotoxicosis associated with hypermetabolism, anxiety, and stress.     

High affinity thyroid hormone binding sites on purified rat liver plasma membranes.

Two orders of saturable binding sites for L-T₃ were detected on purified rat liver plasma membranes--a high affinity, low capacity binding site with a Kd of 3.2 ± 0.5 nM, and a lower affinity, higher capacity site with a Kd of 220 ± 50 nM. Competition-inhibition studies revealed that both D-T₃ and L-T₄ (two compounds with lower biological potencies than L-T₃) were also less potent than L-T₃ in competing for these binding sites. The present studies demonstrate, therefore, the presence of specific thyroid hormone binding sites on rat liver plasma membranes. In addition, they suggest that these sites may have a role both in mediating the known effects of thyroid hormones on membrane functions, and in regulating the entry of thyroid hormones into target cells.     

Thyroid hormone receptors in brain and liver during ageing

The binding properties--binding capacity (MBC) and affinity (Ka)--of T3 nuclear receptors were analyzed in cortex, cerebellum and liver of rats aged 3, 6, 12 and 24 months. A slight but not significant decrease of Ka was observed in different tissues of normal rats. In hypothyroid animals the Ka in cortex at 24 months was significantly lower than at 3 months. During ageing the MBC of brain receptors decreased whereas hepatic receptors were not altered. Hypothyroidism did not further affect the MBC of the receptors. The data indicate that during ageing the T3 nuclear receptors behave differently in brain and liver. The difference in MBC suggests selectivity in organ sensitivity to thyroid hormones.     

Effects of dysthyroidism on central catecholaminergic neurons

After 15 years of research, it is clear that alterations in thyroidal status affect catecholaminergic neurons in the developing as well as in the adult brain. Experiments on fetal catecholaminergic brain areas grafted into the anterior eye chamber of adult thyroidectomized rat have shown the thyroid hormone dependency of the morphological differentiation of catecholaminergic neurons originating from the substantia nigra and the locus coeruleus. Furthermore, thyroid hormones also affect the metabolism of catecholaminergic neurons. Neonatal hypothyroidism induced either by ¹³¹I or by an antithyroid drug decreases the concentration of dopamine, noradrenaline and the activity of tyrosine hydroxylase at least in whole brain studies. Treatments with l-thyroxine of neonatally thyroidectomized rats reverse these neurochemical changes in a both time and dose dependent manner. These presynaptic modifications are associated with a decrease in the number of catecholaminergic receptors in different brain areas. On the opposite, experimental neonatal hyperthyroidism induced by daily administration of l-triiodothyronine increases the synthesis as well as the utilization of catecholamines. These changes are also associated with an alteration of catecholaminergic receptors. Despite numerous studies, there is, so far, no clear conclusion on the effects of neonatal dysthyroidism on the development of each catecholaminergic group. However, from these studies, it appears that the intensity of neonatal dysthyroidism greatly varies, depending of the monoamine and the brain area studied. The utilization of fetal brain cell cultures growing in a chemically defined medium has permitted to demonstrate the direct effect of thyroid hormones on fetal brain cells and the morphological effects of triiodothyronine on the size and the neurite length and arborization of fetal hypothalamic dopaminergic neurons.In the adult brain, hypothyroidism induced by surgical thyroidectomy, decreases the rate of catecholamines synthesis, decreases the number of alpha noradrenergic receptors and has no effect on striatal dopaminergic receptors. In contrast, hyperthyroidism increases the rate of catecholamines synthesis and induced an hypersensitivity of noradrenergic receptors. The intensity of the effects of dysthyroidism seems to be dependent on the monoamine and the brain area studied.In conclusion, it can be proposed that in the neonate thyroid hormones act on CA neuron activity mostly through a morphogenetic effect whereas in the adulthood they directly affect CA metabolism.     

Goitres in rats fed polychlorinated biphenyls.

Rats fed a polychlorinated biphenyl (PCB) mixture in a high- or low-iodine diet (HID or LID respectively) for 15 days had thyroid enlargement, low serum thyroxine (T4), and high serum thyrotropin concentrations. Although binding of thyroid hormones to serum proteins was reduced in PCB-fed animals, the free T4 index (reflecting free T4 in serum) was less in these rats. Both serum triiodothyronine (T3) and the free T3 index were elevated in rats fed PCB in HID. LID-maintained rats elevated serum T3 concentrations but the free T3 index was similar to that in HID-fed rats, owing to enhanced binding of thyroid hormone to serum proteins. Addition of PCB to LID reduced serum T3 levels but did not alter the free T3 index because binding was less. In rats fed HID containing PCB, thyroid 131I uptake was increased.     

Effect of Thyroid Hormones on Benzodiazepine Receptors in Neuron-Enriched Primary Cultures

The effect of administration of thyroid hormones on central benzodiazepine receptors was investigated using neuron-enriched primary cultures obtained from the neopallium of 16-day-old embryonic rats. Addition of L-triiodothyronine for 3 days decreased the maximal number of benzodiazepine receptor binding sites without any change in affinity at 10(-5) and 10(-6) M. L-Thyroxine administered for 3 days had the same effect at 10(-5) M. No significant change was observed over periods of less than 3 days, a finding indicating that this inhibition was not a direct in vitro effect. This down-regulation seems to be a direct modulatory effect of thyroid hormones on cerebral cortical neurons. Addition for 3 days of D-thyroxine and D-triiodothyronine, which are physiologically inactive isomers of the thyroid hormones, did not induce any significant alterations in benzodiazepine receptors. The decrease in number of cerebral cortical neuronal benzodiazepine receptors due to L-isomers of thyroid hormones may be related to the convulsions and anxiety observed in thyrotoxicosis in humans.