Diane W Dynia

Glucocorticoid-Thyroid Hormone Interactions in Fetal Rat Lung

Summary: Previous studies have shown that triiodothyronine (T3) enhances the effect of dexamethasone on phosphatidylcholine (PC) synthesis in organ cultures of fetal rat lung. The aim of this study was to investigate whether similar interactions occurred in vivo and to explore possible mechanisms for this phenomenon.Injection of 7.0 mg/kg T3 into pregnant rats on d 18 and 19 of gestation resulted in a mean fetal serum T3 level of 2380 ng/dl on d 20 (control, 84 ng/dl) and in maximal (34%) stimulation of choline incorporation into PC. Injection of 1.0 mg/kg betamethasone using the same protocol as for T3 resulted in maximal stimulation of 33% and administration of both hormones together produced a 69% increase, an additive affect. The percentage of PC that was disaturated was increased with betamethasone, but decreased with T3. Betamethasone treatment resulted in an increase in the whole lung disaturated PC content, but treatment with T3 did not. Betamethasone administration also increased fetal serum T3 levels, but T3 injection did not produce elevated fetal serum corticosterone levels.Injection of T3 in vivo, or exposure of explants of 18-d fetal lung to 100 nm T3 for up to 48 h did not result in an increase in cytoplasmic glucocorticoid binding or nuclear translocation of the receptor steroid complex. Exposure of explants to glucocorticoid or T3 in vivo or in culture (dexamethasone, 100 nM and T3, 100 nM; for 48 h) resulted in a significant increase in the activity of cholinephosphate cytidylyltransferase, an enzyme in the choline incorporation pathway of PC synthesis. Exposure of explants to the combination of hormones resulted in stimulation that was equal to the sum of that produced by the single hormones but was not statistically significantly different from the glucocorticoid effect. The activities of other enzymes of phospholipid synthesis were not increased by exposure to either hormone, in vivo or in vitro.The additive effects of T3 and glucocorticoid with regard to choline incorporation into PC in fetal rat lung suggest that combined hormone therapy may be useful for the prevention of respiratory distress syndrome in humans. Further animal studies are required, however, before clinical use can be considered.

Influence of epidermal growth factor on fetal rat lung development in vitro.

ABSTRACT. Epidermal growth factor (EGF) has been shown to enhance cell multiplication or differentiation in a number of developing tissues. We have examined the effects of this growth factor on the biochemical development of explants of fetal rat lung, cultured in serum-free medium for 48 h. EGF enhanced the rate of choline incorporation into phosphatidylcholine and disaturated phosphatidylcholine in a dose dependent fashion. Half maximal stimulation occurred at a concentration of 1.0 nM, similar to the Kd for EGF binding to rat lung cell membranes. There was also significant stimulation of acetate incorporation into all phospholipids, particularly phosphatidylglycerol (539%), and increased distribution of radioactivity from acetate in this phospholipid fraction. Exposure to EGF stimulated PC synthesis in 18- and 19-day explants (term is 22 days) whereas maximal enhancement of DNA synthesis occurred after this time. This sequence differs from that observed during early embryonic development when EGF initially enhances cell multiplication. An additive interaction with regard to enhancement of PC synthesis was observed with EGF and thyroid hormone, but not EGF and dexamethasone. EGF had no effect on the activity of the enzymes of the choline incorporation pathway of phosphatidylcholine synthesis or on the activity of enzymes involved with acidic phospholipid synthesis. Fetal lung EGF content and EGF binding capacity were not increased by glucocorticoid treatment and similarly glucocorticoid binding capacity was not increased by EGF. These data indicate that EGF enhances fetal rat lung phospholipid synthesis in a dose-dependent manner and suggest that this is a direct effect on the lung tissue mediated by specific receptors.

Glucocorticoid stimulation of choline-phosphate cytidylyltransferase activity in fetal rat lung: receptor-response relationships.

A number of previous studies using in vivo and cultured fetal lung models have shown that the activity of choline-phosphate cytidylyltransferase, the enzyme which catalyzes a rate-limiting reaction in de novo phosphatidylcholine synthesis, is increased by glucocorticoids and other hormones which accelerate fetal lung maturation. To examine the mechanism of this glucocorticoid action further, we examined the effect of dexamethasone on cytidylyltransferase activity in cultured fetal rat lung explants and related it to specific dexamethasone binding. Dexamethasone stimulated cytidylyltransferase activity in the homogenate, microsomal and 105,000 X g supernatant fractions. The hormone did not alter the subcellular distribution of the enzyme, however; the bulk of the activity was in the supernatant fraction in both the control and dexamethasone-treated cultures. The dose-response curves for stimulation of cytidylyltransferase activity in the supernatant fraction and specific nuclear binding of dexamethasone were similar and both plateaued at approx. 20 nM. The EC50 for cytidylyltransferase stimulation was 6.6 nM and the Kd for dexamethasone binding was 6.8 nM. The relative potencies of various steroids for stimulating choline-phosphate cytidylyltransferase and for specific nuclear glucocorticoid binding were the same: dexamethasone greater than cortisol = corticosterone = dihydrocorticosterone greater than progesterone. The stimulation by dexamethasone of cytidylyltransferase activity and of choline incorporation into phosphatidylcholine were both abolished by actinomycin D. These data show that the stimulatory effect of dexamethasone on fetal rat lung choline-phosphate cytidylyltransferase activity is largely on the enzyme in the supernatant fraction and does not involve enzyme translocation to the microsomes as has been reported for cytidylyltransferase activation in some other systems. This effect of dexamethasone is a receptor-mediated process dependent on RNA and protein synthesis.

Culture of differentiated and undifferentiated type II cells from fetal rat lung

We have developed a relatively simple and reproducible method for the isolation and culture of both differentiated and undifferentiated type II cells from fetal rat lung. The technique involves an initial period of explant culture in serum and hormone free medium, followed by enzymatic dissociation of the explants, differential adhesion to remove fibroblasts, incubation of the cell pellet to promote aggregation of the type II cells and monolayer culture of the type II cells. The type II cells form clusters which are surrounded by scattered fibroblasts. When the technique was performed with three differential adhesion steps, cultures contained 86.0 +/- 1.4% type II cells. To obtain a higher degree of purity and greater yield, two differential adhesions followed by gentle trypsinization of the cultures which selectively removes the isolated fibroblasts was performed. This resulted in cultures with 89.4 +/- 1.7% type II cells. The differentiated fetal type II cell cultures were prepared from 19-day fetal rat lungs which were initially maintained in explant culture for 48 h. These differentiated cells demonstrated the characteristic morphologic features of type II cells including lamellar bodies and microvilli. Undifferentiated fetal cells were prepared in a similar manner from 18-day fetal rat lung maintained in explant culture for 24 h. These cells did not contain intracellular osmiophilic granules; the appearance of these granules could, however, be induced by hormones. For this reason they are considered to be pre-type II cells. The viability of the cultured cells was 97%. Both the differentiated and undifferentiated fetal type II cells specifically bound the Maclura pomifera lectin, a type II cell surface marker. The phospholipid profile of the fetal cells was similar to that of adult rat type II cells; the differentiated fetal cells, however, synthesized less phosphatidylcholine than the adult cells did, but more than the undifferentiated fetal cells. The differentiated fetal cells secreted phosphatidylcholine at a basal rate of 0.6% +/- 0.1% during a 90-min incubation. There was dose-dependent stimulation of phosphatidylcholine secretion after exposure to terbutaline. Maximum stimulation (76%) was observed at a concentration of 10 microM. This culture system provides a valuable model for studies of the maturation of the undifferentiated fetal type II cell and surfactant metabolism and secretion in the differentiated fetal type II cell.

Initiation of Fetal Rat Lung Phospholipid and Surfactant-Associated Protein A mRNA Synthesis

ABSTRACT: To determine whether the initiation of fetal lung surfactant phospholipid production and the activation of the gene for the 35-kD surfactant-associated protein are dependent on circulating corticosteroids, we cultured dexamethasone- responsive explants of 15- to 17-d fetal rat lung in medium with 1 % FCS (controls), charcoal-stripped 1% FCS, or a variety of glucocorticoid antagonists. The steroid antagonist RU 486 almost completely abolished specific cytoplasmic and nuclear dexamethasone binding in the explants but had no glucocorticoid-agonist activity. There was a significant increase in disaturated phosphatidylcholine synthesis during 7 d in culture in control explants (78%) and in those cultured with Charcoal-stripped serum (83%), RU 486 (82%), or the other glucocorticoid antagonists—clotrimazole, cortexelone, and 11-ketoprogesterone. Specific mRNA for surfactant-associated protein A was not detectable in preculture 17-d lung tissue, but accumulated to the same extent in cultures with or without RU 486 in the medium. These findings support the view that expression of the genes responsible for the synthesis of the various components of surfactant is not induced by glucocorticoids, but by signals contained within the lung tissue itself. The role of circulating hormones is later acceleration and modulation of surfactant production.

DO EGF AND GLUCOCORTICOIDS ACT AT THE SAME METABOLIC SITES IN FETAL RAT LUNG

EGF enhances the morphological and physiological maturation of fetal lung in vivo and it has been suggested that it mediates T4 action on developing mouse skin. We examined the effects of EGF on the biochemical development of explants of 18 day fetal rat lung, maintained in a serum free organ culture system for 48h, in order to compare the effects of this peptide to those of T3 and dexamethasone (dex). EGF stimulated choline incorporation into phosphatidylcholine (PC) in a dose dependent fashion with half the maximal effect occurring at 1.0±0.1 nM (6 ng/ml). The effect of EGF on choline incorporation decreased with increasing gestational age whereas stimulation of thymidine incorporation into DNA increased, suggesting that EGF accelerates maturation of undifferentiated cells, but enhances multiplication of differentiated cells. EGF and dex significantly increased the distribution of radioactivity from acetate into the phosphatidylglycerol fraction, but T3 did not (PG as a % of total phospholipid: control, 1.8±0.2%; EGF, 4.4±0.4%; dex, 6.1±0.4%; T3, 2.0±0.4%). In mixing experiments using optimal concentrations, EGF (10 nM) produced 35% stimulation of choline incorporation; dex (100 nM), 47%; T3 (100 nM), 34%; EGF + T3, 75%; and EGF + dex, 43%. The fact that EGF + dex did not produce a greater effect than dex alone suggests that these 2 agents, but not T3, act at the same metabolic sites. We speculate that EGF may partially mediate the effects of dex on fetal lung maturation.

270 IS EGF A MEDIATOR OF GLUCOCORTICOID ACTION ON DEVELOPING FETAL LUNG

EGF is believed to enhance fetal lung maturation. We have examined the interactions between EGF and corticosteroids in ex-plants of fetal rat lung in a serum free organ culture system. EGF stimulated the incorporation of choline into phosphatidylcholine (PC) and disaturated PC (DSPC) in a dose dependent fashion with half the maximal effect occurring at 1.03 nM (6.31 ng/ml). There was also a 6 fold increase in acetate incorporation into phosphatidylglycerol. T3 and EGF together had an additive effect on choline incorporation into DSPC, whereas exposure to saturating doses of EGF plus dexamethasone (dex) had no greater effect than did dex alone, suggesting that the 2 agents act at similar metabolic sites.We further explored the interaction between EGF and dex. Dex increased the activity of cholinephosphate cytidylyltransferase, the rate limiting enzyme of PC synthesis, but EGF had no effect on this enzyme. We also examined the influence of dex on EGF binding and separately that of EGF on glucocorticoid binding. EGF had no effect on specific cytoplasmic or nuclear glucocorticoid binding, but exposure to dex resulted in a 2 fold increase in specific EGF binding capacity.One of the ways in which corticosteroids stimulate lung maturation may be by increasing EGF binding capacity with subsequent amplification of EGF action.

Butyrate decreases homeobox gene expression in fetal rat lung in culture.† 256

Homeobox transcription factors influence the expression of multiple genes, thereby regulating patterns of differentiation and cell fate. The Hoxa5, b5 and Hex homeobox genes are actively expressed in fetal rat lung, but little is known of their regulation.