The effects of glucocorticoid hormone on the expression of c-jun

The effects of glucocorticoid hormone on the expression of c-jun in the fibroblasts were studied. The expression of c-jun was repressed by dexamethasone in the NIH3T3 cells, but not in the transformed B104-1 or EJ-Ras cells. The repression was not relieved by the addition of cycloheximide.     

Role of lysosomotrophic reagents in glucocorticoid hormone action

Administration of (10 mg/200 g) methylamine or chloroquine to adrenalectomized rats for 2 days followed by a single injection of either cortisol (2.5 mg/200 g) or dexamethasone (0.5 mg/200 g) resulted in a significant enhancement of the tyrosine aminotransferase enzymatic activity in rat liver versus rats given a single injection only of either steroid. Lysosomotrophic reagents were unable to induce tyrosine aminotransferase when administered alone. Cytosols from rat liver treated with lysosomotrophic reagents in vivo had approx. 20-30% more specific binding to [3H]dexamethasone as compared to the control, untreated rats. This enhanced binding was due to an increase in the concentration of the receptor rather than a change in the affinity of the hormone for the receptor. Rat livers perfused with and homogenized in 10 mM Tris-HCI/0.25 M sucrose buffer (pH 7.5) containing about 5 mM lysosomotrophic reagents showed optimum stabilization of the steroid unbound glucocorticoid receptor in vitro at both 4 degrees C and 25 degrees C. These reagents had no effect on in vitro transformation of [3H]dexamethasone-receptor complex or on the binding of the thermally transformed receptor to the nuclei. It is concluded from these studies that lysosomotrophic reagents enhance tyrosine aminotransferase induction by glucocorticoids and stabilize unbound glucocorticoid receptor both in vivo and in vitro without any effect on in vitro transformation of the steroid-receptor complex.     

Progestin-induced enhancement of dexamethasone dissociation from glucocorticoid hormone receptors

Several groups have reported that progesterone accelerates the rate of steroid dissociation from the agonist site of the glucocorticoid receptor. It has been proposed that this enhancement reflects the binding of progestins to a second steroid-binding site. Since progestins are frequently antagonists of glucocorticoid hormone action, we decided to characterize this site more fully. In particular, in this study, we investigated whether the cytosolic preparations of four separate glucocorticoid target tissues from the same species all contained this second site and whether it was similar in each case. Cytosolic extracts of rat heart, liver, kidney, and pancreas were examined. In each case it was found that the rate at which prebound tritiated dexamethasone dissociated from the glucocorticoid receptor was faster in the presence of nonradioactive progesterone. The magnitude of this effect was essentially the same in each case. These results indicated that the second site was present in each preparation. To determine if the site was similar in each extract, we studied the steroid specificity of the enhancement of dissociation. This was determined by quantitating the degree to which each of a series of test steroids could cause augmentation of dissociation. Progesterone, R-5020, medroxyprogesterone, deoxycorticosterone, 17-OH-progesterone, and cortexolone were evaluated. The results for all four cytosolic preparations showed that either progesterone or R-5020 was the most potent steroid while both cortexolone and 17-OH-progesterone were essentially without effect. Medroxyprogesterone and deoxycorticosterone were usually of intermediate potency. These results suggest that the cytosolic extracts of all glucocorticoid target tissues have a similar second steroid-binding site which demonstrates a preference for progestins and that interaction with this site causes the glucocorticoid receptor to decrease the affinity with which it binds agonists.     

Spontaneous oscillations of intracellular calcium and growth hormone secretion

A novel combination of two single cell assays allowed the simultaneous measurement of intracellular calcium concentration and hormone secretion in normal pituitary cells. [Ca2+]i was recorded using the fluorescent Ca2+ indicator fura-2 and digital imaging microscopy. This technique was combined with a reverse hemolytic plaque assay for growth hormone in order to identify somatotropes and quantitate the amount of hormone released. A dynamic profile of rhythmic calcium oscillations was found in spontaneously secreting somatotropes. Each somatotrope displayed a distinct frequency (one pulse every 5-30 s) and amplitude (range 50-450 nM) generated asynchronously from cell to cell. The amount of growth hormone (GH) released correlated directly with both the frequency and amplitude of calcium oscillations at the level of single GH cells. Furthermore, calcium excursions in somatotropes were rapidly suppressed by either (i) removal of extracellular calcium, (ii) somatostatin (1 mM), or (iii) the calcium channel blockers cobalt (2 mM) and verapamil (100 microM). These observations demonstrate that spontaneous calcium oscillations are characteristic for normal somatotropes. These oscillations are related to spontaneous hormone secretion and due to influx through calcium channels in the membrane. Somatostatin, the physiologic inhibitor of GH secretion, suppresses calcium transients. These findings suggest that the intracellular signaling information may be encoded both in the frequency and amplitude of calcium oscillations.     

Steroid-affinity purification of the rat liver glucocorticoid hormone receptor complex

The molybdate-stabilized GHRC was isolated from rat liver cytosol with a 9000-fold purification and 46% yield. The major purification step was achieved using an affinity matrix consisting of an agarose support coupled to a dexamethasone ligand via an aliphatic spacer arm. Spacer arms containing disulfide bridges were found to be unsuitable due to their instability in cytosol. To reduce the non-specific binding properties of the affinity matrix, underivatized amino groups were acetylated, since the receptor was found to bind avidly to such groups thus evading elution by the ligand. Sodium molybdate present during biospecific elution from the gel stabilized the steroid-binding activity of the receptor. The use of denaturing and sulfhydryl modifying reagents (NaSCN, DMSO, Mersalyl) during elution led to partial or complete irreversible loss of steroid-binding activity of the unoccupied receptor. Efficient biospecific elution occurred at competing concentration of high affinity steroid in the presence of sodium molybdate. The ligand specific eluate was further purified by DEAE-Sephacel chromatography resulting in additional purification of 3.2-fold. The GHRC eluted from the DEAE-Sephacel column at a salt concentration characteristic of the untransformed GHRC. Molybdate was removed from the purified untransformed GHRC in the ligand eluate by DEAE-Sephacel chromatography in the absence of molybdate, for subsequent heat transformation.     

On the mechanistic origin of damped oscillations in biochemical reaction systems

A generalized reaction scheme for the kinetic interaction of two reactants in a metabolic pathway has been examined in order to establish what minimal mechanistic patterns are required to support a damped oscillatory transient-state kinetic behaviour of such a two-component system when operating near a steady state. All potentially oscillating sub-systems inherent in this scheme are listed and briefly characterized. The list includes several mechanistic patterns that may be frequently encountered in biological system (e.g. involving feedback inhibition, feed-forward activation, substrate inhibition or product activation), but also draw attention to some hitherto unforeseen mechanisms by which the kinetic interaction of two metabolites may trigger damped oscillations. The results can be used to identify possible sources of oscillations in metabolic pathways without detailed knowledge about the explicit rate equations that apply.     

Different incorporation of glucocorticoid hormone into diploid and tetraploid liver nuclei

Tetraploid parenchymal rat liver nuclei incorporate about twice as much (³H)dexamethasone as diploid parenchymal nuclei both in vivo and in vitro. This suggests that the ability of hepatic nuclei to incorporate glucocorticoid hormone is influenced by the number of copies of the genome in these nuclei.     

Adrenergic origin of very low-frequency blood pressure oscillations in the unanesthetized rat

Spectral analysis of cardiovascular signals has been extensively used to investigate circulatory homeostatic mechanisms. However, the nature of very low-frequency (VLF) fluctuations remains unclear. Because we previously observed enhanced VLF fluctuations in blood pressure (BP) in the sympathectomized rat (a model characterized by markedly increased plasma epinephrine levels), the aims of our study were to assess whether the genesis of VLF fluctuations in BP depends on circulating catecholamines and to determine which adrenergic receptor(s) and which membrane ion channel(s) are involved. We used continuous intra-arterial BP recordings from unanesthetized unrestrained rats to compute the power of VLF fluctuations in BP in the intact condition, during acute ganglionic blockade with hexamethonium, and after restoration of BP levels by infusion (in addition to hexamethonium) of adrenergic agonists (epinephrine, norepinephrine, and clonidine) or nonadrenergic vasoconstrictors (vasopressin). Effects of infusion of specific adrenergic receptor blockers (propranolol, prazosin, and yohimbine) with hexamethonium and catecholamines and infusion of various membrane ion channel blockers on VLF fluctuations in BP were also evaluated. Our results are as follows. 1) Ganglionic blockade drastically reduced BP levels and VLF fluctuations. 2) All vasoconstrictors restored BP levels, but only adrenergic vasoconstrictors generated striking VLF fluctuations in BP. 3) Catecholamine-induced fluctuations were abolished by alpha2-, but not alpha1- or beta-, adrenergic receptor blockade and by Ba2+-sensitive K+ channel or L-type Ca2+ channel, but not by other ion channel, blockers. We conclude that, in the conscious, unrestrained ganglion-blocked rat, catecholamine infusion generates VLF fluctuations in BP through stimulation of alpha2-receptors and activation of Ba2+-sensitive K+ channels. These fluctuations may have (patho)physiological relevance under conditions of disrupted circulatory homeostasis.     

Ginsenoside Rg1, a novel glucocorticoid receptor agonist of plant origin, maintains glucocorticoid efficacy with reduced side effects

Glucocorticoids (GCs) are widely used to treat inflammatory diseases. However, they cause debilitating side effects, which limit the use of these compounds. In the past decade, many researchers have attempted to find so-called dissociated GCs that have separate distinct transactivation and transrepression activities. Anti-inflammation of GCs is a result of glucocorticoid receptor (GR)-mediated transactivation and transrepression in some tissues, similar to their side effects; therefore, the goal to discover a compound that has anti-inflammatory properties, but lacks the negative side effects seen with GCs, has yet to be achieved. In the present study, we introduce a plant-derived compound, ginsenoside Rg1, which possesses GC and estrogen-like activities. In this study, we show that Rg1 downmodulates LPS-induced proinflammatory cytokine release and inhibits NF-κB nuclear translocation and DNA binding activity. The negative effects on NF-κB activation are due to a decrease in IκB phosphorylation and protein stabilization. Furthermore, the inhibitory effect of Rg1 on NF-κB is GR-dependent, as small interfering RNA knockdown of GR abrogated this function. Rg1 also displayed profound inhibitory effects on LPS-induced MAPK activation. Importantly, Rg1 did not impair proliferation or differentiation of mouse osteoblasts. Finally, we show that Rg1 can effectively inhibit acute and chronic inflammation in vivo, but it does not cause hyperglycemia or osteoporosis as seen with dexamethasone. These results suggest that ginsenoside Rg1 may serve as a novel anti-inflammatory agent and may exhibit a potential profile for therapeutic intervention in inflammatory diseases.     

Developmentally regulated primary glucocorticoid hormone induction of chick retinal glutamine synthetase mRNA

We have characterized the glucocorticoid hormone induction of glutamine synthetase mRNA in embryonic chick retinal organ cultures by quantitative dot hybridization using a cDNA clone derived from chick retinal RNA. Hydrocortisone (Kapp = 3-4 nM) and dexamethasone (Kapp = 1-2 nM) produce an approximate 30-fold increase in glutamine synthetase mRNA after incubation of organ cultures derived from embryonic day 12 retinae with either hormone for 3 hr. Progesterone is a poor inducer. The glucocorticoid-mediated rise is rapid (t1/2 = 2-3 hr) and occurs in the presence of either of the protein synthesis inhibitors cycloheximide or puromycin, indicating that the induction is a primary or direct response to the hormone. However, the magnitude of the hormonal response observed in culture increases markedly during retinal development. These observations, coupled with the previously reported absence of a hormonal induction in embryonic liver, raise the possibility of a synergistic mechanism, involving tissue-specific regulatory molecules in addition to the glucocorticoid hormone receptor, to explain the retinal-specific primary glucocorticoid hormone induction of glutamine synthetase mRNA.     

Mercury inhibits rat liver and kidney glucocorticoid receptor hormone binding activity

The present study was focused on the influence of mercury on the rat liver and kidney glucocorticoid receptor (GR) binding properties. The time-course and dose-dependence of mercury effects, as well as possible involvement of thiol groups were examined after in vivo and in vitro administration of the metal in the form of HgCl2. Mercury led to reduction of the liver and kidney GR hormone binding capacity. In both examined tissues maximal reduction was noticed 4 h after administration of the metal at 2 and 3 mg Hg/kg bw, but the effect was more prominent in kidney as compared to liver. On the other hand, binding affinity in the two tissues was similar. The complete reversal of mercury effects on GR binding capacity by 10 mmol/L DTT was achieved in liver and partially in kidney. The reversal by DTT suggested that mercury caused the decrease of GR binding activity by interacting with thiol groups. The difference in the response of the two tissues reflected the fact that kidney contained a higher mercury concentration and a lower thiol content in comparison to liver. The implicated thiols probably belong to GR, since when applied in vitro at 0 degrees C, mercury produced reduction of the receptor binding activity similar to that observed in vivo. GR protein level examined by quantitative Western blot was either unchanged, when determined by polyclonal antibody, or reduced, when determined by BuGR2 antibody, suggesting that Hg might affect BuGR epitope availability.     

Induction of tryptophan oxygenase in primary rat liver cell suspensions by glucocorticoid hormone

The effect of dexamethasone phosphate on tryptophan oxygenase activity has been studied in suspensions of enzymatically prepared rat liver cells. The hormone was found to induce a 2–3 fold increase in enzyme activity after incubating the cells for 7 h in Krebs-Ringer phosphate solution at 37 °C. Purified parenchymal cells and a mixture of parenchymal cells and reticuloendothelial cells gave comparable results, indicating that the hormonal effect was on the parenchymal cells.