Beta-adrenergic receptors and cAMP response increase during explant culture of human fetal lung: partial inhibition by dexamethasone.

We studied beta-adrenergic receptors and responses in human fetal lung (15-25 wk gestation) maintained in explant culture with and without added dexamethasone. To determine beta-adrenergic receptor concentration, we performed radioligand binding assays with [125I]-iodocyanopindolol. We also examined the ability of isoproterenol to stimulate cAMP generation as a measure of response to beta-adrenergic receptor occupancy. In control cultures, beta-receptor concentration increased significantly from d 0 to 3 of culture and thereafter remained stable. The kd (approximately 24 pM) of [125I]-iodocyanopindolol did not change with time in culture. The ability of isoproterenol to stimulate cAMP generation over basal levels increased in controls throughout the 5 d in explant culture. Addition of dexamethasone (10 nM) to the culture medium partially blocked the increase in beta-receptor concentration and decreased both cAMP content and generation (basal and stimulated) in a dose-dependent manner (median effective concentration approximately 1 nM). In these same explants, dexamethasone increased the activity of fatty acid synthetase, an enzyme important in surfactant synthesis, more than 2-fold. Our results indicate that beta-adrenergic receptors and isoproterenol stimulation of cAMP generation increase spontaneously in human fetal lung grown in explant culture. Dexamethasone, which accelerates other aspects of human lung development in vitro, decreases beta-adrenergic receptor concentration and inhibits beta-adrenergic responses.

Isolation and characterization of differentiated alveolar type II cells from fetal human lung.

A method has been developed for isolating differentiated type II cells from human lung of 18-24-week gestation. The procedure involves an initial 4-day culture of lung explants in the presence of dexamethasone (10 nM) and triiodothyronine (2 nM). Type II cells (and fibroblasts) are isolated by trypsin digestion of the explants, two differential adherence steps and incubation overnight in primary culture. This method provides a high yield of type II cells ((50 +/- 15) X 10(6) cells/g wet weight of explant) with a purity of 85 +/- 5% in 16 experiments. The type II cells contain numerous perinuclear granules which stain darkly with toluidine blue and Papanicolaou stain; electron microscopy showed these inclusions to be lamellar bodies with tightly stacked, well defined lamellae. Type II cells, but not fibroblasts, were positive by immunofluorescence histology for surfactant apoprotein and binding of Maclura pomifera lectin which binds to the surface of type II but not type I cells in vivo. The rate of both [3H]acetate and [3H]choline incorporation into phosphatidylcholine (PC) was several-fold greater in type II cells than fibroblasts; the saturation of PC was 36.2 and 25.9%, respectively. Release of saturated PC was stimulated by terbutaline, the ionophore A23187, and tetradecanoyl phorbol acetate in type II cells but not fibroblasts. We conclude that differentiated type II cells can be isolated in relatively high yield and purity from hormone-treated explants of fetal human lung.

Thyroid hormone stimulation of phosphatidylcholine synthesis in cultured fetal rabbit lung.

To investigate the mechanism of thyroid hormone action on pulmonary surfactant synthesis, we characterized the effect of triiodothyronine on phosphatidylcholine synthesis in cultured fetal rabbit lung. Since glucocorticoids stimulate surfactant synthesis and reduce the incidence of Respiratory Distress Syndrome in premature infants, we also examined the interaction of triiodothyronine and dexamethasone. The rate of choline incorporation into phosphatidylcholine was determined in organ cultures of rabbit lung maintained in serum-free Waymouth's medium. In 23-d lung cultured for 72 h, the increase in choline incorporation with triiodothyronine alone, dexamethasone alone, and triiodothyronine plus dexamethasone was 50, 62, and 161%, respectively. Both triiodothyronine and dexamethasone also increased incorporation rates with glucose, glycerol, and acetate as precursors, and stimulation with triiodothyronine plus dexamethasone was at least additive. Dexamethasone, but not triiodothyronine, affected distribution of radioactivity from [3H] acetate among phospholipids. Stimulation was first detected 8-12 h after addition of triiodothyronine, and then increased in a linear fashion. With triiodothyronine plus dexamethasone, stimulation was maximal at 48-72 h, and was supra-additive at all times. Exposure of cultured lung to dexamethasone enhanced the subsequent response to triiodothyronine, but not vice versa. When triiodothyronine was removed from cultures, there was no further stimulation and the triiodothyronine effect was partially reversed within 24 h. Half-maximal stimulation of choline incorporation occurred at a triiodothyronine concentration (0.10 nM) very similar to the dissociation constant for triiodothyronine binding to nuclear receptor (0.11 nM). The relative potencies of thyroid hormone analogs for nuclear binding and stimulation of phosphatidylcholine synthesis were also similar: triiodothyroacetic acid greater than triiodothyronine-proprionic acid greater than L-triiodothyronine approximately D-triiodothyronine much greater than thyroxine much greater than 3,5-diethyl-3'-isopropyl-DL-thyronine approximately 3,5-dimethyl-3'-isopropyl-L-thyronine approximately reverse triiodothyronine. The effect of triiodothyronine was blocked by the presence of either actinomycin D or cycloheximide, inhibitors of ribonucleic acid and protein synthesis, respectively. We conclude that triiodothyronine stimulates phosphatidylcholine synthesis by a process involving nuclear receptors and de novo ribonucleic acid and protein synthesis. These findings support the concept that endogenous triiodothyronine has a physiologic role in lung maturation and suggest that a combined antenatal therapy with thyroid hormone and glucocorticoid may be useful for prevention of Respiratory Distress Syndrome in the premature infant.

Corticosteroid binding by fetal rat and rabbit lung in organ culture.

To further characterize glucocorticoid action in fetal lung cells, we investigated corticosteroid metabolism and binding in explants of fetal rat and rabbit lung. Cortisone (E) was concerted to cortisol (F) and bound by receptor with a time course only somewhat slower than for F. Production of F (0.243 pmol/min/mg DNA) was the same in male and female rabbits and was not affected by prior exposure to glucocorticoid in utero or in culture. The t 1/2 for dissociation of nuclear-bound [3H]F was 84 min on changing the culture medium and 21 min on addition of excess non-labeled dexamethasone. Dissociation of [3H]dexamethasone was approx 5-fold slower by both procedures. The KD for nuclear binding of dexamethasone, F, E, and corticosterone in rabbit lung were 0.7, 7.3, 6.8 and 70.6 nM, respectively. In rat lung, the KD for dexamethasone was 6.8 nM. The concentrations of dexamethasone and F required for half-maximal stimulation of phosphatidylcholine synthesis were similar to the KD values. Dexamethasone binding capacity (sites/mg DNA) increased with age in both rat (+103% increase from day 16 to 22) and rabbit (+47% between day 23 and 30). Receptor concentration was the same in both sexes, and there were no developmental changes in non-specific binding, nuclear:cytoplasmic distribution, or KD. In 27-day rabbit fetuses, the rate of choline incorporation was higher in lungs with greater binding capacity. We conclude that (1) E is rapidly converted to F in rabbit lung to become an active glucocorticoid, whereas corticosterone probably has little physiologic activity, (2) there is a species difference in the affinity of dexamethasone binding which is reflected in responsiveness (3) there is no difference between sexes in E conversion, receptor capacity, or phosphatidylcholine synthesis, and (4) the concentration of binding sites per lung cell increases during fetal development. We suggest that developmental increases in both F production and receptor may be important factors in the expression of endogenous glucocorticoid effects.