Anikó Náray

Effect of calcium on chick thymus glucocorticoid receptor

Abstract The effect of Ca 2+ in the millimolar range was investigated on the heat stability of chick thymus receptor in the absence of glucocorticoids. Size and DNA-binding of glucocorticoid-receptor complex was also studied after calcium-treatment. Ca 2+ reduced dramatically and in a concentration-dependent way the heat stability of the receptor at 25°C in the absence of hormone. Binding of triamcinolone acetonide-receptor complex to DNA-cellulose was also strongly decreased when 0.5–8mM Ca 2+ was present during activation of the complex or if cytosol was pre-treated with Ca 2+ before addition of the steroid. Ca 2+ treatment caused a significant reduction in the size of receptor-hormone complex as determined by Sephacryl S-200 chromatography. The results suggest that Ca 2+ may have an in vivo regulatory role decreasing or inhibiting the expression of glucocorticoid hormone action under specific conditions.

The effect of a Ca2+-activated protease on the glucocorticoid receptor of lymphoid tissues

Abstract The effect of 0.5–8 mM Ca2+ on the stability and DNA- or nuclear binding capability of the cytosol glucocorticoid receptors from different lymphoid tissues of chickens and rats was studied. Ca2+ treatment at 25°C significantly increased the inactivation rate of the steroid-free receptor in chick thymus cytosol, but only slightly influenced it in the case of receptors from bursa of Fabricius or from rat thymus cytosol. Ca2+ ion strongly decreased the binding of cytosol hormone-receptor complex to isolated nuclei or DNA-cellulose. The decreased DNA-binding activity of the hormone-receptor complex could be seen in both cases if the Ca2+ -treatment was carried out before or after the formation of hormone-receptor complex. This effect could be observed at much higher Ca2+ concentrations in the case of bursa and rat thymus cytosol than in the case of chick thymus cytosol. The effect of Ca2+ on heat-stability of the receptor was diminished by carbobenzoxy-D-Phe-Pro-Argaldehyde, an inhibitor of thrombin-like enzymes, while the effect on DNA-binding could be abolished with three different types of protease-inhibitors as well as with SH-attacking agents. The results are consistent with the hypothesis that the mechanism of Ca2+ action on the glucocorticoid receptors is probably the activation of some proteolytic enzymes, which are SH-proteases.

Comparative study of glucocorticoid sensitivity and receptors in lymphoid tissues.

Abstract Glucocorticoid sensitivity of different lymphoid tissues of avian and mammalian origin was investigated. As a measure of glucocorticoid effect variation of thymidine kinase activity was selected. By this method an order of sensitivity was established among various species and tissues. Dexamethasone uptake by isolated lymphocytes and specific binding to cytosol receptors was also examined. No direct correlation was found between glucocorticoid sensitivity and either binding capacity (receptor number per cell) or affinity of receptor for hormone. The presence of one homogeneous binding site was detected in each tissue examined both by isolated cell technique and cytosol saturation analysis in the range of 1–40 nM of [3H]-dexamethasone concentration. In all cases about 80% of the bound dexamethasone was associated with cell nucleus in our system. Dexamethasone binding of the hydrocortisone resistant subpopulation of chicken lymphoid tissues was studied after in vivo treatment with high doses of hydrocortisone. The surviving cells contained an amount of dexamethasone receptor cell similar to that of the whole population. A theoretical analysis of the steady-state receptor cycle gives an explanation for the differences between the values of KA determined in intact cells or in cellfree systems.

Molybdate effect on the glucocorticoid receptor in cell-free systems and intact lymphocytes

The effect of sodium molybdate on the stability and activation of the glucocorticoid receptor from chick and rat thymus were investigated. Molybdate, at a concentration range of 1-10 mM, blocked denaturation of the cytosol receptor by elevated (25 and 37 degrees C) temperatures. This effect could be observed only with the aggregated (low-salt) form of the receptor. Molybdate also inhibited transformation of the receptor-hormone complex to the DNA-binding state which occurs either with incubation at 25 degrees C or with salt treatment. The inhibitory effect of molybdate could be observed only on the non-activated receptor; nuclear-and DNA-binding of the activated receptor was not significantly changed by molybdate. Both effects were concentration-dependent. Molybdate had no effect on the activation of the partially purified glucocorticoid receptor. Molybdate effect was also examined using intact lymphocytes. Sodium molybdate had no effect either on the steroid binding of whole cells or on the nuclear transfer of the hormone-receptor complex. While the mechanism of molybdate action remains unknown the results of experiments on purified receptor suggest that molybdate does not act directly on the receptor molecule; rather through some cytosol factor(s). However, these effects could only be seen in cell-free experiments, and not during the conditions of the living cell.

Pyridoxal-5-phosphate affects glucocorticoid receptors in intact lymphocytes similarly as in cell-free systems

Pyridoxal-5-phosphate markedly decreased the heat-stability of the unbound thymus cytosol receptor as well as that of the receptor-[3H]-triamcinolone acetonide complex in cell-free systems. Treatment with pyridoxal phosphate also decreased DNA-binding of the complex, being present either before or after heat- and salt-activation. 5 mM pyridoxal phosphate was required for 50% inhibition of DNA-binding. Pyridoxine, at similar concentrations, had no inhibitory effect. Pre-incubation of intact thymocytes with 10 mM pyridoxine caused also a marked decrease in the binding of [3H]-triamcinolone acetonide by the cells. Treating the cells with liposomes containing 1-100 mM pyridoxal phosphate caused an increase in intracellular pyridoxal phosphate concentration by about 0.1-10 microM and a decrease in [3H]-triamcinolone acetonide binding of the cells by about 50%. The results suggest that 1. pyridoxal phosphate acts not only on the activated but also on the unbound and non-activated forms of glucocorticoid receptor in cell-free systems; 2. pyridoxal phosphate has a similar effect in the intracellular millieu and thus, 3. pyridoxal phosphate might act as a physiologic regulator of the steroid hormone action.

Progesterone receptor in the chick thymus

Abstract Specific, high affinity binding sites for progesterone and promegestone /R-5020/ have been shown to be present in the chick thymus measured by experiments with intact cells or under cell-free conditions. In isolated thymocytes most of the receptor-R-5020 complex is bound to the nucleus. Dissociation constants were determined in thymic cytosol by Scatchard plot analysis and were found to be 3.1 and 2.6 nM for progesterone and R-5020, respectively. On the basis of competition assays the binding sites seemed to be specific for progesterone and R-5020. Glucocorticoids bind only slightly and only at high concentrations. By gel-filtration experiments the thymic R-5020 binding site was shown to be a macromolecule. In vivo treatment of chicks with progesterone or R-5020 caused a significant increase in thymidine kinase activity of the thymus.

STEROID RECEPTORS IN THE LYMPHOID ORGANS OF THE CHICK

Publisher Summary This chapter discusses the steroid receptors in the lymphoid organs of the chick. Two to three week old chickens were injected with various doses of dexamethasone phosphate or triamcinolone acetonide 48 and 24 hour before the experiment. It is shown that glucocorticoids caused a dose-dependent decrease in the weight and in thymidine kinase (TK) activity of lymphoid tissues. The three different organs did not respond in the same extent to hormone treatment. The most pronounced effect was observed on the thymus, somewhat less on the spleen while the bursa of Fabricius was rather resistant to glucocorticoid treatment. It is found that TK activity in the bursa even increased significantly after very small doses of steroid were given. The different lymphoid cell populations showed different sensitivity to glucocorticoids both in vivo and in vitro. The thymus was the most sensitive and the bursa Fabricius the most resistant to glucocorticoid treatment. Responsiveness to glucocorticoids of these lymphocytes does not seem to correlate with their glucocorticoid receptor content or the binding affinity of the receptors. The gluco-receptors from all tissues examined showed similar specificity for steroids.

GLUCOCORTICOID RECEPTORS OF LYMPHOID CELLS OF DIFFERENT STEROID SENSITIVITY

Publisher Summary This chapter discusses glucocorticoid receptors of lymphoid cells of different steroid sensitivity. Lymphoid tissues and cells are targets for glucocorticoid hormones. Glucocorticoids exert their catabolic effects on lymphocytes both in vivo and in vitro . Sensitivity to glucocorticoids varies among lymphoid cells. There are species differences in responsiveness to steroids: most rodent are regarded sensitive, whereas man, guinea-pig, and monkey are regarded resistant species. Even in a sensitive species various lymphocyte populations respond differently to glucocorticoids. Sensitivity changes also with the developmental state of the cell. Recently, several lines of evidence have suggested that receptor number and glucocorticoid sensitivity are not always correlated. The chapter examines in vivo and in vitro responses to glucocorticoids and the number and binding properties of receptors in various lymphoid cells of 5 species. In vivo glucocorticoid sensitivity was monitored by measuring tissue weight and the activity of thymidine kinase (TK); the latter is known to change paralell with the rate of DNA synthesis.