Triamcinolone acetonide regulates glucocorticoid-receptor levels by decreasing the half-life of the activated nuclear-receptor form

Glucocorticoid-receptor activation in GH1 cells results from the conversion of a 10 S oligomeric cytosolic form to a 4-5 S nuclear-binding species (Raaka, B. M., and Samuels, H. H. (1983) J. Biol. Chem. 258, 417-425). In this study, we report that triamcinolone acetonide (9 alpha-fluoro-11 beta, 16 alpha, 17 alpha, 21-tetrahydroxypregna-1,4-diene-3,20-dione 16,17-acetonide) elicits a time- and dose-dependent reduction of total-cell (nuclear + cytoplasmic) receptor. The mechanism of receptor regulation was studied by dense amino acid labeling of receptor using media containing 2H, 13C, and 15N-labeled amino acids. Total cell receptor was extracted with 0.4 M KCl and newly synthesized dense receptor was separated from pre-existing receptor of normal density by centrifugation in gradients of 15-30% sucrose (w/v) in D2O. Receptor levels in cells grown without [3H]triamcinolone acetonide was 260 +/- 19 fmol/100 micrograms of DNA (16,000 molecules/cell), and, with 10 nM [3H]triamcinolone acetonide, this decreased to 130 +/- 14 fmol/100 micrograms of DNA after 30 h. Receptor half-life was 19 +/- 1.9 h in the absence and 9.5 +/- 0.3 h in the presence of triamcinolone acetonide and accounted for the decrease in steady-state receptor levels. Receptor synthesis was 9.7 +/- 0.3 fmol/100 micrograms of DNA/h (580 molecules/cell/h) both in the presence and absence of 10 nM [3H]triamcinolone acetonide. Triamcinolone acetonide reduced the half-life in proportion to the extent of receptor occupancy and activation. During the approach to steady-state conditions, 10 nM [3H]triamcinolone acetonide shortened receptor half-life almost immediately to the value in cells grown with [3H]triamcinolone acetonide for 24 h or longer. Cycloheximide did not prevent the triamcinolone acetonide-mediated decrease in receptor half-life and the shortening of receptor half-life is rapidly reversed by removal of hormone. These studies support a model of receptor regulation in which triamcinolone acetonide converts the unactivated 10 S receptor to the activated 4-5 S form which is degraded at an increased rate by the cell.     

Cross-regulation between G-protein-coupled receptors. Activation of beta 2-adrenergic receptors increases alpha 1-adrenergic receptor mRNA levels.

Stimulation of DDT1 MF-2 vas deferens cells with epinephrine resulted in a time- and dose-dependent loss of alpha 1-adrenergic receptor-specific ligand binding. Regulation of alpha 1-adrenergic receptor mRNA was characterized. In monolayer culture, cells displayed 0.7 +/- 0.05 amol of alpha 1-adrenergic receptor mRNA/microgram of total cellular RNA. Epinephrine, which acts at both alpha 1- and beta 2-adrenergic receptors of DDT1 MF-2 cells, induced a short term (2-8 h) increase (50-70%) in the abundance of alpha 1-adrenergic receptor mRNA. Propranolol, a beta 2-adrenergic receptor antagonist, attenuated the epinephrine-mediated increase in alpha 1-adrenergic receptor mRNA but did not affect the decrease in alpha 1-adrenergic receptor-specific ligand binding. Phentolamine, an alpha 1-adrenergic receptor antagonist, did not attenuate the epinephrine-mediated increase in alpha 1-adrenergic receptor mRNA at 4 h but did block the decrease in alpha 1-adrenergic receptor-specific ligand binding. The half-life of the alpha 1-adrenergic receptor mRNA was approximately 7 h in untreated cells as well as in cells challenged with epinephrine. The epinephrine-promoted increase in alpha 1-adrenergic receptor mRNA was found to result from cross-regulation via beta 2-adrenergic receptors. Cholera toxin, forskolin, as well as the cyclic AMP analog CPT cAMP (8-(4-chlorophenylthio)adenosine 3':5'-cyclic monophosphate) increased the alpha 1-adrenergic receptor mRNA at 4 h, as did epinephrine in the presence of alpha 1-antagonists but not in the presence of a beta-adrenergic antagonist. This is the first report of heterologous up-regulation of mRNA levels of adrenergic receptors. Cross-regulation between alpha 1- and beta 2-adrenergic receptor-mediated pathways at 4 h occurs at the level of mRNA whereas later down-regulation of alpha 1-receptor mRNA and binding proceed via agonist activation of alpha 1-adrenergic receptors.     

Inhibition by dexamethasone of beta 3-adrenergic receptor responsiveness in 3T3-F442A adipocytes. Evidence for a transcriptional mechanism.

Modulation of beta 3-adrenergic receptor (beta 3AR) expression by dexamethasone was investigated in the murine 3T3-F442A adipocytic cell line. In untreated cells, a major population of binding sites (62,000-114,000 sites/cell) of low affinity for (-)-[3H] CGP12177 and (-)-[125I]iodocyanopindolol (corresponding to the beta 3AR subtype) was present along with a minor population (6,500-8,000 sites/cell) of sites of high affinity for the radioligands (corresponding to a mixture of the beta 1 and beta 2AR subtypes). Long-term exposure of the cells to 250 nM dexamethasone led to a sharp decrease in beta 3AR density (less than 5,000 sites/cell) which paralleled a diminished potency of the beta 3AR-selective agonists BRL37344 and CGP12177 to stimulate the production of intracellular cAMP. Analysis of RNA by polymerase chain reaction and nuclear run-on assays indicated that dexamethasone inhibited the synthesis of beta 3AR mRNA, resulting in 4-8-fold decrease in the steady-state levels of this mRNA. The down-regulation of beta 3AR protein and cellular mRNA appeared to be mediated by the receptor for glucocorticoids as assessed by the antagonistic action of the anti-glucocorticoid RU38486.     

Interactions between CRF, epinephrine, vasopressin and glucocorticoids in the control of ACTH secretion

The secretion of ACTH by corticotrophs in the anterior lobe of the rat pituitary gland is under the stimulatory influence of at least three receptors, namely that for peptidic CRF (corticotropin-releasing factor), vasopressin and alpha 1-adrenergic agents. CRF is a potent stimulator of cyclic AMP accumulation as well as adenylate cyclase activity in the rat adenohypophysis, thus suggesting an important role of cyclic AMP as mediator of CRF action on ACTH secretion. Vasopressin causes a 2-fold increase of the stimulatory effect of CRF on ACTH release in rat anterior pituitary cells in culture. The potentiating effects of vasopressin on CRF-induced ACTH release are accompanied by parallel changes of intracellular cyclic AMP levels. Vasopressin, while having no effect on basal cyclic AMP levels, causes a 2-fold increase in CRF-induced cyclic AMP accumulation without affecting the ED50 value of CRF action. ACTH secretion is also stimulated by a typical alpha 1-adrenergic receptor. Epinephrine causes a marked stimulation of ACTH release which is additive to that of CRF. Epinephrine, in analogy with vasopressin, although having no effect alone on basal cyclic AMP levels, causes a marked potentiation of CRF-induced cyclic AMP accumulation. Glucocorticoids cause a near-complete inhibition of epinephrine-induced ACTH secretion within 4 h with the following order of ED50 values: triamcinolone acetonide (0.2 nM) greater than dexamethasone (1.0 nM) much greater than cortisol (11 nM) greater than corticosterone (22 nM). Similar effects are observed for CRF- and vasopressin-induced ACTH release. Although the activity of the pituitary-adrenocortical axis in the rat is highly dependent upon sex steroids, 17 beta-estradiol, 5 alpha-dihydrotestosterone and the pure progestin R5020 have no detectable effect on basal or epinephrine-induced ACTH release, thus illustrating the high degree of specificity of glucocorticoids in their feedback control of ACTH secretion. Moreover, glucocorticoids have no effect on CRF-induced cyclic AMP accumulation, thus indicating that their inhibitory effect is exerted at a step following cyclic AMP accumulation.