Thyroid Hormones Affect the Membrane Dipolar Organization. Is It a General Event in Their Non-genomic Action?

The surface balance technique was employed to study the interactions of 3,5,3',5' tetraiodo L-thyronine, 3,5,3' triiodo L-thyronine, and 3,5-diiodothyronine with monomolecular phospholipid monolayers spread at the air-water interface. With this technique the insertion of thyroid hormones into egg yolk phosphatidylcholine was investigated. An increase of surface pressure and a substantial decrement in surface potential were observed after the injection of these hormones beneath a phospholipid monolayer. The negative dipole contribution upon hormone interaction opposes the well-known positive contribution of phospholipids. These effects correlated with iodo content of the thyroid molecule analogues 3,5,3',5' tetraiodo L-thyronine >3,5,3' triiodo L-thyronine >3,5-diiodothyronine. To our knowledge, these observations suggest a new and surprising effect of thyroid hormones on the regulation of transmembrane dipolar organization.     

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.     

Direct in vitro action of thyroid hormones on mitochondrial RNA-polymerase

The authors show the direct in vitro action of thyroid hormones on RNA-polymerase activity in rat liver mitochondria. 3,5,3 L-triiodothyronine (L-T₃) and 3,5,3,5 L-tetraiodothyronine (L-T₄) stimulate mitochondrial RNA synthesis without either increasing the permeability of preswollen mitochondria or stimulating the synthesis of the triphosphate ribonucleotides (NTP's). Thyroid hormones do not directly depress mitochondrial RNA hydrolysis. Studies carried out with structural analogues of thyroid hormones indicate the structural specifications of the regulating system of the mitochondrial RNA-polymerase. L-T₃ and L-T₄ are also effective in vitro on mitochondria obtained from animals undergoing different hormonal and dietary treatments, with the exceptions of those fed with a hypoprotein diet. Thus, the authors suggest the possible intervention of a specific mitochondrial receptor for L-T₃ and L-T₄.     

Proton nuclear magnetic resonance assignments of thyroid hormone and its analogues

1H NMR data of a series of thyroid hormone analogues, e.g., thyroxine (T4), 3,5,3'-triiodothyronine (T3), 3,3',5'-triiodothyronine (rT3), 3,3'-diiodothyronine (3,3'-T2), 3,5-diiodothyronine (3,5-T2), 3',5'-diiodothyronine (3',5'-T2), 3-monoidothyronine (3-T1), 3'-monoiodothyronine (3'-T1), and thyronine (TO) in dimethylsulfoxide (DMSO) have been obtained on a 300 MHz spectrometer. The chemical shift and coupling constant are determined and tabulated for each aromatic proton. The inner tyrosyl ring protons in T4, T3, and 3,5-T2 have downfield chemical shifts with respect to those of the outer phenolic ring protons. Four-bond cross-ring coupling has been observed in all the monoiodinated rings. However, this long-range coupling does not exist in T4, diiodinated on both rings, and T0, containing no iodines on the rings. There is no evidence that at 30 degrees C these iodothyronines have any motional constraint in DMSO solution. In addition to identification of the hormones, the potential use of some characteristic peaks as probes in binding studies is discussed.     

Stimulation by thyroid hormone analogues of red blood cell Ca2+-ATPase activity in vitro. Correlations between hormone structure and biological activity in a human cell system

Human red blood cell membrane Ca2+-ATPase activity is stimulated in vitro by physiological concentrations (10(-10) M) of L-thyroxine (L-T4) and 3,5,3'-triiodo-L-thyronine (L-T3). This human cell system has been utilized to examine a series of iodothyronine and iodotyrosine analogues for structure-activity relationships. Analogue purity was verified by high pressure liquid chromatography. Analogues were studied at a concentration of 10(-10) M and the stimulatory effect of each analogue was compared with that of L-T4 in this system. Essential to Ca2+-ATPase stimulation were occupation of the 3 and 5 phenyl positions by iodide, bromide, or methyl groups, the L-configuration of the alanine side chain, side chain length equal to that of alanine, and a perpendicular (skewed) conformation of the two rings. The 4'-hydroxyl group is not essential to Ca2+-ATPase stimulation in this model system. T3 was 76% as active as T4 in stimulating Ca2+-ATPase activity. The stimulatory effect of 3,5-dimethyl-3'-isopropyl-L-thyronine and 3,5,3',5'-tetrabromo-L-thyronine approximated that of L-T4. Selected tyrosine analogues also stimulated the enzyme. The bioactivities of hormone analogues in this human model of extra-nuclear thyroid hormone action differ in several ways from results obtained previously in other animal model systems in vitro and in vivo.     

Thyroid hormones and mitochondria

Because of their central role in the regulation of energy-transduction, mitochondria, the major site of oxidative processes within the cell, are considered a likely subcellular target for the action that thyroid hormones exert on energy metabolism. However, the mechanism underlying the regulation of basal metabolic rate (BMR) by thyroid hormones still remains unclear. It has been suggested that these hormones might uncouple substrate oxidation from ATP synthesis, but there are no clear-cut data to support this idea. Two iodothyronines have been identified as effectors of the actions of thyroid hormones on energy metabolism: 3',3,5-triiodo-L-thyronine (T3) and 3,5-diiodo-L-thyronine (T2). Both have significant effects on BMR, but their mechanisms of action are not identical. T3 acts on the nucleus to influence the expression of genes involved in the regulation of cellular metabolism and mitochondria function; 3,5-T2, on the other hand, acts by directly influencing the mitochondrial energy-transduction apparatus. A molecular determinant of the effects of T3 could be uncoupling protein-3 (UCP-3), while the cytochrome-c oxidase complex is a possible target for 3,5-T2. In conclusion, it is likely that iodothyronines regulate energy metabolism by both short-term and long-term mechanisms, and that they act in more than one way in affecting mitochondrial functions.     

High resolution crystal structures of piscine transthyretin reveal different binding modes for triiodothyronine and thyroxine

Transthyretin (TTR) is an extracellular transport protein involved in the distribution of thyroid hormones and vitamin A. So far, TTR has only been found in vertebrates, of which piscine TTR displays the lowest sequence identity with human TTR (47%). Human and piscine TTR bind both thyroid hormones 3,5,3'-triiodo-l-thyronine (T(3)) and 3,5,3',5'-tetraiodo-l-thyronine (thyroxine, T(4)). Human TTR has higher affinity for T(4) than T(3), whereas the reverse holds for piscine TTR. X-ray structures of Sparus aurata (sea bream) TTR have been determined as the apo-protein at 1.75 A resolution and bound to ligands T(3) and T(4), both at 1.9 A resolution. The apo structure is similar to human TTR with structural changes only at beta-strand D. This strand forms an extended loop conformation similar to the one in chicken TTR. The piscine TTR.T(4) complex shows the T(4)-binding site to be similar but not identical to human TTR, whereas the TTR.T(3) complex shows the I3' halogen situated at the site normally occupied by the hydroxyl group of T(4). The significantly wider entrance of the hormone-binding channel in sea bream TTR, in combination with its narrower cavity, provides a structural explanation for the different binding affinities of human and piscine TTR to T(3) and T(4).     

Thyroid hormones and their effects: a new perspective

The thyroid hormones are very hydrophobic and those that exhibit biological activity are 3',5',3,5-L-tetraiodothyronine (T4), 3',5,3-L-triiodothyronine (T3), 3',5',3-L-triiodothyronine (rT3) and 3,5',-L-diiothyronine (3,5-T2). At physiological pH, dissociation of the phenolic -OH group of these iodothyronines is an important determinant of their physical chemistry that impacts on their biological effects. When non-ionized these iodothyronines are strongly amphipathic. It is proposed that iodothyronines are normal constituents of biological membranes in vertebrates. In plasma of adult vertebrates, unbound T4 and T3 are regulated in the picomolar range whilst protein-bound T4 and T3 are maintained in the nanomolar range. The function of thyroid-hormone-binding plasma proteins is to ensure an even distrubtion throughout the body. Various iodothyronines are produced by three types of membrane-bound cellular deiodinase enzyme systems in vertebrates. The distribution of deiodinases varies between tissues and each has a distinct developmental profile. Thyroid hormones. (1) the nuclear receptor mode is especially important in the thyroid hormone axis that controls plasma and cellular levels of these hormones. (2) These hormones are strongly associated with membranes in tissues and normally rigidify these membranes. (3) They also affect the acyl composition of membrane bilayers and it is suggested that this is due to the cells responding to thyroid-hormone-induced membrane rigidificataion. Both their immediate effects on the physical state of membranes and the consequent changes in membrane composition result in several other thyroid hormone effects. Effects on metabolism may be due primarily to membrane acyl changes. There are other actions of thyroid hormones involving membrane receptors and influences on cellular interactions with the extracellulara matrix. The effects of thyroid hormones are reviewed and appear to b combinations of these various modes of action. During development, vertebrates show a surge in T4 and other thyroid hormones, as well as distinctive profiles in the appearance of the deiodinase enzymes and nuclear receptors. Evidence from the use of analogues supports multiple modes of action. Re-examination of data from th early 1960s supports a membrane action. Findings from receptor 'knockout' mice supports an important role for receptors in the development of the thyroid axis. These iodothyronines may be better thought of as 'vitamone'-like molecules than traditional hormonal messengers.     

Effects of thyroid hormones on inner mitochondrial membrane fluidity

Authors studied the effects of thyroid hormones and their diasteroisomers and 3,5-diiodothyronine (LT2) on the fluidity properties of inner mitochondrial membrane (IMM) by specifical fluorescent probe for the internal zone of biological membranes, the 1,6-diphenyl-1,3,5-hexatriene (DPH). The studied parameters are Arrhenius and Perrin plots. The DPH shows a decreased fluorescence quenching in the presence of both T3 and T4. The maximum effect is observed with 2 nM LT2. LT2 is more effective than LT3 in the central zone. The data confirm the selective action of LT3 and LT4 on IMM fluidity.     

Regulation of the mitochondrial anabolism at low concentrations of thyroid hormones and by some of their structural analogues.

The Authors demonstrate that the in vitro stimulation of mitochondrial RNA synthesis produced by thyroid hormones takes place also at physiological levels, equal to those held in the liver cells of experimental animals. Two groups of male rats have been used: normal control animals (N) and animals surgically thyroidectomized on the 25th day of life (T). The animals were fed and kept in standard conditions and killed on the 85th day of life. The purification of mitochondrial samples and the determination of the mitochondrial RNA synthesis were carried out as previously described. The results suggest that the in vitro stimulation of mitocondrial RNA synthesis is already significant at the concentration of lnM. The trends are qualitatively comparable for either N or T animals. The structural analogues TRIAC (3,5,3'-triiodothyroacetic acid) and TRIPROP (3,5,3'-triiodothyropropionica acid) exhibit a clearly stimulatory effect on samples of N animals, while on samples of T animals is significant only for the first analogue. Similar trends are also observed on ADP/O ratio.     

Effects of methy-2[(chloro-4'-benzoyl)-4-phenoxy]-2 propionic acid on the respiratory activity of isolated rat liver mitochondria]

The effects of methyl-2 [(chloro-4' benzoyl)-4 phenoxy]-2 propionic acid (LF 153) on mitochondrial respiration and oxidative phosphorylation are studied in vitro. Its activity is related to that of 3,5,3'-triiodo-L-thyronine (LT3), 3,5,3'-triiodothyroacetic acid (TA3), and to that of clofibrate, LF 153 acts as an inhibitory uncoupler of oxidative phosphorylation. Its uncoupling action is however very intense.     

Stimulation in vitro of rabbit erythrocyte cytosol phospholipid-dependent protein kinase activity. A novel action of thyroid hormone

L-Thyroxine (T4) and 3,3',5-L-triiodothyronine (T3) at 10(-10) M stimulated phospholipid- and Ca2+-dependent protein kinase activity in rabbit red cell cytosol in vitro by 151 and 176%, respectively. Kinase of 30-fold greater specific activity, developed with 0.4 mM NaCl from cytosol applied to DEAE-cellulose, was also stimulated up to 2-fold by thyroid hormone. Hormone enhancement of kinase activity occurred after 60 min of incubation at 37 degrees C prior to enzyme assay. Thyroid hormone analogues triiodothyroacetic acid, 3,5-dimethyl-3'-isopropyl-L-thyronine, D-T3, D-T4, and 3,3',5'-L-triiodothyronine (reverse T3) were inactive. These results support a role for thyroid hormone endogenously in regulation of phospholipid-dependent protein kinase activity.     

Membrane cooperative enzymes. High molecular specificity for blocking action of thyroxine on triiodothyronine effect in rat erythrocyte and Escherichia coli systems.

The molecular specificity for the blocking action of thyroxine on the triiodothyronine effect in the cooperativity of membrane-bound rat erythrocyte acetylcholinesterase and Escherichia coli Ca2+-ATPase was analyzed. Changes in the values of n (Hill coefficient) were obtained at strict physiological levels of these hormones. The structural requirements of the thyroid hormones to modify the membrane-bound systems were studied using various analogues of these hormones. In the erythrocyte system, a very high molecular specificity for triiodothyronine and thyroxine actions was found. The L-alanine side is essential to carry out both the allosteric desensitization and the blocking effects. The blocking ability of thyroxine is characterized by the presence of iodine in the 5' position. The bacterial system presented only specificity for the triiodothyronine allosteric desensitization. A system of membrane-bound enzymes for the study of the actions of thyroid hormones, is presented here.     

Thyroxine-protein interactions. Binding constants for interaction of thyroxine analogues with the thyroxine binding site on human thyroxine-binding globulin.

The binding constants for interaction of various thryoxine analogues with the thyroxine binding site on human thyroxine-binding globulin have been determined. Equilibrium dialysis, at pH 7.4 and 37 degrees C, was used to measure the competitive effects of different iodothyronine compounds on the binding of 125I-labeled thyroxine to highly purified thyroxine-binding globulin. Relative to L-thyroxine, K = 6 . 10(9) M-1, the association constants of some important analogues were D-thyroxine, 1.04 . 10(9) M-1, 3,5-diiodo-3'-isopropyl-L-thyronine, 4.9 . 10(8) M-1; L-triiodothyronine, 3.3 . 10(8) M-1, 3,3',5'-DL-triiodothyronine (reverse triiodothyronine), 3.1. 10(8) M-1; tetraiodothyropropionic acid, 2.7 . 10(8) M-1; tetraiodothyroacetic acid, 2.6 . 10(8) M-1; 3', 5'- diiodo-DL-thyronine, 8.3 . 10(7) M-1; and 3,5-diiodo-DL-thyronine, 7.1 . 10(7) M-1. Calculation of the deltaG0 values for binding of the analogues indicates that a major contribution to the free energy favoring binding is made by the alanine side chain of thyroxine. A change in configuration of the alpha-amino group from the L to D form causes an unfavorable change of 1 kcal/mol in the free energy of binding. Removal of the alpha-amino group as in tetraiodothyropropionic acid causes an unfavorable change of 1.9 kcal/mol in the free energy of binding. With regard to ring substituents, the results indicate that the two inner 3,5-iodines make about the same contribution to binding as the two outer 3', 5'-iodines.     

Effect of membrane fatty acid composition on the action of thyroid hormones on (Ca2+ + Mg2+)-adenosine triphosphatase from rat erythrocyte

The action of L-triiodothyronine, L-thyroxine, and their analogues on the (Ca2+ + Mg2+)-ATPase of erythrocytes from rats fed with two different fat-supplemented diets has been studied. It was found that only L-triiodothyronine and L-thyroxine have effects on the (Ca2+ + Mg2+)-ATPase of both groups, producing inhibition in rats fed with corn oil and activation in rats fed with lard-supplemented diets. The half-maximal effect for L-triiodothyronine and L-thyroxine are, respectively, on the order of 10(-10) and 10(-8) M. These changes were not obtained with analogues of the hormones. It is suggested that the response of this enzymatic system to the hormonal action is conditioned by the fatty acid composition of membrane-bound lipids. This observation is novel in the hormonal research area.     

Studies on the mechanism of action of guinea pig lymphotoxin. II. Increase of calcium uptake rate in LT-damaged target cells.

In the previous paper it was suggested that the primary action of guinea pig lymphotoxin (LT) involved creation of ionic imbalances within the target L cells. The nature of these ionic disturbances is explored in this paper. The exogenous addition of CaCl2, but not KCl or NaCl, inhibited the cytotoxic action of LT. Cellular uptake rates of 45Ca++, but not 86Rb+, increased in LT-damaged L cells. The factor responsible for increasing the 45Ca++ uptake rate cochromatographed on a hydroxyapatite column with the cytotoxic activity of LT. Ouabain prevented the LT-mediated lysis and, concomitantly, depressed the LT-induced increase of 45Ca++ uptake rate. The LT-damaged L cells excluded trypan blue to the same extent as the normal cells. The addition of LT to and LT-resistant L cell mutant affected neither the 45Ca++ uptake rate nor the viability. From these observations, damage to the calcium transport system in the L cell plasma membrane is proposed as a mechanism of LT action.     

Human thyroid cyclic nucleotide phosphodiesterase. Its characterization and the effect of several hormones on the activity.

Cyclic AMP and cyclic GMP phosphodiesterase activities (3',5'-cyclic AMP 5'-nucleotidohydrolase, EC 3.1.4.17) were investigated in the human thyroid gland from patients with hyperthyroidism. Low substrate concentration (0.4 muM) was used. About 60% of the cyclic-AMP and 80% of the cyclic-GMP hydrolytic activities in the homogenate were obtained in the soluble fraction (105 000 X g supernatant). The thyroid gland contains two forms of cyclic-AMP phosphodiesterase, one with a Km of 1.3-10(-5) M and the second with a Km of 2-10(-6) M. Cyclic-AMP and cyclic-GMP phosphodiesterase were purified by gel filtration on a Sepharose-6B column. Cyclic-AMP phosphodiesterase activities were found in a broad area corresponding to molecular weights ranging from approx. 200 000 to 250 000 and cyclic-GMP phosphodiesterase activity was found in a single area corresponding to a molecular weight of 260 000. Cyclis-AMP phosphodiesterase activities were stimulated by the protein activator which was found in human thyroid and this stimulation was dependent on Ca2+. Stimulation of cyclic-AMP phosphodiesterase by the activator was not significant even in the presence of enough Ca2+. The effect of D,L-triiodothyronine, D,L-thyroxine, L-diiodotyrosine, L-monoiodotyrosine, L-thyronine, L-diiodothyronine, thyrotropin, hydrocortisone, adrenocorticotropin, cyclic-AMP and cyclic-GMP on the phosphodiesterase activities was studied. Cyclic-AMP, cyclic-GMP, D,L-triiosothyronine, D,L-thyroxine, adrenocorticotropin and hydrocortisone where found to inhibit the phophodiesterase. Triiodothyronine and thyroxine inhibited cyclic-AMP phosphodiesterase more effectively than cyclic-GMP phosphodiesterase. Thyroxine was a more potent inhibitor than triiodothyronine. The concentration of cyclic AMP producing a 50% inhibition of cyclic-GMP phosphodiesterase activity was 5-10(-5) M, while the concentration of cyclic GMP producing a 50% inhibition of cyclic-AMP phosphodiesterase was 3-10(-3) M. Both cyclic-AMP and cyclic-GMP phosphodiesterase activities in the homogenate of hyperthyroidism, thyroid carcinoma and adenoma were higher than in normal thyroid tissue, when assayed with a low concentration of the substrate (0.4 muM). When a higher concentration (1 mM) of cyclic nucleotides was used as the substrate, cyclic-AMP hydrolytic activity in adenoma tissue was similar to that of normal tissue, while the other activities were higher than normal.     

Thyroid Hormone Enhances the Formation of Synapses Between Cultured Neurons of Rat Cerebral Cortex

1. Thyroid hormones play important roles in the development of the brain. Increasing evidence suggests that the deprivation of thyroid hormones in the early developmental stage causes structural and functional deficits in the CNS, but the precise mechanism underlying this remains elusive. In this study, we investigated the effects of thyroid hormones on synapse formation between cultured rat cortical neurons, using a system to estimate functional synapse formation in vitro. 2. Exposure to 10(-9) M thyroid hormones, 3,5,3'-triiodothyronine or thyroxine, caused an increase in the frequency of spontaneous synchronous oscillatory changes in intracellular calcium concentration, which correlated with the number of synapses formed. 3. The detection of synaptic vesicle-associated protein synapsin I by immunocytochemical and immunoblot analysis also confirmed that exposure to thyroxine facilitated synapse formation. 4. The presence of amiodarone, an inhibitor of 5'-deiodinase, or amitrole, a herbicide, inhibited the synapse formation in the presence of thyroxine. 5. In conclusion, we established a useful in vitro assay system for screening of miscellaneous chemicals that might interfere with synapse formation in the developing CNS by disrupting the thyroid system.     

Inhibition of Ca++-stimulated respiration by uncouplers.

In a phosphate medium at pH 6.6 low concentrations of uncouplers such as p-trifluoromethoxyphenylhydrazone carbonylcyanide and 2,4-dinitrophenol inhibit the oxidation of beta-hydroxybutyrate and succinate, when added during Ca++-accumulation. The inhibition depends on the amount of accumulated Ca++, and is released by N,N,N',N'-tetramethyl-p-phenylendiamine plus ascorbate as substrate. Under identical conditions the uncouplers have no inhibitory effect when added to mitochondria during state 3 respiration or during accumulation of Sr++. Inhibition of respiration by the decrease of transmembranal succinate transport or by accumulation of oxaloacetate can be excluded. It is suggested that accumulation of Ca++ in the presence of phosphate induces structural alteration of the mitochondrial membrane, which on the one hand changes the accessibility or sensitivity of dehydrogenases to uncouplers and causes leakage of accumulated Ca++ on the other.     

Non-genomic effect of L-triiodothyronine on calmodulin-dependent synaptosomal protein phosphorylation in adult rat cerebral cortex

The present study was undertaken to examine calmodulin-dependent effect of thyroid hormones (THs) on synaptosomal protein phosphorylation in mature rat brain. Effect of L-triiodothyronine (L-T3) on in vitro protein phosphorylation was measured in a hypotonic lysate of synaptosomes prepared from adult male rat cerebral cortex, incubated in presence and absence of calcium ion (Ca2+) and calmodulin. L-T3 significantly enhanced incorporation of 32P into synaptosomal proteins as compared to basal level of phosphorylation in the presence of Ca2+ and calmodulin. Under these conditions, increase in protein phosphorylation was 47, 74 and 52% for 10 nM, 100 nM and 1 microM L-T3, respectively. Chelation of Ca2+ using ethylene glycol-bis (2-aminoethylether)-N, N, N', N'-tetraacetic acid (EGTA) inhibited the effects of Ca2+/calmodulin on TH-stimulated protein phosphorylation levels. This study suggests that a high proportion of L-T3-stimulated protein phosphorylation involves Ca2+/calmodulin-dependent pathways in adult rat cerebrocortical synaptosomes.