Regulation of neutral amino acid transport in hepatocytes isolated from adrenalectomized rats

The present report shows that System A-mediated 2-aminoisobutyric acid (AIB) uptake is elevated in hepatocytes isolated from adrenalectomized rats when they are compared to control cells. Although System ASC activity also shows this perturbation, Systems N, beta, L1, and L2 are unaffected. Transport of AIB in both cell types is stimulated by dexamethasone, insulin, and glucagon, yet the hepatocytes from the adrenalectomized rats are much less responsive to these hormones. This apparent decrease in competence is seen for adaptive regulation of System A as well. The in vitro addition of dexamethasone to the hepatocytes from the adrenalectomized animals does not restore fully their ability to respond to hormones or amino acid deprivation. These effects are observed even after the cells have been held in primary culture for 24 hr. The simultaneous addition of glucagon and dexamethasone to either cell type resulted in stimulation of transport to rates significantly greater than the sum of the increases produced by the two hormones when added separately. In contrast, insulin and dexamethasone were additive in their effects rather than synergistic. These results suggest that hepatocytes from adrenalectomized rats are less competent than control cells with respect to regulation of neutral amino acid transport, including stimulation by insulin or amino acid starvation, two processes which appear not to depend on glucocorticoid for maximal response.     

Demonstration that corticotropin-releasing factor binding to rat peripheral tissues is modulated by glucocorticoid treatment in vivo and in vitro

In a recent study we reported the presence of specific binding sites for corticotropin-releasing factor (CRF) in peripheral tissues of the rat (Endocrinology, 116, 2151, 1985). The objective of this study was to determine if CRF binding to peripheral tissues was modified following adrenalectomy and glucocorticoid replacement therapy. Adult male rats were adrenalectomized and CRF binding to liver, spleen and testicular membranes was determined at 5, 7 or 14 days following adrenalectomy. An additional group of adrenalectomized rats received subcutaneous injections of dexamethasone (75 micrograms/day) for 14 days. Adrenalectomy of rats for 14 days increased CRF binding to liver, kidney, testis, spleen and ventral prostate by approximately 65%-125% above sham-control values. CRF binding to membrane preparations obtained from the pancreas of sham-operated rats was undetectable; however, adrenalectomy produced detectable CRF binding in this tissue. Adrenalectomy produced a time-related increase in CRF binding to ventral prostate, spleen and liver tissue. Administration of dexamethasone to adrenalectomized animals prevented increased CRF binding to peripheral tissues observed following adrenalectomy alone. In vitro dexamethasone treatment of prostatic or hepatic homogenates from adrenalectomized rats resulted in a dose-related decrease in CRF binding activity. However, similar in vitro treatment of prostatic or hepatic homogenate with progesterone exhibited no significant effects on CRF binding. Our results suggest that glucocorticoids may be a regulator of peripheral CRF receptors.     

Glucocorticoid regulation of hepatic cytosolic glucocorticoid receptors in vivo and its relationship to induction of tyrosine aminotransferase

Regulation of rat hepatic cytosolic glucocorticoid receptors was studied using our newly developed exchange assay. Injecting 1 mg of dexamethasone or corticosterone into 150-250 g adrenalectomized rats caused a rapid decline in glucocorticoid receptor binding. Glucocorticoid receptor levels were depressed 80-90% in less than 15 min after hormone treatment, and remained low for about 24-48 h after glucocorticoid administration. 80-90% of glucocorticoid receptor binding was regenerated by 48 h, and complete binding was recovered by 72 h. Regenerated glucocorticoid receptor binding (48-72 h after first hormone injection) could be re-depressed by a second injection of the hormone. Similar results were obtained using normal (intact) rats. Optimum induction of tyrosine aminotransferase activity was obtained within 2 h following the first hormonal injection. Induction of tyrosine aminotransferase activity (measured 2 h after a second injection of the glucocorticoid) correlated with glucocorticoid receptor levels. Thus, 1 mg of dexamethasone or corticosterone greatly enhanced the liver tyrosine aminotransferase activity in the adrenalectomized rats (not previously hormone treated) and in adrenalectomized rats previously injected (48-72 h) with 1 mg of the glucocorticoid hormone. Enhancement of tyrosine aminotransferase activity was lowest 16-24 h after the first hormone injection (when receptor levels were extremely low). These results indicate that the induction of liver tyrosine aminotransferase activity by glucocorticoid hormones is correlated with cytosolic glucocorticoid receptor levels.     

Tissue-specific regulation of angiotensinogen mRNA accumulation by dexamethasone

The regulation of angiotensinogen gene expression in response to adrenalectomy and dexamethasone treatment was examined in multiple rat tissues. Angiotensinogen mRNA as quantitated by slot blot hybridization utilizing an angiotensinogen cRNA probe was most abundant in the liver with levels in the brain, kidney, and adrenal of 50, 25, and 10%, respectively. No angiotensinogen mRNA was detected in testes or heart. Although no change in the quantity of angiotensinogen mRNA was found following adrenalectomy and maintenance on 0.9% saline, dexamethasone treatment of both normal and adrenalectomized rats resulted in a time-dependent and tissue-specific accumulation of angiotensinogen mRNA. In normal animals, the hepatic response to treatment was a 4.5-fold increase in angiotensinogen mRNA by 8 h which remained 2.4-fold above basal levels by 24 h. Angiotensinogen mRNA levels in the brains of normal rats treated with dexamethasone increased only 60% by 6 h and returned to basal levels by 24 h. In contrast to the increases seen in brain and liver, angiotensinogen mRNA derived from kidney did not significantly change following dexamethasone treatment. In adrenalectomized animals, the hepatic response to dexamethasone was similar to normal animals with a 3.7-fold increase by 6 h. The accumulation in brain was greater in these animals compared to normals and increased 3-fold by 8 h. Finally, dexamethasone did not significantly increase levels in the kidney. These results clearly demonstrate glucocorticoid regulation of angiotensinogen mRNA levels in liver and brain. In contrast, the kidney, an organ known to contain glucocorticoid receptors, does not respond with increased angiotensinogen mRNA levels following glucocorticoid stimulation. These studies provide the first evidence for tissue-specific differences in the control of angiotensinogen mRNA.     

Acute stimulation by glucocorticoids of gluconeogenesis from lactate/pyruvate in isolated hepatocytes from normal and adrenalectomized rats

Dexamethasone stimulated gluconeogenesis from lactate/pyruvate in suspensions of hepatocytes isolated from both adrenalectomized and normal fasted rats. This stimulation was observed in incubations with 1 mM pyruvate and at a lactate/pyruvate ratio of 25 but not at a ratio of 10-13. At a lactate/pyruvate ratio of 10-13, the stimulation by dexamethasone was progressively enhanced as the pyruvate concentration was decreased to 0.25 mM. Concurrent administration of a maximally stimulating concentration of dexamethasone with angiotensin II or glucagon yielded an additive stimulation at all concentrations of the peptide hormones tested. No potentiating or permissive actions of acute glucocorticoid administration were observed using hepatocytes from either normal or adrenalectomized animals. The acute stimulation by dexamethasone was antagonized by prior addition of progesterone or cortexolone to the hepatocyte suspensions. Triamcinolone and corticosterone also stimulated gluconeogenesis. Concentrations of the active glucocorticoids needed to elicit half-maximal stimulations (Kact) were approximately 100 nM for dexamethasone and triamcinolone and 400 nM for corticosterone. Deoxycorticosterone, 17 alpha-methyltestosterone, and 5 beta-dihydrocortisol did not stimulate. Stimulation of gluconeogenesis by dexamethasone was seen following a lag averaging 9 min after the time of steroid addition. Preliminary evidence suggests that this effect was not dependent upon a stimulation of protein synthesis, but the observed stimulation and inhibition of control rates of gluconeogenesis by cycloheximide and cordycepin, respectively, demonstrate the difficulties of working with such inhibitors in attempting to answer this question.     

On the mechanism of action of dexamethasone in a rat mast cell line (RBL-2H3 cells). Evidence for altered coupling of receptors and G-proteins

Prolonged exposure of rat basophilic leukemia (RBL-2H3) cells, a cultured analog of rat mast cells, to 0.1 microM dexamethasone resulted in global suppression of various stimulatory events in response to Ag and a global enhancement of the same stimulatory events to the adenosine analog, N-(ethylcarboxamide)adenosine (NECA). We had previously shown that Ag and NECA both activate phospholipase C but by different mechanisms; cells that had been treated with cholera or pertussis toxin, for example, responded to Ag but not to NECA with the release of inositol phosphates, increase in levels of cytosolic Ca2+, and secretion. Because the toxins still inhibited the responses to NECA in dexamethasone-treated cells, the effects of dexamethasone may have been exerted at the level of receptor/G-protein coupling rather than at the level of effector systems. Additional evidence for this was the following: 1) NECA-induced hydrolysis of the inositol phospholipids was still enhanced after permeabilizing (with streptolysin O or Staphylococcus alpha-toxin) and washing the cells; 2) the response to the G-protein stimulant, guanosine 5'-(3-O-thio)triphosphate was also enhanced in permeabilized, dexamethasone-treated cells and 3) binding and kinetic studies suggested that the enhanced responsiveness to NECA was attributable in part to an increase in receptor number. The suppressive action of dexamethasone on Ag-induced hydrolysis of inositol phospholipids, however, was readily lost by permeabilizing RBL-2H3 cells. The results indicate, therefore, that treatment with dexamethasone leads to changes in receptor-coupling mechanisms that are either resistant to (i.e., NECA-mediated responses) or reversed by (i.e., Ag-mediated responses) cell permeabilization.     

The effect of hormones on glucocorticoid binding capacity of rat liver cytosol.

The relationship between the glucocorticoid binding capacity of rat liver cytosol and the activity of tyrosine aminotransferase has been studied in adrenalectomized male rats. Bilateral adrenalectomy of male rats caused an increase within 3 days in the level of specific dexamethasone binding of liver cytosol accompanied by a rapid decrease in tyrosine aminotransferase activity. Known inducers of tyrosine aminotransferase were administered in vivo to test their effect on dexamethasone binding capacity, in order to determine whether the induction was by an indirect mechanism involving an increase in glucocorticoid binding capacity. Insulin, adrenalin, glucagon, dibutyryl cyclic AMP and oestradiol caused a significant increase in the activity of the enzyme, with no change in the specific dexamethasone binding. Tetracosactrin, a synthetic analogue of ACTH, had no effect on either parameter. It was concluded that the induction of tyrosine aminotransferase by the compounds tested was not mediated by an increase in glucocorticoid receptor activity.     

Increase in acridine orange (AO) fluorescence intensity of monocytes cultured in plastic tissue culture plates as measured by flow cytometry.

Although the green-red fluorescence of AO is an accepted measure of DNA-RNA content, respectively, it is actually a measure of the fluorescence of dye bound to nucleic acids, and may vary with changes in accessibility to the dye. It has been shown for example that extraction of nuclear proteins results in a marked increase in DNA stainability. Moreover, in certain cell systems the binding of fluorochromes correlates with structural modifications in chromatin that accompany cell differentiation. We report here that changes in green & red fluorescence intensity also occur in long-term monocyte cultures. The increased red fluorescence intensity observed in cultured monocytes may reflect ribosomal RNA synthesis and the increased green fluorescence enhanced AO accessibility to DNA due to changes in chromatin organization. We compared cultured monocytes from bladder cancer patients and healthy donors. The results indicate a small but statistically significantly greater increase in mean green & red fluorescence of cultured monocytes from the cancer patients. These fluorescence variations may indicate differences in the immunologic status of cancer patients and/or be related to disease state.     

Ornithine Decarboxylase Induction by Glucocorticoids in Brain and Liver of Adrenalectomized Rats

Abstract: Ornithine decarboxylase (ODC), the rate-limiting enzyme in the biosynthesis of polyamines, was measured in the brain and the liver of adrenalectomized rats after an acute S.C. treatment with glucocorticoids. The effects of corticosterone and dexamethasone were compared in three brain areas, the cerebral cortex, hippocampus, and cerebellum. These structures have similar concentrations of cytosolic glucocorticoid receptor, as measured by an in vitro exchange assay using a specific glucocorticoid ligand, [³H]RU 26988, but contain different amounts of mineralocorticoid receptor. Corticosterone and dexamethasone increased ODC activity in the liver and brain areas in a dose dependent manner, dexamethasone being more active than corticosterone in all tissues. Moreover, estradiol, progesterone, and testosterone were inactive. Aldosterone, at high doses, increased brain ODC activity. Glucocorticoids, selected for their weak binding, or lack of binding to the mineralocorticoid receptor, were tested and found to be highly active in inducing brain and liver ODC, thus showing that ODC induction by steroids is specific for glucocorticoids. These results are among the first to suggest biochemically a central action of glucocorticoids following an acute treatment and confirm that the brain is a glucocorticoid target organ.     

Effect of glucocorticoid administration in vivo on insulin binding in rat testis

We have studied the effects of adrenalectomy and glucocorticoid injections on insulin binding in membranes from rat testis and liver. Glucocorticoids were administered for 7 days to adrenalectomized rats at daily doses of 30 or 300 micrograms for dexamethasone and 100 or 1000 micrograms for prednisolone. Glucocorticoids, at the selected doses, were associated with 5- to 10-fold increases in basal insulin levels with no significant changes in glucose concentrations. As previously shown by other studies, down-regulation of insulin receptors was observed in liver membrane particularly at the higher dose of steroids. Such an effect was not found in the testis. By contrast, the number of high-affinity sites in the testis was slightly increased with the higher doses of dexamethasone and prednisolone. However, percent 125I-labelled insulin binding was not significantly changed after corticotherapy. These results are in accord with our previous studies and suggest that the testicular receptor for insulin is not affected by mild to moderate changes of insulin concentrations, but that it can be modulated by glucocorticoids through other mechanisms.     

Differences in chromatin thermal lability between thymic and splenic lymphocytes in intact and adrenalectomized rats detected by acridine orange microfluorometry

The sensitivity of chromatin to thermal denaturation was compared between small lymphocytes from the thymus and spleen of intact and adrenalectomized rats. RNA was enzymatically removed from uniformly spread lymphocytes attached to glass slides in low density and chromatin denaturation effected by heating at 90 °C in a solution free of formaldehyde and sodium ions. The preparations were stained with acridine orange following acetylation. Fluorescence emission intensity of the nuclei in individual cells was measured at 530 and 590 nm and the ratio used as a relative index of chromatin denaturation. The data show that the chromatin of small thymus lymphocytes is generally more thermolabile than that of morphologically comparable cells of the spleen in both intact and adrenalectomized animals. This difference between cells of the same morphological type from the two lymphoid organs was not evident from measurements of the amount of dye bound by the cells without denaturation. Removal of the endogenous source of glucocorticoids resulted in an increase in the number of spleen lymphocytes with lower chromatin thermal stability but had no detectable effect on the population of thymus lymphocytes. The results are discussed in terms of the histological organization and immunological role of the thymus and spleen and in relation to the technical aspects of acridine orange microfluorometry as a sensitive cytochemical probe of chromatin structure.     

Time dependent effects of dexamethasone on serum insulin level and insulin receptors in rat liver and erythrocytes

The effects of glucocorticoid excess on regulation of insulin receptors were investigated in dexamethasone-treated rats. Glucocorticoid excess was produced by administration of dexamethasone (0.5 mg/100 g b.w.) 30 min, 4, 12, 18, 24, 42 or 70 h before experiments. This treatment caused time-dependent changes of glucose and insulin concentration in blood, as well as in amounts of specific insulin binding and insulin receptors of liver cells and erythrocytes. The time intervals in which dexamethasone produced the increase in insulin concentration were accompanied with decrease in insulin binding to receptors in membranes of liver cells, while significant changes in insulin binding to receptors of erythrocytes were not observed under the same experimental conditions. The effect is maximal 18 and 42 h after dexamethasone treatment that increase insulin blood level by about 85% and 60%, respectively. Receptor analysis revealed that changes in specific binding of insulin could be due to significant changes in amount of binding sites on cell surface rather than to mild alteration in receptor affinity. These findings suggest that besides the changes in insulin level, the alterations in insulin receptor number and affinity may play a major role in the states of altered insulin sensitivity which accompany glucocorticoid excess.     

The biopotency of dexamethasone at causing hepatic glucocorticoid receptor down-regulation in the intact mouse

The effect of dexamethasone administered intraperitoneally on hepatic glucocorticoid receptor binding capacity was measured in adrenalectomized male Swiss Webster mice. The liver content of dexamethasone was also measured. Within 30 min of a 5 micrograms injection, the hepatic content of dexamethasone reached a maximum and fell quickly thereafter. By 6 h the hepatic content of dexamethasone had decreased to 25% of maximum and by 24 h the liver did not contain detectable dexamethasone. At this 24 h point, the glucocorticoid binding capacity was reduced to 50% of control. This decrease reflected down-regulation. Other studies revealed that only glucocorticoids caused this effect and doses of dexamethasone as low as 0.5-5 ng caused a clear down-regulation in binding capacity. Doses that cause receptor down-regulation are also effective at inducing tyrosine aminotransferase, suggesting that dexamethasone down-regulates its own receptors over a physiologically meaningful dosage range. It is concluded that dexamethasone causes a dose-dependent down-regulation of the glucocorticoid receptor in mouse liver.     

Dexamethasone acts as a negative regulator of epidermal growth factor receptor synthesis in fetal rat lung cells

125I-Epidermal growth factor (EGF) binding capacity in fetal rat lung cells is decreased by approximately 50% following 24-h dexamethasone treatment. Ligand binding assays identified an average of 30,000 receptors per cell in untreated FRL cells, while analysis of dexamethasone treated cells showed a decrease to about 16,000 receptors per cell. No substantial changes in receptor affinities were detected. Immunoprecipitation of 35S-methionine-labeled EGF receptor protein demonstrated a 50% decrease in total EGF receptor protein after 24-h dexamethasone treatment. Brief pulse labeling with 35S-methionine showed that the reduction in total EGF receptor protein content was due to a decrease in EGF receptor synthesis. Receptor synthesis declined about 25% after 1 h of dexamethasone treatment and at 3 h, EGF receptor synthesis was maximally decreased to nearly 50% that of cells not exposed to dexamethasone. Dexamethasone treatment was also effective in reducing EGF receptor synthesis in cells pretreated with retinoic acid, an agent which enhances receptor synthesis. These data are the first to document a dexamethasone-induced decrease in EGF receptor synthesis. Furthermore, these findings may provide a plausible mechanism by which dexamethasone could regulate EGF responsiveness.     

Early nuclear cytochemical changes in regenerating mammalian liver

Various cytochemical parameters were studied cytophotometrically in parenchymal cell nuclei isolated from rat liver at different times following partial hepatectomy. The study was confined to the early period following operation, before DNA synthesis and before mitotic activity ensued. Binding of acridine orange was found to increase approx. 70% over normal controls at 3 h post-hepatectomy followed by a sharp decrease to 60% below controls by 12 h and a return to near control levels at 24 h. These changes were found in both diploid and tetraploid cell nuclei. The initial two-fold difference in AO binding observed between diploid and tetraploid nuclei in normal liver persisted at 3 h but decreased markedly from 6 to 24 h after operation. Chromatin thermal stability, determined by acridine orange microfluorimetry, showed a rapid decrease at 3 h in both diploid and tetraploid cell nuclei followed by an increase above control levels at 6 h which persisted up to 24 h after partial hepatectomy. Parallel measurements of Feulgen dye binding showed no change in the relative DNA content of diploid and tetraploid cells throughout the experimental period. Ratios of specific picric acid and alkaline bromphenol blue dye binding by histone arginine and lysine side chains were found to rise significantly over normal controls at 3 h post-hepatectomy and return to normal levels by 6 h.