Effects of thyroid hormones on the receptor level in estrogen target organs

The influence of thyroid hormones on the turnover of cytoplasmic estrogen receptors in the liver, kidney and uterus of intact and ovariectomized female rats was studied under in vivo conditions. Thyroidectomy had no significant effect on the receptor level in the uterus but caused a substantial reduction of the receptor content in the liver and kidney. In livers of intact and ovariectomized animals receptor values were reduced with 70 and 80%, respectively, 30 days after thyroidectomy. Substitution with triiodothyronine (T3) restored the hepatic estrogen receptor concentration in thyroidectomized rats to the preoperative level. If rats that had been both ovariectomized and thyroidectomized were substituted with thyroid hormone for the same time period, the receptor level was increased but did not reach the level seen in animals that had been ovariectomized only. The effects of thyroid hormone substitution was found to be dose dependent and paradoxical. Thus, a high dose of 50 micrograms/day of triiodothyronine given to intact animals for nine days caused a 30% reduction in the hepatic receptor content. The same level of reduction was seen in the ovariectomized rat given a hormone dose of only 1 micrograms/day. When this type of rats was treated with the higher dose of triiodothyronine the reduction in hepatic estrogen receptors was 50%. These results are discussed in relation to existing information concerning the multihormonal regulation of estrogen receptor concentration in the rat liver.     

Morphological and functional studies during aging at mitochondrial level. Action of drugs

1. Mitochondria show morphological changes on aging: the volume density decreases and the total surface area or volume increase. 2. Biochemical studies indicated a decrease in the rate of oxygen consumption and ATP levels for increasing age. Measurements of calcium transport across the mitochondrial membrane revealed a decrease in accumulated calcium and an alteration in the uptake kinetics of the ion in older animals. 3. Theophylline and calcitonin action were also studied. In both age groups (young and old) theophylline shows an inhibitory effect on all the parameters studied (both morphological and biochemical). 4. Calcitonin showed no effect on morphological and respiratory parameters, although it increased calcium uptake into the mitochondrion to a lesser extent in the 24 month old animals.     

Cardiac mechanics at the cellular level.

The active mechanical properties of heart muscle are load, length, and time-dependent. The capability for investigating cardiac mechanisms at the cellular level may help to distinguish between those properties of the myocardium which arise from myocardial cells and those which arise from the tissue architecture and extracellular matrix of connective fibers. We present here, for the first time, a general approach for subjecting single heart cells to isometric, isotonic, afterloaded, or physiological loading sequences, while obtaining on-line measures of cell force and length. This approach has been implemented and tested on freshly dissociated, adult frog ventricular myocytes. Examples are presented for each of the four loading sequences.     

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.     

Restitution at the cellular level: regulation of the migrating phenotype.

Intestinal epithelial cells migrating across a mucosal defect are generally described as dedifferentiated, a term that suggests a loss of regulatory biology. Since cell biology may be more readily studied in established cell lines than in vivo, a model is developed using the human Caco-2 intestinal epithelial cell migrating across matrix proteins. This resembles in vivo models of mucosal healing in its sheet migration and loss of the brush border enzymes, which are conventional markers for intestinal epithelial differentiation. Immunohistochemical studies of migrating Caco-2 cells suggest, however, that the rearrangements of cytoskeletal, cell-cell and cell-matrix proteins during migration are not random but seem adapted to the migratory state. Indeed, Caco-2 migration may be substantially regulated by a variety of physiologic and pharmacologic stimuli and differentiation, measured by the specific activity of the intestinal epithelial brush border enzymes alkaline phosphatase and dipeptidyl dipeptidase, may be independently pharmacologically programmed during the stimulation or inhibition of cell motility.     

Action of thyroid hormones on the calorigenic effect of adrenaline]

The present investigation was undertaken in order to appraise the interaction between thyroxine and epinephrine in dogs exposed to acute cold. In normal, then successively thyroidectomized, adrenal demedullated and thyroxine restored dogs, epinephrine was infused in basal condition and during acute cold exposure. In thyroidectomized dogs epinephrine lost its calorigenic effects. So it did in thyroidectomized and adrenal demedullated dogs. Conversely, when dogs were restored in thyroxine, epinephrine recovered its calorigenic effect. Evidence for thyroxine-catecholamine interaction can be seen in basal condition as during acute cold. Nevertheless, this interaction is more obvious during shivering thermogenesis.     

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.     

Effect of thyroid hormones and their analogues on the mitochondrial calcium transport activity.

In this paper the authors studied the effects of thyroid hormones and their structural analogues on the mitochondrial calcium transport activities. The thyroid hormones, 3,5,3' L-triiodothyronine (LT3) and 3,5,3'5' L-tetraiodothyronine (LT4) at physiological intracellular concentrations between 7.2 and 9 nM, decouple total Ca++ transport, as well as inhibit the passive transport of Ca++, either due to oxidation of pyruvate, malate or succinate or after inhibition with rotenone. The optical isomers 3,5,3' D-triiodothyronine (DT3) and 3,5,3',5' D-tetraiodothyronine (DT4) are less effective at all the used concentrations. Furthermore the structural analogues 3,3',5' L-triiodothyronine (LrT3), 3,5-dicloro, 3',5' L-diiodothyronine (LDiClT2) and 3,5 L-diiodothyronine (LT2) furnished even less effects on the same activities. The effect of the thyroid hormones and of their structural analogues has revealed that the mitochondrial calcium transport may be influenced both by a stereospecific interaction between hormones and protein ligands and by a lipophilic chaotropic action on the mitochondrial membranes lipids. In this context it is interesting to consider that both thyroid hormones and Ca++ transport activity are interacting with the energetic metabolism by means of phosphorylation and substrate oxidation mechanism.     

The mechanism of the increase in mitochondrial proton permeability induced by thyroid hormones.

Three possible mechanisms by which different levels of thyroid hormones in rats might cause the observed sevenfold change in the apparent proton permeability of the inner membrane of isolated liver mitochondria were investigated. (a) Cytochrome c oxidase was isolated from the livers of hypothyroid, euthyroid and hyperthyroid rats and incorporated into liposomes made with soya phospholipids. There was no difference between the proton current/voltage curves of the three types of vesicles. The hormonal effects, therefore, were not an inherent property of the enzymes, and were not due to different coupling of electron flow through the enzyme to proton transport. (b) The surface area of the mitochondrial inner membrane was shown by three different assays to be greater by a factor of between two and three in mitochondria from hyperthyroid animals than in mitochondria from hypothyroid animals; euthyroid controls were intermediate. This difference in surface area of the inner membrane explains less than half of the difference in apparent proton permeability. (c) The proton permeability of liposomes prepared from phospholipids extracted from mitochondrial inner membranes of hyperthyroid rats was three times greater than the proton permeability of those from hypothyroid rats; euthyroid controls were intermediate. This suggests, first, that the proton permeability of the phospholipid bilayer is an important component of the proton permeability in intact mitochondria and, second, thyroid hormone-induced changes in the bilayer are a major part of the mechanism of increased proton permeability. Such changes may be due to the known differences in fatty acid composition of mitochondrial phospholipids in different thyroid states. Thus we have identified two mechanisms by which thyroid hormone levels in rats change proton flux/mass protein in isolated liver mitochondria: a change in the area of the inner membrane/mass protein and a change in the intrinsic permeability of the phospholipid bilayer.     

Quantification of calcitonin gene expression at the cellular level in medullary carcinoma of the thyroid

Calcitonin mRNA was detected in human medullary carcinoma using an in situ hybridization technique. This specific and reproducible method could lead to a better functional characterization of medullary carcinoma and should allow the definition of new prognosis factors in medullary thyroid cancer.     

Apoptosis: controlled demolition at the cellular level

Apoptosis is characterized by a series of dramatic perturbations to the cellular architecture that contribute not only to cell death, but also prepare cells for removal by phagocytes and prevent unwanted immune responses. Much of what happens during the demolition phase of apoptosis is orchestrated by members of the caspase family of cysteine proteases. These proteases target several hundred proteins for restricted proteolysis in a controlled manner that minimizes damage and disruption to neighbouring cells and avoids the release of immunostimulatory molecules.     

Action of insect hormones at the cellular level : II. Differing sensitivity to β-ecdysone of several lines and clones tof Drosophila melanogaster Cells

1. 1. When checked for their responsiveness to β-ecdysone, several established lines and sublines of Drosophila melanogaster cells displayed varying behaviour: (a) most of them responded with characteristic morphological modifications accompanied by an arrest of cell multiplication; (b) in one line, the cells only stopped dividing; (c) another line reacted with immediate death of all cells; (d) one line was found to be resistant to ecdysone.

2. 2. To establish the possible genetic grounds of such a differential sensitivity to ecdysone, clones were isolated from some of the lines. All clones isolated from one of the sensitive lines displayed the same succession of morphological modifications as the original line, but each of them with its own rate. The specific sensitivity to β-ecdysone of subclones derived from the above clones was established with greater precision. Each of them responded when a certain threshold concentration of β-ecdysone (varying from 0.5×10⁻⁸ M to 5×10⁻⁸ M) was reached in the culture medium. The clones were most sensitive to ecdysone between the 3rd and the 7th month of their development.

We have studied clones displaying a wide range of reactions to ecdysone, from cells sensitive to very low concentrations of the hormone, to completely resistant cells. Comparison of these genetically defined populations of cells might help towards an understanding of the different stages of ecdysone action at the cellular level.     

Influence of mitochondrial DNA level on cellular energy metabolism: implications for mitochondrial diseases

The total amount of cellular mitochondrial DNA (mtDNA) varies widely and seems to be related to the nature and metabolic state of tissues and cells in culture. It is not known, however, whether this variation has any significance in vivo, and to which extent it regulates energy production. To better understand the importance of the cellular mtDNA level, we studied the influence of a gradual reduction of mtDNA copy number on oxidative phosphorylation in two models: (a) a control human cell line treated with different concentrations of 2′, 3′-dideoxycytidine, a nucleoside analogue that inhibits mtDNA replication by interfering with mitochondrial DNA polymerase γ, and (b) a cell line derived from a patient presenting mtDNA depletion. The two models were used to construct biochemical and phenotypic threshold curves. Our results show that oxidative phosphorylation activities are under a tight control by the amount of mtDNA in the cell, and that the full complement of mtDNA molecules are necessary to maintain a normal energy production level.     

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.