High Concentrations Of Dexamethasone Research Articles

Growth-stimulatory effects of androgen, high concentration of glucocorticoid or fibroblast growth factors on a cloned cell line from shionogi carcinoma 115 cells in a serum-free medium

The effects of various kinds of growth factors or steroids on the proliferation of Shionogi carcinoma 115 (SC115) cells were investigated in cell culture. In a serum-free medium [Ham's F-12: Eagle's minimum essential medium (1:1, vol/vol) containing 0.1% bovine serum albumin], the proliferation of SC-3 cells (a cloned cell line from SC115 cells) estimated by [ 3H]thymidine incorporation into DNA and cell number reached a plateau at 10 −8 M testosterone (up to 200-fold), 10 −7 M dexamethasone (up to 30-fold) or 1 ng/ml of fibroblast growth factors (FGF; up to 50-fold). However, the proliferation in the serum-free medium was not significantly stimulated by the addition of low to very high concentrations of progesterone, oestradiol-17β, epidermal growth factor, platelet derived growth factor or insulin; transforming growth factor β slightly stimulated the growth (up to 5-fold) but markedly inhibited the growth stimulation induced by testosterone. Furthermore, an epithelial appearance of SC-3 cells grown in the absence of growth factors or steroids was changed to a fibroblast-like appearance only by the addition of testosterone, high concentrations of dexamethasone or PGF. By investigating various kinds of growth factors or steroids, the present study demonstrates that androgen, high concentration of glucocorticoid or FGF alone significantly stimulates the proliferation of SC-3 cells with a change of morphology in the serum-free medium.

Stimulation and inhibition of pituitary growth hormone release by angiotensin II in vitro.

Rat pituitary cell aggregates cultured in serum-free chemically defined medium, single cells, and hemipituitaries were used in a perifusion system to study the influence of angiotensin II (AII) on GH release. In aggregates the peptide displayed both stimulatory and inhibitory effects on GH release, depending on the hormonal conditions of the culture medium and the age of the animal. When cultured in the absence of glucocorticoid, a modest but statistically significant stimulation was seen in aggregates from immature as well as adult animals. In aggregates from 5-day-old animals, dexamethasone (DEX) strongly enhanced the GH-releasing activity of AII in a dose-dependent way; in aggregates from 14- and 25-day-old rats, the same pattern was found, although the stimulatory action was weaker than the effect in 5-day-old rats. In aggregates from adult animals, the glucocorticoid established an inhibitory effect of AII on GH release, an effect seen with both low and high concentrations of DEX. These age- and DEX-dependent effects were not found for AII stimulation of PRL release. In the presence of DEX, AII also inhibited GRF-induced GH release in aggregates from adult animals, while it was synergistic with GRF in aggregates from developing animals. The effects of AII on GH release disappeared when aggregates were redispersed into single cells. However, in these single cell preparations AII strongly stimulated PRL release. In hemipituitaries from 1-, 5-, and 14-day-old animals, AII also stimulated GH release, but no effect was seen in hemipituitaries from 25-day-old and adult animals. These data indicate that AII has dual effects on GH release depending on the developmental stage of the animal and the hormonal environment. Furthermore, since no effect of AII was seen after redispersion of aggregates into single cells, both stimulatory and inhibitory effects seem to be based on an intercellular signaling system.

Long-term culture of hepatocytes: effect of hormones on enzyme activities and metabolic capacity.

(i) Hepatocytes isolated from adult rats were cultured for 2 to 3 weeks on collagen in a modified, serum-free Waymouth medium containing fatty acids and varying concentrations of glucocorticoid, insulin and glucagon. (ii) In the presence of all three hormones, it was possible to maintain the content of DNA, the activity of glucokinase, pyruvate kinase, hexokinase and lactate dehydrogenase at initial levels for 2 to 3 weeks. The activity of glucokinase and pyruvate kinase was affected by the concentration of insulin. (iii) The activity of alcohol dehydrogenase was stable for 3 days and declined to about 25% of the initial level after 2 weeks of culture, irrespective of the presence of hormones. (iv) Maintenance of albumin secretion was dependent on the presence of glucocorticoid, and glucocorticoid and insulin showed an additive or, at some time points, a synergistic effect on its secretion. (v) The content of cytochrome P-450 could be kept at 65% of the initial level, provided that a relatively high concentration of dexamethasone was present (10(-6) M). (vi) In the absence of hormones, urea synthesis was 70% of initial levels throughout the experimental period. With insulin and glucocorticoid present, a high concentration of glucagon (10(-8) M) was required to maintain the synthesis of urea at this level. (vii) It is concluded that hepatocyte cultures as described in the present study may be a useful, well-defined system for long-term metabolic, pharmacologic and toxicologic studies.

Acetylcholine stores in rat diaphragm are increased by higher concentrations of dexamethasone

In indirectly stimulated hemidiaphragms, dexamethasone (Dex, 2 μM) causes a significant increase in tissue acetylcholine (ACh) content. No increase in tissue ACh is found with 0.2, 0.6 or 6 μM Dex. Physostigmine (Physo, 15 μM) also causes an increase in tissue ACh, which is even greater in the presence of Dex (6–25 μM). There is no increase in ACh due to Dex, with 50 μM Dex plus Physo. The order of addition is important, as the Dex-induced increased in ACh is only found when Dex is added before Physo. No increase in twitch tension is found with any of these treatments. No Dex-induced increase in ACh is found with unstimulated hemidiaphragms. Similar increases in ACh are also found with other glucocorticoids (plus Physo), namely prednisolone (6–9 μM), corticosterone (2 μM) and hydrocortisone (2 μM). The mineralocorticoid aldosterone (2 μM), and other types of steroids cause no increase in tissue ACh. The increases in hemidiaphragm ACh are not found in a Na +-depleted medium, or in a medium containing 20 mM Mg 2+ extra. The increase in ACh due to Physo is Ca 2+-dependent, even though an increase in ACh due to Dex plus Physo is found in the absence of Ca 2+. No increase in ACh due to either Physo, or Dex plus Physo are found in the presence of the nicotinic antagonists (+)-tubocurarine (5 μM) or alpha-cobrotoxin (5 μg/ml), while the muscarinic ligands atropine or oxotremorine (10 μM) abolish the extra increase in ACh due to Dex. Perhaps the glucocorticoid-specific increase in ACh stores may be caused by a direct interaction of the corticosteroids with an ACh receptor (possibly presynaptic) which may regulate ACh synthesis and/or output.

Experimental study on drug therapy of "traction retinal detachment" after posterior penetrating eye injury in the rabbit.

In an experimental study on rabbits, a standardized eye injury was created by using the "pars-plana incision model." Subsequently, the effect of intravenous application of dexamethasone and penicillamine on traction retinal detachment was investigated. The two drugs were applied in varying concentrations and combinations (single and combined use in varying intervals), followed by a 3-month control period without medication. Clinical and histological findings showed that intravitreal instillation of 1.2 mg dexamethasone reduces the incidence of retinal detachment from 46% to 27%. Higher concentrations of dexamethasone, as well as the use of penicillamine or a combination of both substances, proved to enhance traction retinal detachment.

Activation of alpha-fetoprotein synthesis in rat hepatoma cells with reduced sensitivity to dexamethasone

The Faza 967 ‘differentiated', dexamethasone (DEX)-sensitive cell line of Reuber rat hepatoma cells does not synthesize detectable amounts of alpha-fetoprotein (AFP), whereas it does produce albumin. AFP production was activated in ‘differentiated’ variants of Faza 967 cells with reduced glucocorticoid sensitivity upon culture for several months in the presence of high concentrations of dexamethasone. The stability of AFP production differed among the variants, while albumin synthesis did not change, thus indicating that the regulation of these two genes is not co-ordinated. Using molecular hybridization techniques, we found that the AFP message could not be detected in the non-AFP-producing cells, suggesting that the lack of AFP synthesis most probably originates from a tran-scriptional block of the AFP gene. AFP-producing and non-AFP-producing variants of Faza 967 cells constitute a valuable model system for studying the regulatory mechanisms involved in the activation and inactivation of the gene coding for the oncodevelopmental protein, AFP.

Effects of glucocorticoids on eosinophil colony growth

The in vivo and in vitro effects of glucocorticoids on eosinophilopoiesis were examined with soft agar cultures of bone marrow and peripheral blood cells. Prednisone, 10 mg four times daily for three days, administered to normal volunteers, caused a significant drop in circulating eosinophil numbers ( p < 0.005) but did not decrease eosinophil colony numbers in cultures of bone marrow or peripheral blood. A patient with peripheral blood eosinophilia demonstrated a larger percentage decrease in mean eosinophil colony numbers (50%) after prednisone than did any normal volunteer. Incubation of bone marrow cells for 1 hour with either high concentrations of hydrocortisone, up to 3.3 × 10 −4 mol/L, or with postinfusion plasma from volunteers administered intravenous hydrocortisone, plasma levels to 1.6 × 10 −5 mol/L, did not decrease the numbers of eosinophil colonies. At a concentration of 3.3 × 10 −6 mol/L of hydrocortisone, there was a slight but statistically significant stimulation of eosinophil colony numbers. In contrast, incubation with high concentrations of dexamethasone, 3.3 × 10 −4 mol/L or 3.3 × 10 −6 mol/L, significantly reduced eosinophil colony numbers. Prednisone caused a significant reduction in plasma levels of Charcot-Leyden crystal protein but not of eosinophil granule major basic protein. The results indicate that (1) soft agar assay of eosinophil colony growth by blood or bone marrow cells cannot be used to model the in vivo eosinopenic effect of glucocorticoids, (2) levels of dexamethasone in excess of levels commonly administered in clinical practice are required to inhibit eosinophilopoiesis in vitro, and (3) patients with peripheral blood eosinophilia may be more susceptible to the eosinopenic effects of glucocorticoids than normal subjects.

Dexamethasone and retinyl acetate similarly inhibit and stimulate EGF- or insulin-induced proliferation of prostatic epithelium.

Prostatic epithelium proliferates in a defined medium consisting of basal medium RPMI1640 containing transferring (1 microgram/ml), EGF (10 ng/ml), and insulin (3.7 micrograms/ml or 0.1 IU/ml). Although neither dexamethasone nor retinyl acetate affected the proliferation of prostatic epithelium in RPMI1640 containing transferrin alone, they modify the mitogenic effect of EGF and insulin. Dexamethasone at 10(-10) M or retinyl acetate at about 3 X 10(-9) M inhibits proliferation stimulated by EGF. Higher concentrations of dexamethasone (10(-8) - 10(-6) M) or retinyl acetate (3 X 10(-8) - 10(-7) M) enhance the mitogenic activity of EGF. Dexamethasone had a similar effect in the presence of insulin. However, retinyl acetate stimulated, but did not significantly inhibit, proliferation in the presence of insulin. These results suggest that both dexamethasone and retinyl acetate, and possibly other glucocorticoids and retinoids, may regulate the proliferation of prostate epithelium by a dose-dependent modification of the activity of insulin and EGF.

Regulation of prostaglandin synthesis during differentiation of cultured mouse myeloid leukemia cells.

Mouse myeloid leukemia cells (Ml) were induced to differentiate into mature macrophages and granulocytes by various inducers. The differentiated Ml cells synthesized and released prosetaglandins, whereas untreated Ml cells did not. When the cells wee prelabelled with [14C]arachidonate, the major prostaglandins released into the culture media were found to be prostaglandin E2, D2, and F2 alpha in an early stage of differentiation, but the mature cells produced predominantly prostaglandin E2. The synthesis and release of prostaglandins were completely inhibited by indomethacin. Dexamethasone, a potent inducer of differentiation of Ml cells, did not induce production of prostaglandins in resistant Ml cells that could not differentiate even with a high concentration of dexamethasone. These results suggest that production of prostaglandins in Ml cells is closely associated with differentiation of the cells. Homogenates of dexamethasone-treated Ml cells converted arachidonate to prostaglandins, but this conversion was scarcely observed with homogenates of untreated Ml cells. Dexamethasone and the other inducers stimulated the release of arachidonate from phospholipids. Therefore, induction of prostaglandin synthesis during differentiation of Ml cells may result from induction of prostaglandin synthetase activity and stimulation of the release of arachidonate from cellular lipids. Lysozyme activity, which is a typical biochemical marker of macrophages, was induced in Ml cells by prostaglandin E2 or D2 alone, as well as by inducers of differentiation of the cells, but it was not induced by arachidonate or prostaglandin F2 alpha. These results suggest that prostaglandin synthesis is important in differentiation of myeloid leukemia cells.

Properties of a cell-culture line derived from lymphosarcoma p1798

A cell culture line has been derived from lymphosarcoma P1798. Cultures of this line grow exponentially with a doubling time of about 20–24 h. Cells will not divide in the presence of 10 −8 M dexamethasone. Half-maximal inhibition of proliferation occurs at about 10 −9 M. Half maximal inhibition of [ 3H] thymidine incorporation occurs at somewhat higher concentrations of dexamethasone. When cells are cultured in the presence of 2 × 10 −7 M dexamethasone they remain 88% viable after 5 days. No decrease in cell number occurs. These cells can be rescued by in vivo passage, and they responded in culture to dexamethasone in exactly the same fashion as do untreated cells. Both dexamethasone rescued and untreated cultured cells contain about 10 4 dexamethasone receptor sites which bind with a dissociation constant of 4 × 10 −9 M. Cultured cells retain the ability to proliferate both subcutaneously and in ascites. The properties of such tumors resemble those of the parental tumor cell line.

Effects of dexamethasone on tumor-induced brain edema and its distribution in the brain of monkeys.

A human choriocarcinoma was successfully adapted to grow in the brain of monkeys (Macaca mulatta), thus providing a model of tumor-induced brain edema. Four animals were given dexamethasone (3 mg/kg/day) during 3 to 5 days after the onset of clinical signs, and the other five received no treatment for the same period. Tissue water and electrolyte content of treated and untreated animals were compared in cortex and white matter at various distances from the edge of the tumor. In untreated animals, 67.9% and 23.6% swelling was detected in adjacent and remote white matter, respectively, but only 11.8% swelling was noted in adjacent cortex. In animals treated with dexamethasone these percentages of swelling were improved to 32.4% and 11.9% in the corresponding white matter, and to 4.9% in adjacent cortex. The electrolyte changes shown in edematous brain of control animals also demonstrated significant improvement in the dexamethasone-treated group. Tissue radioactivity of 3H-dexamethasone at 60 minutes after intravenous injection was high in the periphery of tumor, adjacent cortex, and white matter, but low in the center of tumor, remote cortex, and white matter. The sites with high concentrations of dexamethasone also showed significant improvement of brain edema after dexamethasone treatment, suggesting that dexamethasone may act directly at these loci.

Biochemical actions of glucocorticoids on macrophages in culture. Specific inhibition of elastase, collagenase, and plasminogen activator secretion and effects on other metabolic functions.

The effects of glucocorticoids on biochemical functions of macrophages from man, mouse, rabbit, and guinea pig were examined. Secretion of plasminogen activator by human peripheral blood monocytes was decreased by 50% with 1 nM dexamethasone. Differentiation of murine monocytic and granulocytic colonies in agar from bone marrow precursors was decreased by 50% at 7 days with 20 nM dexamethasone. Secretion of elastase, collagenase, and plasminogen activator by resident and thioglycollate-elicited mouse peritoneal macrophages was decreased by dexamethasone, cortisol, and triamcinolone acetonide (1--1,000 nM), but not by progesterone, estradiol, and dihydrotestosterone (1,000 nM); in contrast, secretion of lysozyme was not affected by glucocorticoids or other steroids. The inhibition of macrophage secretion by dexamethasone was both time and dose dependent. Effects were detected within 1--6 h after addition of the glucocorticoids, became maximum by 24 h, and were reversed during a similar time period after removal of the hormones. The extent of inhibition of macrophage secretion increased with increasing glucocorticoid concentration. Half-maximum inhibition of secretion of elastase, collagenase, and plasminogen activator was seen at dexamethasone concentrations (1--10 nM) similar to those that half-saturated the specific glucocorticoid receptors in these cells. At high concentrations of dexamethasone (100--1,000 nM) the secretion of plasminogen activator was inhibited to a greater extent (greater than 95%) than the secretion of elastase (60--80%). Progesterone alone had no effect on secretion, but it blocked the inhibitory effects of dexamethasone and cortisol. Secretion of collagenase, neutral proteinases, and plasminogen activator by elicited rabbit alveolar macrophages was inhibited with glucocorticoids (0.1--100 nM) but not with progesterone or sex steroids. Secretion of a neutral elastinolytic proteinase by guinea pig alveolar macrophages was also inhibited by dexamethasone. These data support the regulatory role of glucocorticoids on macrophage functions at physiological concentrations.

Production of differentiation-stimulating factor in cultured mouse myeloid leukemia cells treated by glucocorticoids

Mouse myeloid leukemic cells (Ml) could be induced by glucocorticoids to produce a factor(s) stimulating their own differentiation to macrophage- and granulocyte-like cells. The differentiation-stimulating factor(s) (DSF) was not due to a contaminating steroid, although glucocorticoids were effective to induce differentiation of the cells. DSF seemed to be a glycoprotein(s) with a molecular weight of 20 000–40 000 D, since it was susceptible to a treatment of proteases of glycosidases. The inducing ability of corticoids to produce DSF was correlated with their glucocorticoid activity, which was in parallel with the inducing activity of cell differentiation. Moreover, glucocorticoids were unable to stimulate the production of DSF in a dexamethasoneresistant Ml cells which could not differentiate even in a high concentration of dexamethasone. These results suggest that production of DSF in Ml cells was closely associated with differentiation of the cells. Differentiation of Ml cells by DSF was confirmed by morphological, functional and biochemical evidences.

Glucocorticoid binding and mechanism of resistance in some clones of mouse myeloid leukemic cells resistant to induction of differentiation by dexamethasone

Several clones of dexamethasone-resistant cells, which could not differentiate even in a high concentration of dexamethasone, were isolated from glucocorticoid-sensitive myeloid leukemic cells. Some of them were shown to be deficient in steroid binding to specific cytoplasmic receptors, while the others contained glucocorticoid-specific cytoplasmic receptors that might be the same as those in sensitive cells. One of the resistant clones was found to be almost completely deficient in nuclear acceptor sites for cytoplasmic steroid-receptor complexes. The remaining clones were also characterized by significantly reduced amounts of nuclear-bound glucocorticoid. These results suggest that resistibility to glucocorticoids in the resistant clones of myeloid leukemic cells is due mainly to a defect in some steps of intracellular transfer of the steroid. Dexamethasone-sensitive cells, which could differentiate in the presence of dexamethasone, could be also induced to differentiate by protein factor(s) in ascitic fluid. Although all the resistant cells showed a low response to ascitic fluid, some of them showed 10-fold enhancement of phagocytic activity which is a typical character of differentiated cells. These results suggest that response to steroids is not directly correlated with that to protein inducer(s).

Glucocorticoids and fetal lung development

Acceleration of fetal lung development by administration of glucocorticoid hormones has been demonstrated in a number of mammalian species. Dexamethasone 10 −5 M significantly depressed protein content per explant in the presence and absence of insulin. Insulin did not alter the protein content but significantly increased radioactive choline incorporation. However, dexamethasone provided no added stimulation above that observed with insulin alone. The incorporation of radioactive choline and specific activities in isolated phosphatidylcholine were almost identical in control and dexamethasone (10 −7 M) treated explants. The data presented demonstrate a modest but significant increase in choline incorporation using a concentration of dexamethasone of 10 −9 M. At higher concentrations of dexamethasone, the incorporation was significantly depressed using an 8 day exposure time. Although choline incorporation increases as a function of gestational age with a burst in rate on the last day of fetal life, dexamethasone suppresses this activity at all developmental ages past 19.5 days. The only conclusion that appears valid at this time is the rat fetal lung system reported here differs in an unknown way when compared to the rabbit monolayer system, the human organ culture, and the in vivo situation.

Glucorticoid-induced differentiation of cultured mouse myeloid leukemia cells.

Mouse myeloid leukemia cells were induced by some corticoid hormones to migrate in agar, phagocytize, and change into forms which were morphologically similar to macrophages and granulocytes. Inducing ability of corticoids was correlated with glucocorticoid activity, not mineralocorticoid activity. Other steroids were ineffective to induce differentiation of myeloid leukemia cells. Glucocorticoid-resistant cells, which could not differentiate even in a high concentrations of dexamethasone, were selected from steroid-sensitive cells by stepwise increase in concentrations of dexamethasone in culture medium.

Kinetics of glucocorticoid-receptor complexes in rat thymus cells

Using cortisol and dexamethasone, we have studied the kinetic behavior of glucocorticoid receptors in intact rat thymus cells at 37°C. As reported previously, translocation of the cortisol-receptor complexes from “cytoplasmic” to nuclear-bound form takes about 1 min. At 26°C this time is doubled. Consistent with these observations and with the current view that formation of nuclear-bound complex must be preceded by formation of cytoplasmic complex, on addition of hormone to cells at 37°C the initial time-course of formation of cytoplasmic complex precedes that of nuclear complex by about 30 s. After cells have achieved a steady state with [ 3H]-dexamethasone, however, a “chase” of unlabelled dexamethasone lowers nuclear levels of [ 3H]-dexamethasone several minutes before cytoplasmic levels, suggesting formation of nuclear complex by direct reaction of hormone with nuclear-bound receptors. Following depletion of cytoplasmic receptor by incubation with a high concentration of dexamethasone and then sudden lowering of steroid concentration by dilution, the time-course of replenishment of cytoplasmic receptors appears to lag significantly behind the time-course of removal of nuclear-bound steroid. The lag is roughly the same magnitude as the time-constant of dissociation of the glucocorticoid used, and is much shorter with cortisol. The pathway of replenishment thus may be different from simple reversal of the pathway of depletion of cytoplasmic receptor, and may depend on the dissociation rate or affinity constant of the steroid.

Effect of glucocorticoid hormones on fatty acid mobilization and re-esterification in rat adipose tissue.

1. The effect of dexamethasone and cortisol on fatty acid mobilization and re-esterification has been studied in intact adipose tissue and isolated fat cells of the rat. 2. Dexamethasone added in vitro inhibited both the re-esterification of mobilized free fatty acids and the esterification of palmitate in the medium. 3. Under several conditions (low concentrations of dexamethasone, cortisol at a high concentration, with tissue from starved animals), steroid-induced release of free fatty acids could be accounted for by decreased re-esterification only, overall lipolytic activity remaining unmodified. At higher concentrations of dexamethasone, however, stimulation of lipolytic activity also occurred. 4. Decreased re-esterification produced by dexamethasone was observed in the total absence of glucose from the incubation medium. Further, dexamethasone stimulated the disappearance of prelabelled [(14)C]glycogen from the tissue. 5. The evidence presented suggests that mobilization of free fatty acids induced by glucocorticoid hormones under physiological conditions is primarily due to a decrease of the re-esterification rate rather than to lipase activation.