Glucocorticoids inhibit estradiol-mediated uterine growth: possible role of the uterine estradiol receptor

Stress-related activation of the hypothalamic-pituitary-adrenal axis (HPA) is associated with suppression of the reproductive axis. This effect has been explained by findings indicating that corticotropin-releasing hormone suppresses hypothalamic gonadotropin-releasing hormone (GnRH) secretion via an opioid peptide-mediated mechanism, and that glucocorticoids suppress both GnRH and gonadotropin secretion and inhibit testosterone and estradiol production by the testis and ovary, respectively. To evaluate whether glucocorticoids suppress the effects of estradiol on its target tissues, we examined the ability of dexamethasone to inhibit estradiol-stimulated uterine and thymic growth in ovariectomized rats. Estradiol alone, given daily for 5 days, caused dose-dependent uterine and thymic growth. Dexamethasone alone, given daily for 5 days, caused a dose-dependent decrease in body weight gain and in thymic growth. When estradiol and dexamethasone were administered simultaneously, however, body weight gain and thymic growth were also inhibited (p less than 0.05). Dexamethasone decreased estradiol-induced uterine cytosolic and nuclear estrogen receptor concentrations (E2 R0, p less than 0.05; E2nR0, respectively), but had no effect on estradiol-induced progesterone receptor concentrations (P4R0, p greater than 0.05). Levels of uterine glucocorticoid receptors were not affected by estrogen and/or dexamethasone treatment. These findings suggest that stress levels of glucocorticoids, administered over a 5-day interval, block the estradiol-stimulated growth of female sex hormone target tissues. This effect may be partially mediated by a glucocorticoid-induced decrease of the estradiol receptor concentration. Thus, another mechanism by which the HPA may influence reproductive function during stress is by a direct effect of glucocorticoids on the target tissues of sex steroids.     

Altered functional and immunophenotypical properties of neutrophilic granulocytes in postpartum cows associated with fatty liver

The intention of the study was to analyze the relationship between liver triacyl glycerol content (liver TAG content) and immunophenotypical and functional properties of polymorphonuclear neutrophilic granulocytes (PMN) of dairy cows in the peripartum period. We investigated characteristics of bovine PMN from the blood and uterus of clinically healthy cows in the periparturient period. The numbers of circulating leukocytes and segmented granulocytes continuously increased until parturition and declined afterwards to starting values. This was independent of the liver TAG content and mainly affected neutrophils. The liver TAG content exceeded 40 mg/g liver, the reference value, in 12 of 19 cows in the first two weeks postpartum. Increased liver TAG content, > 40 mg/g, went in parallel with a reduced expression of function-associated surface molecules on blood neutrophils (e.g. CD11b/CD18 = CR3 and CD11c/CD18 = CR4). Moreover, in cows with high liver TAG levels the antibody-independent and -dependent cellular cytotoxicity (AICC, ADCC) of blood PMN was markedly reduced. PMN also were less capable of ROS generation after stimulation with Phorbol Myristate Acetate (PMA). In comparison with contemporarily harvested blood PMN, neutrophils recovered from the uterine lumen showed a decreased expression of 4/6 examined surface structures. Only the expression densities of CR3 molecules and those detected by mAb IL-A110 were enhanced on uterine PMN. The cytotoxic capacity and the ROS generation were significantly lower for uterine PMN than for blood PMN. The results suggest that increased liver TAG content in the first and second week after calving is associated with decreased functional capacities of PMN derived from blood and uterus. This may help to explain why cows who are too fat at calving (who therefore have an increased liver TAG content) have a higher incidence of infectious diseases such as endometritis     

Effect of 1,25-dihydroxyvitamin D-3 on phosphate uptake into chick intestinal brush border membrane vesicles

The production of collagenase by human skin explants in culture is prevented by 10(-8) M dexamethasone, 5 . 10(-4) M dibutyryl cyclic AMP, or 2.5 . 10(-3) M theophylline. Decreases in collagenase activity are paralleled by reductions in the degradation of explant collagen during the culture period. Progesterone, which effectively inhibits collagenase production in rat uterine explant cultures, has no effect on human skin explants. The inhibition by cyclic AMP is nucleotide specific. When partially inhibitory concentrations of dexamethasone and dibutyryl cyclic AMP, or dexamethasone and theophylline, are added to culture medium together, the resultant inhibition is that predicted by additivity. Synergistic inhibition, as observed in rat uterus between progesterone and dibutyryl cyclic AMP, fails to occur. Dexamethasone inhibits the production of collagenase by cultured explants of rat uterus, with complete inhibition occurring at 10(-7) M steroid. Synergism between glucocorticoids and dibutyryl cyclic AMP or between dexamethasone and progesterone could not be demonstrated in the uterine culture system. These results suggest the existence of three regulatory systems for the control of collagenase production in mammalian tissues, and that cooperativity between systems may occur on a tissue-specific basis.     

Glucocorticoid suppression of the sympathetic nervous system and adrenal medulla

Studies were performed to evaluate the effects of glucocorticoids on the activity of the sympathetic nervous system and adrenal medulla. Plasma concentrations of norepinephrine and epinephrine were measured in rats in which endogenous glucocorticoids were removed by bilateral adrenalectomy and in rats to which exogenous glucocorticoids were administered. In intact rats, dexamethasone (2.5, 25 or 250 micrograms) pretreatment suppressed ether vapor-induced elevations of norepinephrine and epinephrine concentrations in plasma. Corticosterone (3 mg/kg), similar to dexamethasone, attenuated the elevation of plasma concentrations of norepinephrine and epinephrine in rats exposed to ether vapor. Glucocorticoids did not alter the elevation of plasma catecholamines stimulated by intracerebroventricular injections of corticotropin-releasing factor or calcitonin gene-related peptide, thus demonstrating functional integrity of the sympathetic nervous system and adrenal medulla. Adrenalectomy resulted in elevation of basal plasma norepinephrine levels and accentuation of ether vapor-induced elevations of plasma norepinephrine concentrations in rats. Dexamethasone (25 ug) administration blunted the effects of adrenalectomy on both basal and ether vapor-stimulated levels of plasma norepinephrine. It is concluded that glucocorticoids acting at as yet undefined sites may be involved in the regulation of sympathetic nervous system and adrenal medullary function.     

Development and comparison of in vivo and in vitro models for endometritis in cows and mares

In order to investigate pathogenic mechanisms of acute endometritis in cows and mares, we established an in vivo model in both species. Based on the results of an in vitro transmigration system, human recombinant interleukin-8 (rhIL-8; 1.25 microg per mare and 5 microg per cow in 50 ml phosphate-buffered saline) was used to attract polymorphonuclear neutrophil granulocytes (PMNs) into the uteri. Peak numbers of uterine neutrophils were attracted after 6h, in both cows and mares. On average, mares responded more sensitively than cows, with 15 times higher numbers of rhIL-8-attracted uterine neutrophils (72+/-8 x 10(7)cells). In contrast to in vitro studies, in vivo migrated neutrophils (uterine neutrophils) of both species displayed a significantly reduced MHC class I expression. Expression of the CD11a molecule was significantly enhanced on equine uterine neutrophils but downregulated on bovine cells. Compared with untreated autologous peripheral neutrophils, both uterine and in vitro migrated neutrophils showed no alteration of phagocytic capacity. The ability to generate reactive oxygen species (ROS) was significantly upregulated in bovine and equine uterine neutrophils. This was also observed after in vitro migration of equine neutrophils, whereas ROS generation by bovine neutrophils was significantly depressed. In summary, the concept of inducing endometritis directly by local application of human interleukin-8 has been reliably successful in cows and mares. The model permits the analysis of PMN migration into the uterus under defined and controlled conditions. The observed differences between cows and mares with respect to phenotypical and functional characteristics of in vivo attracted uterine cells point to species-related features of neutrophil migration. In vitro transmigrated bovine and equine cells partially differ in phenotype and function from uterine neutrophils. Therefore, the in vitro transmigration assay cannot completely represent the in vivo endometritis model described here.     

Hormonal interactions in mammalian collagenase regulation: Comparative studies in human skin and rat uterus

The production of collagenase by human skin explants in culture is prevented by 10^?8 M dexamethasone, 5·10^?4 M dibutyryl cyclic AMP, or 2.5· 10^?3 M theophylline. Decreases in collagenase activity are paralleled by reductions in the degradation of explant collagen during the culture period. Progesterone, which effectively inhibits collagenase production in rat uterine explant cultures, has no effect on human skin explants. The inhibition by cyclic AMP is nucleotide specific. When partially inhibitory concentrations of dexamethasone and dibutyryl cyclic AMP, or dexamethasone and theophylline, are added to culture medium together, the resultant inhibition is that predicted by additivity. Synergistic inhibition, as observed in rat uterus between progesterone and dibutyryl cyclic AMP, fails to occur.Dexamethasone inhibits the production of collagenase by cultured explants of rat uterus, with complete inhibition occurring at 10^?7 M steroid. Synergism between glucocorticoids and dibutyryl cyclic AMP or between dexamethasone and progesterone could not be demonstrated in the uterine culture system. These results suggest the existence of three regulatory systems for the control of collagenase production in mammalian tissues, and that cooperativity between systems may occur on a tissue-specific basis.     

Down-regulation of plasmin receptors on human sarcoma cells by glucocorticoids

After incubation of confluent monolayer cultures of human HT-1080 fibrosarcoma cells with purified native human plasminogen in plasminogen-depleted serum-containing medium, bound plasmin activity could be specifically eluted from the cells with tranexamic acid, an analogue of lysine. Dexamethasone reduced the amount of recoverable bound plasmin activity in a dose-dependent manner. Dexamethasone was also found to induce a time- and dose-dependent decrease in the ability of the cells to bind added plasmin. Untreated HT-1080 cells bound added plasmin with a high capacity (600,000 molecules bound per cell), and this decreased to an undetectable level after treatment with 100 nM dexamethasone. The kinetics of the loss of plasmin binding by the dexamethasone-treated sarcoma cells, a clear decrease after 4 h, correlated with those for the loss of cell-bound urokinase (u-PA) activity. Plasmin was not, however, bound to the active site of u-PA: an anti-catalytic monoclonal antibody to u-PA had no effect on plasmin binding. Other glucocorticoids, such as hydrocortisone and corticosterone, had a similar effect to dexamethasone on plasmin binding to HT-1080 cells. The effect of glucocorticoids on the plasmin receptor seemed to occur at least partly via a decrease in the affinity for plasmin, since the Kd for plasmin with untreated cells was 5.4 x 10(-9) M, and with cells treated with 5 nM dexamethasone, the Kd value for plasmin was 1.2 x 10(-7) M. These results show that glucocorticoids induce down-regulation of plasmin receptors on the surface of HT-1080 cells: a novel mechanism, in addition to the known effects of glucocorticoids on u-PA and PA inhibitors, by which human tumor cells may be disarmed of their pericellular proteolytic activity.     

The role of MAP kinase phosphatase-1 in the protective mechanism of dexamethasone against endotoxemia

AIMS: We have previously shown that glucocorticoids induce the expression of MAP kinase phosphatase (Mkp)(a)-1 in innate immune cells. Since Mkp-1 is a critical negative regulator of the innate immune response, we hypothesize that Mkp-1 plays a significant role in the anti-inflammatory action of glucocorticoids. The specific aim of the present study is to understand the role of Mkp-1 in the anti-inflammatory function of glucocorticoids. MAIN METHODS: Wild-type and Mkp-1(-/-) mice were treated with different doses of dexamethasone and then challenged with different doses of lipopolysaccharide (LPS). The survival and blood cytokines were assessed. The effects of dexamethasone on cytokine production in wild-type and Mkp-1(-/-) primary macrophages ex vivo were also examined. KEY FINDINGS: We found that dexamethasone induced the expression of Mkp-1 in vivo. Dexamethasone treatment completely protected wild-type mice from the mortality caused by a relatively high dose of LPS. However, dexamethasone treatment offered only a partial protection to Mkp-1(-/-) mice. Dexamethasone attenuated TNF-alpha production in both wild-type and Mkp-1(-/-) mice challenged with LPS, although TNF-alpha production in Mkp-1(-/-) mice was significantly more robust than that in wild-type mice. Dexamethasone pretreatment shortened the duration of p38 and JNK activation in LPS-stimulated wild-type macrophages, but had little effect on p38 or JNK activation in similarly treated Mkp-1(-/-) macrophages. SIGNIFICANCE: Our results indicate that the inhibition of p38 and JNK activities by glucocorticoids is mediated by enhanced Mkp-1 expression. These results demonstrate that dexamethasone exerts its anti-inflammatory effects through both Mkp-1-dependent and Mkp-1-indepent mechanisms.     

Inhibition of vesicular stomatitis virus replication in dexamethasone-treated L929 cells

We previously demonstrated that dexamethasone treatment of L929 cells inhibited plaque formation by vesicular stomatitis virus (VSV), encephalomyocarditis virus, or vaccinia virus. We now have characterized the antiviral effects of glucocorticoids in L929 cells. Dexamethasone did not directly inactivate VSV nor did steroid treatment of L929 cells affect virion adsorption or penetration. The VSV yield in L929 cells treated with dexamethasone for a period of only 4 or 8 hr was decreased by 50% when cells were infected the day following steroid treatment. Treating L929 cells with dexamethasone for a longer period resulted in greater inhibitions of virus synthesis. Interferon activity (less than 5 units/ml) was not detected in L929 cell culture fluids and cell sonicates from steroid-treated cells and the addition of antiserum to murine alpha/beta-interferon had no effect on the ability of dexamethasone to inhibit VSV replication. Dexamethasone treatment of L929 cells did not induce the production of double-stranded RNA-dependent protein kinase but did result in a slight elevation of 2-5A oligoadenylate synthetase activity, two enzymatic activities associated with the antiviral state induced by interferon. However, the elevated 2-5A synthetase activity was not associated with an inhibition of VSV RNA accumulation in dexamethasone-treated L929 cells. By contrast, the synthesis of all five VSV proteins was reduced by 50-75% in dexamethasone-treated L929 cells as early as 4 hr after infection. Thus, the dexamethasone-mediated inhibition of VSV replication in L929 cells is associated with decreased production of VSV structural proteins.     

Inhibition of basophil histamine release by anti-inflammatory steroids. II. Studies on the mechanism of action

The glucocorticosteroids inhibit the IgE-dependent release of histamine by human basophils with an order of potency that very closely parallels that found in vivo (i.e., triamcinolone acetonide greater than dexamethasone greater than beta-methasone greater than prednisolone greater than hydrocortisone much greater than progesterone approximately tetrahydrocortisone approximately 0). The effect is seen after a 24-hr preincubation with nanomolar to micromolar concentrations of glucocorticoid. In contrast, release of histamine stimulated by the formyl methionine containing peptide f-met-leu-phe, the calcium ionophore A23187, and the tumor-promoting phorbol diester 12-O-tetradecanoylphorbol-13-acetate was not inhibited by 24-hr incubation with the potent glucocorticoid dexamethasone. Dexamethasone inhibited anti-IgE-induced histamine release without altering its rate, suggesting that the glucocorticoids do not inhibit histamine release by elevating the intracellular level of cAMP. Dexamethasone did not consistently alter either the total or occupied basophil IgE Fc receptor number, and therefore the glucocorticoid effect does not appear to be due to the modulation of cell surface Fc epsilon receptor content. These data indicate that steroid hormones inhibit basophil IgE-dependent activation through a specific glucocorticoid receptor. The mechanism by which they do so appears not to involve an elevation of cAMP or a shedding of cell surface Fc epsilon receptors. Further, because the glucocorticoids did not inhibit release initiated by the PLA2-dependent stimuli f-met-leu-phe, A23187 and TPA, the inactivation of IgE-dependent histamine release by glucocorticoids may not be the result of PLA2 inhibition.     

Paradoxical stimulation of both lipocortin and prostaglandin production in human amnion cells by dexamethasone

Glucocorticoids inhibit prostaglandin biosynthesis by inducing the formation of lipocortins. In human amnion cells dexamethasone elicited a concentration-dependent increase in prostaglandin production and raised intracellular lipocortin 1 concentrations. Dexamethasone could also potentiate the epidermal growth factor (EGF)-induced stimulation of prostaglandin production. EGF alone or in combination with dexamethasone increased lipocortin 1 formation in amnion cells. Human amnion cells may provide a unique insight into interactions between glucocorticoids, lipocortin and eicosanoid biosynthesis.     

Regulation of vasopressin messenger RNA levels in the small cell lung carcinoma cell line GLC-8: interactions between glucocorticoids and second messengers.

The role of glucocorticoids and second messenger systems in the regulation of the vasopressin (VP) gene was studied in the human small cell lung carcinoma cell line GLC-8. Small cell lung carcinoma GLC-8 cells express VP mRNA and contain both glucocorticoid and mineralocorticoid receptors. Treatment with the synthetic glucocorticoid dexamethasone when added alone at 10(-8) M had no effect on the VP mRNA level and decreased the level by 30% at 10(-6) M. However, the effect of dexamethasone changed to positive when cells were simultaneously treated with cAMP-enhancing agents. VP mRNA levels, which were elevated by 1.5- to 2-fold by the cAMP-enhancing agents alone, increased a further 1.5- to 3-fold by dexamethasone. Thus, the combined effect of dexamethasone and cAMP stimulation was a 3- to 7.5-fold increase in VP mRNA levels. Long term treatment with the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) reduced the VP mRNA level by 75%. The TPA-suppressed VP mRNA levels could be up-regulated about 6-fold by simultaneous treatment with 8-bromo-cAMP. Dexamethasone did not alter the TPA-suppressed VP mRNA levels. These results indicate that both cAMP and protein kinase-C pathways as well as glucocorticoid receptors are involved in the regulation of VP mRNA levels and that these factors interact. This leads to a negative or positive response of VP gene expression to glucocorticoids in a state-dependent manner. The interactions may be of significance in a physiological context and relate to the different regulation of VP-expressing systems in the brain.     

Glucocorticoid-mediated Inhibition of Lck Modulates the Pattern of T Cell Receptor-induced Calcium Signals by Down-regulating Inositol 1,4,5-Trisphosphate Receptors

Glucocorticoids are potent immunosuppressive agents that block upstream signaling events required for T cell receptor (TCR) activation. However, the mechanism by which glucocorticoids inhibit downstream responses, such as inositol 1,4,5-trisphosphate (IP₃)-induced calcium signals, is not completely understood. Here we demonstrate that low concentrations of dexamethasone rapidly convert transient calcium elevations to oscillations after strong TCR stimulation. Dexamethasone converted the pattern of calcium signaling by inhibiting the Src family kinase Lck, which was shown to interact with and positively regulate Type I IP₃ receptor. In addition, low concentrations of dexamethasone were sufficient to inhibit calcium oscillations and interleukin-2 mRNA after weak TCR stimulation. Together, these findings indicate that by inhibiting Lck and subsequently down-regulating IP₃ receptors, glucocorticoids suppress immune responses by weakening the strength of the TCR signal.     

Excess of glucocorticoids impairs whole-body antioxidant status in young rats. relation to the effect of dexamethasone in soleus muscle and spleen.

The action of glucocorticoids in high doses is catabolic, but not much is known about the accompanying effects on antioxidative capacity of the entire body. Animals were treated (or not) with dexamethasone (Dex) 2 mg/kg b.w. d-1 during 5 consecutive days followed by recovery, during which an additional group received 3-hydroxy-3-methylbutyrate (40 mg/kg b.w.). Animals were killed after treatment with Dex, and after 5 days of the recovery period. Dexamethasone treatment decreased appetite almost twofold (from 20 g/day to 10 g/day, P < 0.001). Feed restriction, however, seemed to have only minor impact on the effects observed since body weight loss of pair-fed rats after the 5th day of treatment was only 2% and Dex-treated rats decrease in body weight was 22% (P < 0.05). In turn, wet weight of the soleus muscle (expressed per body weight) did not significantly decrease after Dex treatment, suggesting relative resistance of oxidative type muscles to the catabolic action of dexamethasone. Spleen wet weight expressed per body weight dropped by 65% (P<0.001). Additionally, there was a 46% reduction (P<0.001) of blood glutathione (GSH/Hb), and 36% (P < 0.001) of muscle glutathione (GSH/tissue wet weight). This suggests that dexamethasone directly and/or indirectly impaired antioxidant reactions. This was further confirmed by a significant (49%) decline of SOD-1 activity in erythrocytes isolated from the group treated with dexamethasone. Another index of lipid peroxidation (TBARS) was also significantly increased. Activity of blood plasma CK increased by 73% (P<0.001) in Dex-treated rats, indicating moderate injury of muscle tissue. In conclusion, young growing rats were sensitive to the dosage of dexamethasone, but in contrast to lymphoid tissue, could easily compensate the outcomes of impaired antioxidative defence within 5 days of recovery.     

Recombinant human interleukin-1 inhibits the induction by dexamethasone of alkaline phosphatase activity in murine capillary endothelial cells

A mutual antagonism exists between interleukin-1s (IL-1s) as pro-inflammatory and glucocorticoids as anti-inflammatory mediators. This report examines the effects of IL-1 on the induction by dexamethasone of alkaline phosphatase in LEII murine endothelial cells. Dexamethasone increases the specific activity of alkaline phosphatase in a time- and dose-dependent fashion (maximum 14-fold induction at 10(-6) M, IC50 = 10(-8) M), and this induction can be completely inhibited by simultaneous incubation with picomolar concentrations of recombinant human IL-1 alpha or IL-1 beta. This IL-1-mediated antagonism of dexamethasone activity is not due to a down-regulation of glucocorticoid receptors in the cell line used, because the number of receptors and their affinity for dexamethasone is unchanged in IL-1-treated cells. However, induction of alkaline phosphatase by dexamethasone in LEII cells is receptor-mediated, since it can also be inhibited by glucocorticoid-receptor antagonists.     

Role of interleukin-15 and interleukin-18 in the secretion of sIL-6R and sgp130 by human neutrophils

BACKGROUND: Available data indicate that neutrophils (PMN) produce a wide range of cytokines with the potential to modulate immune response. Recent investigation have shown that interleukin (IL)-15 and IL-18 potentiated several functions of normal neutrophils. It has been reported that IL-18-induced cytokine production may be significantly enhanced by coincident addition of IL-15. AIMS: In the present study we compared the effect of recombinant human (rh)IL-15 and rhIL-18 as well as effect of a rhIL-15 and rhIL-18 combination on the induction secretion of sIL-6Ralpha and sgp130 by human neutrophils. METHODS: PMN were isolated from heparinized whole blood of healthy persons. The PMN were cultured for 18 h at 37 degrees C in a humidified incubator with 5% CO(2). rhIL-15 and/or rhIL-18 and lipopolysaccharide were tested to PMN stimulation. The culture supernatants of PMN were removed and examined for the presence of sIL-6R and sgp130 by human enzyme-linked immunosorbent assay kits. Cytoplasmic protein fractions of PMN were analysed for the presence of sIL-6R and sgp130 by western blotting using monoclonal antibodies capable of detecting these proteins. Cells were lysed and cytoplasmic proteins were electrophoresed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The resolved proteins were transferred onto nitrocellulose and incubated with the primary monoclonal antibodies anti-sIL-6R and anti-sgp130. The membranes were incubated at room temperature with alkaline phosphatase anti-mouse immunoglobulin G. Immunoreactive protein bans were visualized by an AP Conjugate Substrate Kit. RESULTS AND CONCLUSIONS: The results of our investigation revealed that IL-15 alone, similarly to IL-18, has no significant ability for the regulation of both soluble IL-6 receptors, sIL-6R and sgp130, released by human neutrophils. It is interesting to note that the secretion of sgp130 was changed after PMN stimulation with rhIL-15 in the presence of rhIL-18. The combination of rhIL-15 and rhIL-18 was shown to induce PMN to secretion relatively higher amounts of sgp130 compared with the stimulation of PMN with rhIL-15 alone and rhIL-18 alone. The results obtained suggest that IL-15 and IL-18, belonging to the inflammatory cytokines, through the regulation of sgp130 secretion must be also considered as anti-inflammatory mediators that may influence the balance reactions mediated by the IL-6 cytokine family.     

Effect of glucocorticoids on the glucose transport system of isolated fat cells.

Glucocorticoids inhibit glucose utilization by fat cells. The possibility that this effect results from altered glucose transport was investigated using an oil-centrifugation technique which allows a rapid (within 45 s) estimation of glucose or 3-O-methylglucose uptake by isolated fat cells. At high concentration (greater than 25 muM), dexamethasone inhibited glucose uptake within 1 min of its addition to fat cells. Efflux of 3-O-methylglucose was also impaired by 0.1 mM dexamethasone. However, diminished glucose uptake was not a specific effect of glucocorticoids; high concentrations (0.1 mM) of 17beta-estradiol, progesterone, and deoxycorticosterone produced a similar response in adipocytes. At a more physiologic steroid concentration (0.1 muM), glucocorticoids inhibited glucose uptake in a time-dependent manner (maximum effect in 1 to 2 hours). This effect was specific for glucocorticoids since, under these conditions, glucose uptake was not changed by the non-glucocorticoid steroids. Lineweaver-Burk analysis showed that 0.1 muM dexamethasone treatment produced a decrease in Vmax for glucose uptake but did not change the Ku. Hexokinase activity and ATP levels were not altered by this treatment, suggesting that processes involved in glucose phosphorylation were not affected. Dexamethasone treatment also caused a reduction in uptake of 3-O-methylglucose when assayed using a low sugar concentration (0.1 mM). At a high concentration (10 mM), uptake of the methyl sugar was only slightly less than normal in treated cells. Stimulation by insulin markedly enhanced uptake of glucose and 3-O-methylglucose by both treated and untreated cells. At a low hexose concentration (0.1 mM) and in the presence of insulin, sugar uptake by dexamethasone-treated cells was slightly less than control cells. Stimulation by insulin did however completely overcome the alteration in hexose uptake when larger concentrations of sugars (greater than 5 mM) were used. There was no detectable change in total protein synthesis during incubation of fat cells with dexamethasone. However, actinomycin C blocked the inhibitory effect of dexamethasone on glucose uptake. Cycloheximide, which caused a small inhibition in glucose uptake, prevented the full expression of the inhibitory effect of dexamethasone on glucose transport. These results indicate that dexamethasone alters the facilitated transport of glucose and, secondly, suggest that synthesis of RNA and protein is needed for glucocorticoid action.     

Inhibition by glucocorticoids of phosphate transport in primary cultured renal cells

The regulation by glucocorticoids of phosphate transport in primary cultured chick renal cells was examined. Dexamethasone inhibited the Na+-dependent phosphate uptake system. Na+-independent phosphate uptake and Na+-dependent uptakes of alpha-methylglucoside and L-proline were unaffected. The mineralocorticoid aldosterone did not alter phosphate uptake. The inhibition of Na+-dependent phosphate uptake by dexamethasone was concentration-dependent, exhibited an induction period, was blocked by inhibitors of RNA and protein synthesis, and was rapidly reversed when the steroid was removed. Following reversal, the cells could respond a second time to the glucocorticoid. However, this time the response was rapid, could be evoked at least for 24 h after glucocorticoid withdrawal, and might be prevented by actinomycin D and cycloheximide. These findings demonstrate that glucocorticoids act on renal cells to modulate phosphate transport and suggest that the renal cell system provides an attractive model to examine the mechanism by which glucocorticoids control gene expression and regulate plasma membrane transport function.