Enhanced sensitivity of pituitary cells to corticosteroids in neonatal rats]

The effects of corticosteroids on pituitary cells, obtained from developing rats of different ages, were studied during long-term (3-4 days) incubation. Dexamethasone, cortisol and aldosterone inhibited the rate of DNA synthesis in primary cultures of pituitary cells from neonatal and pup rats more efficiently than in cell cultures of prepubertal and adult animals. Moreover, the above mentioned corticosteroids inhibited total protein biosynthesis in pituitary cells of infantile rats and did not change significantly this index of functional activity in cells of prepubertal and adult animals. In separate experiments steroid specificity of glucocorticoid action was examined in detail: estradiol and testosterone did not change the rates of macromolecule synthesis in neonatal pituitary cells whereas progesterone possessed a weak inhibitory effect that was, however, far less marked as compared to the effect of corticosterone. It has been concluded that neonatal rat pituitary cells demonstrate enhanced sensitivity to anti-proliferative and catabolic action of corticosteroids.     

Regulation of pancreatic islet cell replication: an inquiry into the controversy regarding the effects of steroid hormones

The controversial issue of the effects of prednisolone and 17 beta-estradiol on replication of fetal rat pancreatic islets in culture was studied using 32P and [3H]thymidine as probes for studying DNA synthesis. DNA synthesis was not affected by the steroid hormones, as was evident from the rate of incorporation of 32P into total DNA. Decreased incorporation of [3H]thymidine into DNA found in islets treated with either of these steroids seemed to reflect an inhibitory effect of these hormones on thymidine kinase, leading to decreased phosphorylation of labeled thymidine. In addition, the hormones stimulated the activity of thymidylate synthetase, thus enhancing the endogenous synthesis of thymidine and thereby diluting the specific activity of the [3H]thymidine added to the cultured islets. Further support for a lack of inhibition of growth of islet cells treated with steroid hormones was provided by the observation that prednisolone increased uridine kinase activity and RNA biosynthesis, both of which may participate in the growth of cells preceding mitosis and (the latter) in protein hormone biosynthesis.     

The sex difference in type II cell surfactant synthesis originates in the fibroblast in vitro

Lung from 19 days' gestation male and female rat fetuses was grown in mixed monolayer culture. Cultures from female lungs synthesized twice as much 3H-saturated phosphatidylcholine (3H-SPC) from 3H-choline as cultures from male lungs. Preincubation with cortisol caused a doubling in 3H-SPC synthesis by cultures from both sexes. Study of sex-specific fibroblast cultures revealed significantly higher 11-oxidoreductase activity and basal and cortisone- or cortisol-stimulated fibroblast-pneumonocyte factor activity in female cultures. In contrast to the findings in the fibroblast cultures, sex-specific type II cell cultures showed no difference in basal 3H-SPC synthesis or in response to cortisol-stimulated fibroblast-conditioned medium. Furthermore, the type II alveolar cells had no 11-oxidoreductase activity. These data indicate that there are sex differences in both cortisol metabolism and fibroblast-pneumonocyte factor production by the fetal lung fibroblast. These differences would lead to a sex difference in the synthesis of pulmonary surfactant.     

Testosterone stimulation of a rapidly labeled, low-molecular-weight RNA fraction in human hepatic erythroid cells in culture.

The addition of erythropoietin to cell cultures of erythroid cells of human fetal liver resulted in an increased incorporation of thymidine, adenine, and uridine into trichloroacetic acid-insoluble cell fractions and in an increased uptake of adenine and uridine into the cell. Although the effects of testosterone and erythropoietin on heme synthesis in these cells are known to be very similar, there was no effect of testosterone on the total incorporation of radioactive precursors into DNA or RNA. The RNA synthesized after short pulses of radioactive uridine, when analyzed on sucrose gradients containing 1% sodium dodecyl sulfate, consisted of a homogeneous peak sedimenting at 10 plus or minus 2 S, which is quite different from the heterogeneous, high-molecular-weight RNA synthesized under identical conditions in primary cultures of human fetal lung, kidney, or liver parenchymal cells. Addition of testosterone to liver erythroid cells in cultures for 5 hr followed by a 1-hr uridine pulse resulted in a 3-fold increase of RNA species with an average sedimentation coefficient of 14 plus or minus 3 S. The similarity with the sedimentation coefficient of the globin mRNA described in other systems and the high degree of specialization of the erythroid cells suggest that this RNA may be a stable intermediate involved in the synthesis of hemoglobin.     

Effects of corticosteroids, prostaglandin E2, and beta-agonists on adenylate cyclase activity in fetal rat lung fibroblasts and type II epithelial cells

The effect of glucocorticoids on the response of adenylate cyclase in fetal rat lung fibroblast and Type II epithelial cell cultures to beta-agonists and prostaglandin E2 (PGE2) was investigated. There was significant stimulation of cyclic AMP synthesis by adrenalin in both fibroblasts and Type II cells, which was potentiated in a dose-dependent manner by cortisol. Stimulation of adenylate cyclase by PGE2 (10-1000 nM) was demonstrated in fibroblasts but not in Type II cells. The response to PGE2 was stimulated by pretreatment with cortisol only in fibroblasts (p less than .01), and no latent response to PGE2 was evident in Type II cells after cortisol treatment. These experiments suggest that both cell types isolated from late gestation fetal lung contain active beta-agonist and glucocorticoid receptors that synergize in raising intracellular cyclic AMP, which has multiple effects, including surfactant secretion from Type II cells. Since the adenylate cyclase response to PGE2 and its enhancement by glucocorticoids occurred only in fibroblasts, it is concluded that the reported effects of PGE2 on surfactant release are not mediated via raised intracellular cyclic AMP in Type II cells.     

Stimulation of DNA and Protein Synthesis in Fetal-Rat Liver Cells by Serum from Partially Hepatectomized Rats

Primary cultures of differentiated liver cells from fetal rats respond to serum from partially hepatectomized rats by an increased uptake of [(3)H]thymidine and [(3)H]leucine as compared with cultures incubated with normal rat serum. These findings are discussed in relation to the mechanism of induction of DNA synthesis and growth in liver cells during liver regeneration.     

Sex hormones influence growth and surfactant production in fetal lung explants

Sex-related differences may be present during fetal lung growth and at the onset of surfactant synthesis. In this study we investigated the role of dihydrotestosterone (DHT) and estrogen (EST) on cell division and on labeled palmitate incorporation into disaturated phosphatidylcholine (DSPC) at various times of gestation. Using organ cultures of fetal rat lung from sexed littermates, it was shown that both DHT and EST reduce DNA synthesis only in tissue taken during the rapid growth phase from day 16 to 19 of gestation. From autoradiographic counts, epithelial cell division was most affected. Both hormones reduced DSPC synthesis in explants prepared at day 18, when levels are normally low. At day 19, DHT reduced palmitate incorporation into DSPC of female explants to the male level; subsequently DHT had no effect on any tissue. In contrast, the addition of EST stimulated DSPC synthesis 40% above controls in both male and female explants taken at day 20 only. The results suggest that sex differences seen in late fetal lung development may arise from the combined effects of slowed epithelial growth induced by these hormones followed by inhibition of DSPC synthesis by DHT and acceleration by EST at the crucial period when surfactant synthesis begins.     

Glucocorticoid-thyroid synergism in lung maturation: a mechanism involving epithelial-mesenchymal interaction.

We studied the effects of cortisol and triiodothyronine (T3) on 20-day fetal rat lung cell cultures. Cortisol enhanced the production of surfactant-associated saturated phosphatidylcholine while T3 did not. However, T3 potentiated the cortisol effect. We observed that T3 enhanced the response of cultures enriched with alveolar type H cells to fibroblast-pneumonocyte factor (FPF). Intracellular cAMP was increased by exposure of these cultures to FPF, and T3 potentiated this increase. Unlike cortisol, T3 had no effect on production of FPF by fetal lung fibroblasts, as determined by bioassay of fractions of fibroblast-conditioned medium partially purified by column chromatography. The time course of cortisol action on mixed (fibroblast/epithelial) cultures was in keeping with the proposed mechanism: glucocorticoid induction of FPF in fibroblasts, followed by FPF induction of cAMP in epithelia and, finally, by enhanced production of saturated phosphatidylcholine. Thus, glucocorticoid acting on mesenchyme and thyroid hormone acting on epithelium have a synergistic effect on expression of differentiated epithelial function.     

Glucocorticoid Receptors and the Role of Glucocorticoids in Fetal Lung Development

The cellular mechanism of glucocorticoid effects upon fetal lung was examined in studies of specific binding activity for corticosteroids. Cytoplasm of fetal rabbit lung contains receptor sites for [(3)H]dexamethasone at a concentration of 0.43 +/- 0.04 pmol/mg of cytosol protein, and the apparent dissociation constant for the binding reaction is 2.7 +/- 0.4 nM. The ability of various steroids to compete with labeled dexamethasone for binding to receptor correlates with their biologic potency. The hormone-receptor complex formed in vitro at 0 degrees binds with high affinity at 20 degrees to isolated lung nuclei. It is estimated that there are 9500 nuclear binding sites and 12,000 cytoplasmic receptor sites per fetal lung cell. During the last 12 days of gestation in a rabbit, the concentration of cytoplasmic receptor in lung is constant and is 2- to 5-times greater than receptor-site concentration in fetal skin, kidney, heart, muscle, gut, liver, brain, thymus, and placenta. These findings demonstrate that the early steps in the mechanism of glucocorticoid action in target tissues are present in lung cells, and suggest that these hormones accelerate fetal lung differentiation and surfactant production in animals by the induction of new protein synthesis mediated by receptor.     

High performance liquid chromatography of rat globin chains of fetal liver during the switch from embryonic to adult hemoglobin

A reversed-phase high performance liquid chromatography (HPLC) method for the direct separation of globin chains from cell lysates of peripheral blood and fetal liver from rat fetuses is described. Partial amino acid analysis of the globins eluted from the HPLC columns as well as comparison with the known elution positions of the adult globin chains in carboxymethyl cellulose chromatography indicated that alpha-chains are eluted first, followed by adult beta chains. The last chains to be eluted are considered as embryonic globins because of their absence in adult rats and their rapid disappearance from the liver after the 14th day of gestation. Liver erythroid cells isolated from rat fetuses on day 14 of gestation mainly synthesized alpha-chains and embryonic globin chains, whereas cells prepared from 16-day old fetuses synthesized almost exclusively alpha-chains and adult beta chains. When the fetal rat liver cells were cultured for 20h with erythropoietin there was a significant stimulation in the synthesis of alpha-globins and adult beta chains but not on the synthesis of embryonic globin chains. It is concluded that HPLC can be useful for the study of rat globin chain synthesis during fetal development, because it separates the globin chains in the three groups of globins, namely alpha, beta and embryonic chains which are important in the switch occurring in the liver.     

Opposite effects of ACTH and glucocorticoids on adrenal DNA synthesis in vivo.

Administration of ACTH to rapidly growing weanling rats results in an increase of DNA synthesis in adrenal and a decrease in liver. Dexamethasone administration decreases both adrenal and liver DNA synthesis. When both hormones were administered to the same animals, the liver DNA synthesis was similar to that observed with dexamethasone alone, but the DNA synthesis in adrenal was lower than that obtained with ACTH alone, yet higher than that observed with dexamethasone. The plasma levels of corticosterone were similar in animals treated with ACTH or with ACTH plus dexamethasone. Aminoglutethimide stimulated adrenal DNA synthesis, but less than ACTH. This substance overcame partially the inhibitory effects of dexamethasone on liver DNA synthesis but did not in adrenal. When both ACTH and aminoglutethimide were given simultaneously, adrenal DNA synthesis was higher than that observed with each substance alone. In all experiments in which adrenal cytosol DNA polymerase was studied, the activity varied in the same direction as DNA synthesis. These results indicate opposing effects of ACTH and glucocorticoids on adrenal DNA synthesis. The finding of a glucocorticoid effect on the adrenal is supported by the demonstration of a glucocorticoid specific binding protein in adrenal cytosol. Cycloheximide blocks the stimulatory action of ACTH on both steroidogenesis and DNA synthesis. Actinomycin D, as well as dexamethasone, blocks only the DNA synthesis-promoting action of ACTH. This latter result suggests some differences in the metabolic pathways by which ACTH controls steroidogenesis and growth in the adrenal cell.     

Biosynthesis of the low molecular weight carrier protein for insulin-like growth factors in rat liver and fibroblasts

Biosynthesis of the low mol wt (Mr) carrier protein for insulin-like growth factors (IGFs) was studied in the BRL-3A rat liver cell line, rat embryo fibroblasts (REFs), and fetal rat liver by biosynthetic labeling of intact cells and cell-free translation of extracted RNA. [35S]Cysteine-labeled carrier protein precursors were immunoprecipitated using antibodies raised to the approximately 33,000 Mr carrier protein from BRL-3A cells that recognize the IGF carrier protein present in fetal and neonatal rat serum, but not in adult rat serum. The IGF carrier protein is synthesized as a 35,000 Mr precursor in a reticulocyte lysate translation system directed by RNA from BRL-3A cells or REFs. Supplementation of the translation incubation with microsomal membranes decreases the size of the precursor to 33,000 Mr, presumably by removal of a signal peptide. In continuous labeling or pulse-chase experiments of intact BRL-3A cells or REFs, the 33,000 Mr protein is labeled within 10 min intracellularly, appears in the medium after 40 min, and persists in the medium for 24 h without a change in size. The intracellular carrier protein was biosynthetically labeled in BRL-3A cells with [3H]leucine, [3H]phenylalanine, [3H]arginine, or [35S]cysteine and purified, and its NH2-terminal amino acid sequence was determined. Eleven of 34 residues were identified and correspond to those of mature unlabeled carrier protein purified from conditioned medium, indicating that after removal of the signal peptide, the carrier protein undergoes no detectable further processing at its NH2-terminus. These results establish that although they are regulated coordinately, IGF-II and the fetal IGF carrier protein are synthesized as separate proteins. Finally, RNA extracted from fetal, but not adult, rat liver directs the synthesis of the 35,000 Mr carrier protein precursor, suggesting that the developmental regulation of the carrier protein may occur at the level of RNA abundance.     

Ontogeny of corticosteroid-binding globulin biosynthesis in the rat

In plasma, glucocorticoids are transported by corticosteroid-binding globulin (CBG), which is synthesized primarily in the liver. Plasma levels of maternal and fetal CBG fluctuate during gestation, and this may be due to changes in the biosynthesis and/or clearance of the protein. We have, therefore, studied the ontogeny of CBG biosynthesis in the rat by using a solution hybridization assay to measure hepatic CBG mRNA levels. The results indicate that the concentration of CBG mRNA is exceptionally high in 15-day-old fetal livers (55.1 pg CBG mRNA/micrograms total RNA), but declines to very low levels at birth (day 21). During the same period, CBG mRNA levels in maternal livers remained relatively constant (18.9-23.1 pg CBG mRNA/micrograms total RNA). Hepatic CBG mRNA levels were barely detectable 1 week after birth, and a sex difference was apparent by 2 weeks of age, with higher levels in female livers. Although adult CBG mRNA levels were attained by 3 weeks of age, serum CBG concentrations did not reach adult values for an additional 3 weeks. To determine whether age-related differences in the clearance of CBG are responsible for this, CBG from infant or adult animals was radiolabeled and administered iv to infant and adult rats. When this was done, the half-life of CBG in infants (approximately 6.9 h) was consistently less than that in adults (approximately 14.5 h) regardless of the source of the labeled CBG, and we conclude that variations in CBG biosynthesis and clearance may influence glucocorticoid action during fetal and postnatal development.     

Insulin-like growth factor (IGF) binding proteins modulate the glucocorticoid-dependent biological effects of IGF-II in cultured fetal rat hepatocytes

The role of insulin-like growth factor binding proteins (IGFBPs) in regulation by IGF-II of glycogenesis and DNA synthesis was investigated in hepatocytes isolated from fetal rat livers at days 15 and 18 of gestation and grown in the presence or absence of cortisol. IGFBP-1 was clearly revealed by Western ligand blot and immunoblot analysis of IGFBPs secreted into conditioned media. Its production and cellular messenger RNA (mRNA) were positively regulated by cortisol and increased in older cells. In the absence of IGFBP (fresh medium), glycogenesis, and DNA synthesis were stimulated by IGF-II and insulin. In each case, cortisol enhanced this stimulation. In the presence of IGFBPs (cell-conditioned media), IGF-II stimulation of DNA synthesis and to a lesser extent glycogenesis was inhibited. The degree of inhibition was directly related to IGFBP-1 production. IGFBPs had no effect on stimulation of glycogenesis and DNA synthesis by des(1-6)IGF-II, a structural analog of IGF-II that does not bind to IGFBPs. Insulin, whose biological effects were not modified by conditioned media, inhibited IGFBP-1 production. Comparison of the dose dependence of the two bioactivities showed that DNA synthesis was more sensitive to IGF-II than glycogenesis. Our results suggest that in the case of DNA synthesis the effects of IGF-II are mediated via the IGF-I receptor and those of insulin via the insulin receptor, whereas in the case of glycogenesis both are mediated via the insulin receptor. In conclusion, IGF-II and insulin stimulation of glycogenesis and DNA synthesis in cultured fetal hepatocytes depends on the presence of glucocorticoid and the stage of development. IGF-II action is negatively regulated by IGFBP-1 whose synthesis increases in the presence of glucocorticoids.     

Nuclear-cytoplasmic interactions affecting DNA synthesis during induced cardiac muscle growth in the rat

Nuclear-cytoplasmic interactions affecting DNA synthesis during induced cardiac muscle growth were examined in 29 to 46 day old rats. DNA synthesis was examined in vitro using isolated nuclei from rat heart and adult X. laevis spleen. Cytoplasmic extract (CE) was obtained from a 105 000 g supernatant of rat heart and fetal liver homogenates. To measure DNA synthesis we utilised DNA within the isolated quiescent nucleus as the template and measured the effect of CE on the incorporation of 3H-TTP into an acid precipitable product. In a homologous system of rat heart nuclei from weanling rats and CE from cardiac muscle undergoing induced growth, no stimulation of 3H-TTP incorporation was observed. Cardiac muscle CE however, did possess stimulatory factor(s) since quiescent X. laevis nuclei could be stimulated with the rat heart CE. Furthermore, CE from hearts undergoing induced growth had greater activity than extract from control hearts. While cardiac muscle nuclei were not stimulated by heart CE, they showed substantial stimulation by CE from fetal rat liver, which contains a large population of proliferating cells. Stimulation by fetal rat liver was greater with nuclei obtained from hearts undergoing induced growth than from control hearts. Stimulatory factor(s) in CE was distinct from DNA polymerase-alpha activity, as shown by separation of the two activities on a 5 to 15% glycerol gradient.     

Differentiation-arrested rat fetal lung in primary monolayer cell culture. II. Dexamethasone, triiodothyronine, and insulin effects on different gestational age cultures

Differentiation-arrested and hormone-depleted monolayer cultures were developed from rat fetal lungs of 18-, 19-, 20-, and 22-days gestation. Incorporation of [3H]-choline into saturated phosphatidylcholine increased, whereas the rate of cell division decreased, with advancing gestational age. Both functions were modified by physiological concentrations of glucocorticoids, thyroid hormones, and insulin. Dexamethasone (0.055-5.5 nM) increased [3H]-choline incorporation into total saturated phosphatidylcholine in immature cultures only, but caused secretion in mature (day-22) cultures. Triiodothyronine (0.055-5.5 nM) increased [3H]-choline incorporation into total and secreted saturated phosphatidylcholine at all gestational ages. Insulin (5-50 microU/ml) inhibition of [3H]-choline incorporation into saturated phosphatidylcholine was evident only in mature cultures. Dexamethasone (0.55 nM), triiodothyronine (5.5 nM), and insulin (50 microU/ml) also had gestation-dependent effects on cell division.     

Increase in nuclear thyroid and glucocorticoid receptors and growth hormone production during the deoxyribonucleic acid synthesis phase of the cell growth cycle

We have previously described a significant increase in cellular DNA and nuclear T3 receptor concentration in S phase cultures of GC cells, a rat pituitary cell line that produces GH. We have now measured GH production and some aspects of GH regulation in asynchronous cells and in cultures that were synchronized in early S phase by 25-h treatment with 2 mM thymidine (dT). Cellular DNA and both cellular and medium GH increased significantly at the end of dT treatment and for 3-6 h after removal of dT (S phase). Pulse-labeling experiments with [3H]leucine followed by specific immunoprecipitation of rat GH suggested that an S phase-associated 87% increase in GH could be attributed predominantly to a 101% increase in the GH synthesis rate. The relative GH synthesis rate (GH synthesis divided by total protein synthesis) increased from 0.85% to 1.70% during the S phase. Since cortisol (115 nM) augmented GH production in both asynchronous and S phase cultures, studies of glucocorticoid receptor concentration were also carried out. In comparison with asynchronous cultures, a significant increase in glucocorticoid receptor was identified in the nuclear fraction but not in the cytosol of cultures synchronized in the S phase. Studies with cells grown in media which were selectively depleted or repleted with T3 and/or cortisol and experiments with the glucocorticoid antagonist 17 alpha-methyltestosterone suggested that T3 was primarily responsible for augmented GH during the S phase and that cortisol modulated the amplitude of this response. Our studies suggest that increases in DNA, nuclear receptors for T3 and glucocorticoids, and GH synthesis occur in GC cell cultures synchronized in the S phase. We conclude that the GH production rate observed in asynchronous cultures may be an integrated value for all phases of the cell cycle, each of which may have a distinctive rate of hormone synthesis.