Glucocorticoid receptors in ageing rats.

The role of the glucocorticoid receptor (GR) in senescence was studied in rats of increasing age. Statistically significant changes in the number of GRs from rat liver were detected, whereas the affinity for the ligand triamcinolone acetonide (TA) did not change with increasing age, and was in the range of 1-2 nM. In all cases the number of receptors was lower in rats treated with hormone in vivo relative to untreated animals. In addition, we have found changes in GR activation, as measured by the binding to DNA cellulose in the mentioned age groups. Furthermore, expression of the glucocorticoid hormone (GH)-inducible gene, tyrosine amino transferase (TAT) also showed age-related alterations. We conclude that receptor function shows oscillatory changes during ageing. In addition, response to GH generally declines towards the older age. This specific periodicity in functional characteristics of the GR may reconcile conflicting results about the receptor number and properties during the ageing process, and marks particular age at which individual organism shows the highest or the lowest sensitivity to the actions of GH.

The mitochondrion as a primary site of action of glucocorticoids: the interaction of the glucocorticoid receptor with mitochondrial DNA sequences showing partial similarity to the nuclear glucocorticoid responsive elements.

Six mitochondrial genome sequences, showing strong similarity to the glucocorticoid responsive element consensus sequence (GRE), four localized within the cytochrome c oxidase (COX) subunit I and II genes (GREs I-IV) and two within the D-loop region (GREs a and b) have been examined as binding sites of glucocorticoid receptor (GR) from rat liver cytosol. Purified GR from rat liver cytosol binds with high specificity to all potential mitochondrial GREs, as shown by filter retention and gel shift assays. Specific binding of protein(s), present in a mitochondrial extract from dexamethasone-induced mice, to all six putative mitochondrial GREs was also documented by the same methodology. Both purified GR and protein(s) from mitochondrial extract give the same band in the gel retardation assay. Using monospecific anti-glucocorticoid receptor polyclonal antibody (EP), a supershift of the gel retarded protein-DNA band was obtained. These results demonstrate that the mitochondrial genome sequences examined have characteristics of GREs, since they show the capacity to specifically bind the respective receptor protein. These findings support the hypothesis that the mitochondrial genome is a primary site of action of steroid and thyroid hormones (Sekeris C.E.: The mitochondrial genome: a possible primary site of action of steroid hormones, In vivo 4 (1990) 317-320).

Import of the glucocorticoid receptor into rat liver mitochondria in vivo and in vitro.

Administration of inducing doses of dexamethasone (10 microg/100 g) to adrenalectomized rats results, within 2-5 min, in import of the glucocorticoid receptor from liver cytoplasm into mitochondria, as demonstrated by Western blotting and by electron microscopy. Furthermore, glucocorticoid receptor (GR) synthesized in an in vitro reticulocyte system programmed with the respective mRNA, enters within minutes to added rat liver mitochondria in the form of intact GR, as demonstrated by Western blotting using either monoclonal or polyclonal antibodies against different domains of GR. In vitro studies show that the import is dependent on temperature and/or activation of the hormone-GR complex. These results, in connection with the presence in the human and rodent mitochondrial genome of sequences showing partial homology to the nuclear glucocorticoid response elements, support the hypothesis that the well documented effects of glucocorticoids on mitochondrial functions result from a direct interaction of the GR complex with the mitochondrial genome.

Steroid receptors in nonmalignant and malignant kidney tissue of patients with endemic (Balkan) nephropathy.

The presence, affinity, binding capacity, structure and function of receptors for estrogen (ER), progesterone (PR) and glucocorticoid (GR) were investigated in 24 autologous pairs of control and neoplastic kidney tissues of patients with endemic (Balkan) nephropathy. In control tissue, all the three steroid receptors were absent in 20.8% and present in 25.0% of samples, whereas in malignant tissues the percentage of negative samples increased to 37.5% and that of positive ones decreased to 20.8%. Ten patients had identical receptors in both, control and cancer tissues. Due to malignant transformation nine patients lost one or more receptors, while five patients acquired them. Wide ranges of values were obtained when evaluating receptor affinity (Kd) and binding capacity (N). The structure and function of steroid receptors were investigated by determining the sedimentation coefficients (S) of steroid-receptor complexes before and after the activation. The unactivated GR-complex (8 S) was detected in two of control samples only, whereas in the remaining control tissues, as well as in malignant tissues only the activated form (4 S) was found regardless of the activation. PR and ER complexes were detected at 4 S region only. These results show that in endemic nephropathy the structure of steroid receptors may be altered often in both, non malignant and malignant kidney tissue, suggesting that the analysis of receptor structure may be worthwhile for the prediction of the success of eventual hormone therapy.

Steroid receptors in human uterine carcinoma. Their biological importance and therapeutic implications.

Surgical material of primary uterine carcinoma from postmenopausal patients was analyzed with regard to cytosol content of estradiol (ER) and progesterone (PR) receptors, as well as their binding sites in nuclear compartment. A dextrancoated charcoal technique was used and binding data were calculated from Scatchard plots. In all examined specimens intracellular high-affinity, low-capacity estrogen - and progesterone-binding proteins (which have the characteristics of steroid receptors), with equilibrium dissociation constants (Kd's) of 10(-10)M and 10(-9)M respectively, have been observed in detectable levels. DNA binding ability of steroid-receptor complexes was examined and clearly demonstrated even at 4 degrees C by using DNA-cellulose slurry. Preheating of complexes at 25 degrees cause increased DNA binding ability in some specimens, but significant loss of this ability in other tumors. The relationship between the nuclear retention of steroid-receptor complexes and their interaction with nuclear components (giving a response) is discussed on the basis of steroid-responsive and unresponsive systems. Present investigation indicate that described steroid analysis may become an important tool in predicting the value of endocrine therapy in human uterine carcinoma.