Search

CTP:phosphocholine cytidylyltransferase catalyses a rate regulatory step in the de novo synthesis of surfactant phosphatidylcholine in alveolar type II cells. To investigate if cytidylyltransferase can be regulated by cAMP-dependent protein kinase, we first studied the ontogeny of cAMP-dependent protein kinase activity in type II cells of fetal rat lung. Total cAMP-dependent protein kinase activity, measured in the presence of 10 microM cAMP, as well as endogenous activity, measured without cAMP, increased with advancing gestation. Phosphocholine cytidylyltransferase activity showed a similar developmental profile. This temporal relationship between cAMP-dependent protein kinase and cytidylyltransferase supports a potential role for cAMP-dependent protein kinase in regulating cytidylyltransferase phosphorylation. Cytidylyltransferase purified from adult rat lung was, indeed, phosphorylated in vitro by cAMP-dependent protein kinase. Despite the phosphorylation, however, no change in cytidylyltransferase activity was noted. Pre-incubation of fetal type II cell cytosol with ATP and Mg2+ did not affect cytidylyltransferase activity. Addition of either cAMP, dibutyryl-cAMP or the catalytic subunit of cAMP-dependent protein kinase to the pre-incubation medium did also not alter cytidylyltransferase activity. Furthermore, neither cAMP-dependent protein kinase inhibitor peptide, nor H8, a cyclic nucleo-dependent protein kinase inhibitor, affected cytidylyltransferase activity in fetal type II cell cytosol. Treatment of intact fetal type II cells with either cAMP, dibutyryl-cAMP or 8-[4-chlorophenylthio]cAMP activated cAMP-dependent protein kinase activity but did not alter cytidylyltransferase activity. We conclude that the increase in cytidylyltransferase activity in fetal type II cells at late gestation is not regulated by the developmental activation of cAMP-dependent protein kinase.

Phosphatidylcholine synthesis increases in fetal rat type II cells during late gestation, as demonstrated by an increased incorporation of radiolabeled palmitate, glycerol, acetate, and choline into phosphatidylcholine. However, the percentage of phosphatidylcholine present in the saturated form remains essentially constant. The developmental profile of the enzymes of the CDP-choline pathway suggests that CTP:choline-phosphate cytidylyltransferase catalyses a rate regulatory step in de novo phosphatidylcholine synthesis by fetal type II cells. When cytidylyltransferase activity is assayed in different subcellular fractions, the greatest increase, as a function of development, is found in microsomes. This developmental increase is accompanied by a shift in subcellular distribution of cytidylyltransferase activity from cytosol to microsomes in fetal type II cells during late gestation. This shift is evident even when cytidylyltransferase activity is assayed in the presence of 0.5 mM phosphatidylcholine/oleic acid (1/1 molar ratio) vesicles. We speculate that either a subcellular translocation of CTP:phosphocholine cytidylyltransferase from cytosol to microsomes or an increase in cytidylyltransferase gene expression are responsible for the developmental increase of de novo phosphatidylcholine synthesis by fetal type II cells.

The developmental profiles of glycogen and surfactant-associated saturated phosphatidylcholine were investigated in type II cells isolated from fetal rat lung. Incorporation of radiolabeled glucose into glycogen and type II cell unlabeled glycogen content decreased as a function of gestational age. Conversely, an increase was noted in radioactive choline incorporation into saturated phosphatidylcholine and in the content of unlabeled saturated phosphatidylcholine as a function of gestational age. Type II cells from days 19 and 21 of gestation were also studied by electron microscopy. Temporal relationships similar to those noted biochemically were observed by morphometric analysis. A decrease in glycogen content and an increase in lamellar bodies (the storage organelles for the pulmonary surfactant) were noted as gestation progressed. These studies biochemically and morphologically demonstrate a temporal relationship between glycogen degradation and saturated phosphatidylcholine synthesis in type II cells isolated from fetal rat lung. These findings provide further support for the use of such type II cell preparations for studies of development at the cellular level.

Fetal lung cells from 28 day gestation rabbits cultured in the presence of cortisol (5.5 times 10 - minus 6M) or dexamethasone (5.5 times 10- minus 8M) incorporated [3-H] choline into lecithin to a significantly greater extent than did control cultures. 11-Deoxycortisol, 21-deoxycortisol and 11beta-hydroxyprogesterone, at a concentration of 5.5 times 10- minus 5 M, had no effect on lecithin synthesis. However, when lung cells were simultaneously exposed to these steroids and to cortisol at the concentrations quoted, [3-H] choline incorporation was reduced to control values. Cortisone (5.5 times 10- minus M) also enhanced lecithin synthesis, the activity of the steroid likely being related to the capacity of the lung cells to convert cortisone to cortisol. This hypothesis was supported by the observations that 11-ketoprogesterone (1.3 times 10- minus 5M), which totally inhibited the conversion of cortisone to cortisol and which had no effect of its own on [3-H] choline incorporation, inhibited the effect of cortisone on lecithin synthesis but not that of cortisol. These data support the view that glucocorticoids affect lung cell maturation in a manner comparable to the interaction of other steroid hormones with their target tissues. The capacity of the fetal lung to convert cortisone to cortisol may be physiologically significant in light of the high concentration of 11-oxo-steroids in the fetal circulation throughout pregnancy.

The effect of cortisol (5.5 muM) on primary monolayer cultures of trypsin-dispersed lung cells from rabbit fetuses of 20-28 days gestation was monitored with respect to (a) cellular growth as determined by DNA content after 72 h, at which time all cultures were in the exponential phase of growth, and (b) cellular maturation as reflected by the incorporation of [(14)C]-palmitate into saturated lecithin and its release into the culture medium. Cortisol significantly increased growth in cultures prepared from 20 day (control: 59.8+/-8.9 nmol DNA/flask; cortisol: 118.7+/-15.7, P < 0.001) and 22 day (control: 69.2+/-17.2; cortisol: 106.7+/-13.3, P < 0.001) fetuses but had no effect on the growth of cells from 24 or 26 day fetuses. At 28 days, the effect was reversed, cortisol reducing growth by a factor of two (control: 42.0+/-8.5; cortisol: 19.3+/-4.0, P < 0.001). Incorporation of palmitate into lecithin was expressed as picomoles incorporated per micromole DNA per flask, thus correcting for differences in the number of cells. Cortisol had no effect on palmitate incorporation until day 26, at which time it caused a slight increase (control: 51.2+/-5.5: cortisol: 72.8+/-16.2, P < 0.01) which became very striking by day 28 (control: 19.7+/-3.1; cortisol; 286.8+/-47.0, P < 0.001). The proportion of recovered radiolabeled lecithin that was disaturated rose with gestational age from 72% at 20 days to 98% at 28 days. Saturated lecithin made up over 90% at the two gestational ages (26 and 28 days) where cortisol increased palmitate incorporation. In contrast, cortisol had no effect on the incorporation of palmitate into sphingomyelin at any of the gestational ages studied.The results suggest that cortisol may increase fetal pulmonary cellular growth in early gestation while enhancing maturation and slowing growth as term approaches.

Lung and liver glycogen and corticosteroid 11-reductase activity were studied in fetal and neonatal rats. Glycogen content peaked in the lung on day 20 of gestation, while hepatic glycogen content was generally lower and peaked later. Both pre- and postnatally, betamethasone administration resulted in lowered pulmonary and elevated hepatic glycogen. In both lung and liver, corticosteroid 11-reductase activity showed an inverse developmental pattern to glycogen content. Betamethasone elevated pulmonary corticosteroid 11-reductase activity and lowered hepatic activity. In lung and liver, glycogen content and corticosteroid 11-reductase activity are tightly linked during development and following glucocorticoid therapy.

Alveolar type II cells that were isolated from organotypic cultures of fetal rat lung and maintained in primary monolayer culture were analyzed. Morphologically the cells retained many characteristics of type II cells in vivo. Biochemical analysis revealed that the cells contained and synthesized phosphatidylcholine with a high degree of saturation. Furthermore, the cells released saturated phosphatidylcholine and responded to adrenergic stimulation with increased release. The type II cells retained their ability to proliferate and responded to fibroblast-pneumonocyte factor. These data indicate that primary cultures of fetal type II cells will be useful to elucidate mechanisms modulating type II cell proliferation, surfactant synthesis, and surfactant secretion.

The prenatal and postnatal therapeutic management of surfactant insufficiency are reviewed. Prenatal maternal glucocorticoid therapy promotes lung maturation and enhances lung surfactant levels in the neonate, but a minimum of 24 hr treatment is required and the therapy is of limited effectiveness even under optimal conditions. Relatively few women in premature labour are good candidates for glucocorticoid therapy. Research into combinations of glucocorticoids with hormones (e.g. thyroid), and adrenergic agents in progress. The authors are studying the effects of fibroblast-pneumonocyte factor (FPF) on the fetal lung surfactant system. Postnatal therapy with insufflated natural and artificial surfactants has been studied in several centres with varying degrees of success. Currently, the risk:benefit ratios favour attempts to reduce the risks of respiratory distress syndrome (RDS) by both prenatal surfactant induction and postnatal replacement therapy. Greater understanding of the underlying mechanisms should permit the establishment of more satisfactory treatment.

Chronic maternal administration of diazepam resulted in a modification of fetal respiratory-like activity (FRLA) in the sheep. An over-all increase in this activity occurred. This increase was probably due to a rebound effect following periods of acute suppression with each administration of diazepam. Loss of central control in the regulation of FRLA was implied. A complete reversal of a circadian rhythm of FRLA was observed in animals receiving diazepam. These changes have implications with respect to the antenatal monitoring of the fetus and sequelae in the newborn infant.