The effect of cortisol on rabbit fetal lung maturation in vitro

Explants of lung tissue from 19-day gestational age fetal rabbits were maintained in organ culture in medium with or without fetal calf serum for 1 to 11 days. Based on the results of biochemical and morphological studies it was apparent that the type II pneumonocyte differentiated in vitro at a time similar to that which occurs with maturation in vivo. The epithelial cells of the presumptive alveoli were undifferentiated at the start of incubation, but within 9 days developed increased amounts of Golgi apparatus and rough endoplasmic reticulum, many microvilli on the luminal surface and numerous lamellar bodies. Secreted lamellar bodies and tubular myelin figures were observed in the lumina of cultured explants. The incorporation of [³H]choline into phosphatidylcholine by lung tissue explants maintained in medium containing 10% fetal calf serum remained relatively constant for 7 days of incubation but thereafter increased two-fold. When explants were maintained in fetal calf serum-containing medium and cortisol (10^?7M) or betamethasone (10^?7M), the incorporation of choline into phosphatidylcholine was two to three times greater than that of explants maintained in serum-containing medium without cortisol. When explants of fetal lung tissue were incubated in the presence of cortisol without fetal calf serum there was no stimulatory effect of cortisol on phosphatidylcholine biosynthesis. Therefore, serum cofactors are necessary for the stimulatory effects of cortisol on fetal lung development. The specific activity of phosphatidate phosphohydrolase (PAPase) increased to very high levels during the culture period. In the presence of serum, cortisol or betamethasone had no effect on the specific activity of phosphatidate phosphohydrolase.     

The phospholipids of lamellar body material from fetal rabbit lung tissue in culture. Unusual phosphatidylcholine fatty acid profile

Tissue pieces of rabbit fetal lung, 23 days gestation, were cultured for 7 days in serum-free medium to obtain lamellar body material for phospholipid analysis. Cultures were maintained in culture medium without serum and (1) with no added hormones (control cultures), (2) with thyroxine (1 x 10(-7) M), (3) with cortisol (1 x 10(-7) M) and (4) with thyroxine plus cortisol (1 x 10(-7) M each). The hormonal response was evaluated by measuring the quantity of lamellar body material isolated from the tissue pieces after the 7-day culture period. Compared to control cultures, more lamellar body material was recovered from cultures treated with cortisol (180% of control) and with thyroxine plus cortisol (250% of control). Cultures treated with thyroxine alone yielded the same amount of lamellar body material as the controls. Hormone treatment produced only minor changes in the glycerophospholipid profile of the lamellar body material. A small but significant increase in the percentage of phosphatidylglycerol and a small but significant decrease in phosphatidylinositol were found in lamellar body material from cultures treated with thyroxine and thyroxine plus cortisol. The disaturated phosphatidylcholine content of the lamellar body material from culture was 28% of the total lamellar body phospholipid and was not affected by hormone treatment. This disaturated phosphatidylcholine content was low compared to the disaturated phosphatidylcholine of lamellar body material from adult lung (46%). The low proportion of disaturated phosphatidylcholine was due to the unusual presence of palmitoleic acid (16:1(cis-9)), which was more than one-fourth of the total fatty acid of the lamellar body phosphatidylcholine. It is possible that an abnormal delta 9 fatty acid desaturation activity was expressed in the lung tissue in vitro, which resulted in the high incorporation of the 16:1 fatty acid into lamellar body phosphatidylcholine.     

Effect of cortisol on human fetal lung in organ culture

Human fetal lung tissue obtained during the second trimester was cultured as organ culture with or without cortisol. The effect of cortisol on the phospholipid metabolism, as related to the appearance of osmiophilic lamellar bodies and the localisation of newly incorporated choline, was studied. In cortisol-treated explants, the concentration of saturated lecithins and the incorporation of (Me-3H)-choline into saturated lecithins increases significantly concomitantly with an increased number of osmiophilic lamellar bodies. The labelled choline is predominantly associated with these bodies. The obtained results indicate that cortisol accelerates the synthesis of pulmonary surfactant in the human fetal lung as early as in the second trimester.     

The cellular mechanism of glucocorticoid acceleration of fetal lung maturation. Fibroblast-pneumonocyte factor stimulates choline-phosphate cytidylyltransferase activity

The cellular mechanism by which glucocorticoids stimulate phosphatidylcholine biosynthesis has been studied in the fetal rat lung in vivo and in cultured fetal rat lung cells of varying levels of complexity. Administration of dexamethasone to pregnant rats at 18 days gestation resulted in a significant increase in saturated phosphatidylcholine content in fetal lung 24 h after injection. Dexamethasone administration increased the activity of fetal lung choline-phosphate cytidylyltransferase by 34%. It had no effect on the activities of fetal lung choline kinase and choline phosphotransferase. Exposure of fetal lung type II cells in organotypic cultures (which contain both type II cells and fibroblasts) to cortisol resulted in a 1.6-fold increase in the incorporation of [Me-3H]choline into saturated phosphatidylcholine. The activities of the enzymes in the choline pathway for the de novo biosynthesis of phosphatidylcholine were not significantly altered except for a 105% increase in choline-phosphate cytidylyltransferase activity. Treatment of monolayer cultures of fetal type II cells with cortisol-conditioned medium from fetal lung fibroblasts resulted in a 1.5-fold increase in saturated phosphatidylcholine production. This effect correlated with a doubling of choline-phosphate cytidylyltransferase activity. Additional evidence that this stimulatory action is mediated by fibroblast-pneumonocyte factor, produced by fetal lung fibroblasts in response to cortisol, was obtained. The factor was partially purified from cortisol-conditioned medium of fetal lung fibroblasts by gel filtration and affinity chromatography. Based on biological activity, a 3000-fold purification was obtained. Stimulation of saturated phosphatidylcholine synthesis in type II cells by fibroblast-pneumonocyte factor was maximal within 60 min of incubation. Pulse-chase experiments indicated that the stimulatory effect was correlated with an increased conversion of choline phosphate into CDP choline. Moreover, the enhanced phosphatidylcholine formation by fetal type II cells in response to fibroblast-pneumonocyte factor was accompanied by decreased levels of cellular choline phosphate. These findings further support the concept that glucocorticoid action on surfactant-associated phosphatidylcholine synthesis occurs ultimately at the level of the alveolar type II cell and involves fibroblast-pneumonocyte factor which stimulates the activity of choline-phosphate cytidylyltransferase.     

Sex-specific differences in rabbit fetal lung maturation in response to epidermal growth factor.

Males and females exhibit different stages of lung development at the same gestation with males lagging behind. We hypothesized that one of the mechanisms responsible for the sex-specific difference in fetal lung maturation is a delay in the onset of epidermal growth factor (EGF) activity in the male fetal lung. EGF influences growth and differentiation during development. We studied the effects of EGF on the incorporation of glycerol into lamellar body disaturated phosphatidylcholine (DSPC) in sex-specific fetal rabbit lung explants prepared at 21 and 24 days gestation (term 31 days). The explants were maintained in Waymouth's media + 10% stripped fetal calf serum with or without EGF (10 ng/ml). The incorporation of [1,3-14C]glycerol into lamellar body DSPC was assessed after 3, 5, or 7 days of culture. Female lung explants prepared at 21 days of gestation had increased incorporation of glycerol into DSPC over time in response to EGF treatment. Male lung explants prepared at 21 days did not respond to EGF treatment. In explants prepared at 24 days gestation, baseline glycerol incorporation into DSPC was higher in female as compared to male fetal lung explants. EGF-responsiveness was also sex-specific in these more mature explants, with the male explants now responding to EGF with a consistent increase in the incorporation of glycerol into lamellar body DSPC. We conclude that one of the mechanisms responsible for the lag in male fetal lung development is a delay in the onset of EGF activity.     

Regulation of the synthesis of the major surfactant apoprotein in fetal rabbit lung tissue

Antibodies directed against the major apoprotein of rabbit lung surfactant, a 29-36-kDa glycoprotein, were used to study changes in the levels of translatable surfactant apoprotein mRNA in rabbit lung tissue during development, as well as the effects of cortisol and cyclic AMP analogues on the levels of surfactant apoprotein and its mRNA in fetal rabbit lung tissue in organ culture. The major surfactant apoprotein and its mRNA were undetectable in lung tissues of 21-day gestational age fetal rabbits. Translatable mRNA specific for the major surfactant apoprotein was first detectable in lung tissues of 26-day fetuses, increased 25-fold on day 28, reached peak levels at day 31, and declined after birth. Incubation of 21-day fetal rabbit lung explants with cortisol in serum-free medium resulted in an increase in the specific content of the 29-36-kDa apoprotein. Cyclic AMP analogues and forskolin, an activator of adenylate cyclase, also caused a marked increase in the accumulation of surfactant apoprotein. When fetal lung explants were incubated with cortisol and dibutyryl cyclic AMP in combination, the specific content of the surfactant apoprotein was increased to levels greater than that of explants treated with either cortisol or dibutyryl cyclic AMP alone. These effects of dibutyryl cyclic AMP and cortisol on surfactant apoprotein accumulation were associated with comparable changes in the levels of translatable surfactant apoprotein mRNA. Thus, we have shown for the first time that the induction of pulmonary surfactant apoprotein synthesis during differentiation in vitro and in vivo is associated with an increase in the level of translatable mRNA and that cortisol and cyclic AMP increase both the accumulation of the major surfactant apoprotein and the corresponding mRNA in fetal rabbit lung tissue in vitro.     

The development of surfactant synthesis in fetal rabbit lung organ culture exhibits a sex dimorphism

Sex differences in amniotic fluid and lung lavage surfactant have been found. Although these studies suggest that augmented fetal surfactant synthesis occurs earlier in the female fetus, there is little direct evidence for a sex difference in fetal surfactant synthesis. We studied the synthesis of surfactant by evaluating the appearance of labelled phospholipids in lamellar bodies recovered from sex-specific organ culture of fetal rabbit lungs. Furthermore, we studied the ability of dexamethasone to stimulate surfactant synthesis in male and female fetal lungs. Organ culture was begun on day 21 of gestation. After 5 days the incorporation of [1,3-14C]glycerol into phosphatidylcholine (PC), disaturated phosphatidylcholine, phosphatidylinositol (PI), and phosphatidylglycerol was studied. Female lungs in organ culture synthesized more disaturated PC per milligram protein than male lungs. In the presence of dexamethasone (10(-8) M) and dihydrotestosterone (10(-8) M) an increased synthesis was noted in the female cultures of PC (270%), disaturated PC (234%), PI (281%), and phosphatidylglycerol (754%). No significant increase in the synthesis of PC or disaturated PC was observed in the male cultures. However in the male cultures smaller increases in the synthesis of PI (193%) and of phosphatidylglycerol (360%) were observed. Overall, dexamethasone stimulated synthesis in females but not in males such that significant differences in the synthesis of all phospholipids were found in the presence of 10(-8) M dexamethasone. These studies show that the synthesis of surfactant in the fetal lung is sexually dimorphic, as is the ability of dexamethasone to regulate synthesis. An understanding of the mechanism which causes these differences may provide important insight into the control of the developmental clock which regulates the orderly progression of development.     

The effect of betamethasone and fetal sex on the synthesis and maturation of lung surfactant phospholipids in rabbits

In the present study we investigated the maturation of the surfactant phospholipids and the role of fetal sex on the effect of betamethasone in male and female rabbit fetuses. Betamethasone was administered to the doe (0.2 mg/kg intramuscularly) 42 and 18 h prior to killing. The fetuses were studied at 27 and 28 days from conception. Results from the alveolar lavage show that male fetuses tended to have a lower disaturated phosphatidylcholine/sphingomyelin ratio and lower levels of phosphatidylinositol. Phosphatidylglycerol was detected in trace amounts. This was apparently due to the high extracellular levels of myo-inositol inhibiting the synthesis of surfactant phosphatidylglycerol while increasing the synthesis of surfactant phosphatidylinositol. Betamethasone increased the recovery of disaturated phosphatidylcholine and phosphatidylinositol from the lung lavage in both sexes. As studied in lung slices in vitro, the betamethasone treatment decreased the incorporation of glucose into phospholipids, including into the fatty acid moiety of disaturated phosphatidylcholine, although it had no significant effect on the incorporation of glucose into the glycerol moiety of disaturated phosphatidylcholine. However, the addition of palmitate increased the incorporation of glucose into the glycerol moiety of disaturated phosphatidylcholine. The betamethasone treatment did not increase the incorporation of [1-14C]pyruvate into disaturated phosphatidylcholine. Following betamethasone administration, the availability of fatty acids may become rate-limiting for the synthesis of surfactant phospholipids. Betamethasone increased the activities of phosphatidic acid phosphohydrolase and phosphatidate cytidyltransferase in a fraction of microsomal membranes. The present evidence suggests that the glucocorticoid-induced lung maturation and the maturation of the normal lung are associated with an increase in the activity of the enzymes which are involved in metabolizing phosphatidic acid to neutral and acidic surfactant secretion of the male fetus was not explained by possible sex-related differences in the biosynthesis of the phospholipids.     

The effect of human urogastrone on lung phospholipids in fetal rabbits

Previous in vivo studies have demonstrated that mouse epidermal growth factor (EGF) can enhance fetal lung maturation. We have examined the effect of urogastrone, the human equivalent of mouse EGF and a related growth factor, on the phospholipid profile of fetal rabbit lung lavage and its action on fetal rabbit Type II pneumocytes in culture. Urogastrone (1 or 8 micrograms) given i.p. to fetal rabbits on day 25 of gestation resulted in increased total phospholipid, phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine contents, increased phosphatidylinositol and phosphatidylethanolamine as a proportion of phospholipid and decreased sphingomyelin as a proportion of phospholipid in lung lavages on day 28. These changes were unaccompanied by alterations in body weight or lung weight, DNA or protein concentrations. Urogastrone (16 micrograms) resulted in increased fetal deaths. Phospholipid profiles on day 27 were unchanged after fetal administration of urogastrone (1 microgram) on day 25. Urogastrone (0.01 and 0.1 ng/ml) added to fetal rabbit Type II pneumocytes in culture for 24 h enhanced the incorporation of radiolabelled choline and thymidine into phosphatidylcholine and DNA respectively. These findings indicate that human urogastrone can alter the phospholipid composition of the rabbit lung in a similar manner to that which occurs during maturation of the lung surfactant system in late pregnancy. This effect can be achieved, at least in part, by a direct action on Type II pneumocytes.     

Isolation of alveolar type II cells from fetal rat lung by differential adherence in monolayer culture

Type II alveolar epithelial cells were isolated from fetal rat lung by differential adherence in monolayer culture. The preparation had a high degree of purity, as assessed by phase contrast microscopy and immunocytochemistry. Purity, based on reactivity with specific anti-adult lung serum (SAALS), which recognizes only type II cells, was 91% for cells isolated from 19-day fetal lungs and 79% for cells isolated from 21-day fetal lungs. The lower purity of type II cells in cultures derived from 1-day postnatal rat lungs (51% cells reactive with SAALS) is probably due to a lower tendency of the type II cells from neonatal rats to adhere to culture dishes than of type II cells from fetal rats. Type II cells isolated from 21-day fetal lungs contained a higher percentage phosphatidylglycerol and incorporated [Me-3H]choline faster into phosphatidylcholine (PC) than type II cells isolated from 19-day fetal lungs. Moreover, in cell preparations derived from lungs at fetal day 21, a higher percentage of epithelial cells contained lamellar bodies than in preparations derived from lungs at fetal day 19. The observation of these differences in the stage of maturation indicates that these differences, which are typical features of the original material, are not obliterated by differentiation during the culture. Type II cells isolated according to the present procedure were capable of synthesizing PC with a high percentage of the disaturated species. This method for the isolation of fetal type II cells may be a useful tool in studies concerning surfactant synthesis and its regulation in the fetal lung.     

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.     

A comparison of the specificity of phosphatidylcholine synthesis by human fetal lung maintained in either organ or organotypic culture.

Human fetal lung (14-18 weeks gestation) was maintained in either organ or organotypic culture. By 4 days in organ culture or 14 days in organotypic culture, epithelial cells within both culture systems exhibited well-developed apical microvilli and possessed numerous intracellular lamellar bodies characteristic of surfactant phospholipid stores. However, analysis of the pattern of synthesis of individual molecular species of phosphatidylcholine by [14C]choline incorporation and reversed-phase h.p.l.c. showed that this apparent maturation was not paralleled by an increased synthesis of the dipalmitoyl species in either culture system. By contrast, the fractional synthesis of dipalmitoyl phosphatidylcholine, expressed as a percentage of total [14C]choline incorporation, decreased with time in both organ and organotypic culture. Moreover, these fractions were not significantly different from those measured in parallel monolayer cultures of mixed human fetal lung cells that displayed mainly fibroblast morphology. These results suggest that the synthesis pattern of phosphatidylcholine species by lung cells in culture is determined principally by their incubation conditions and not by their state of apparent maturation.     

Regulation of phospholipid synthesis by intracellular phospholipases in fetal rabbit type II pneumocytes

Exposure of fetal type II pneumocytes to phospholipase A2 inhibitors led to significantly reduced choline uptake and decreased synthesis of total and disaturated phosphatidylcholines from both [methyl-14C]choline and [9,10(n)-3H]palmitate precursors. The percentage of the total synthesized phosphatidylcholine recovered as disaturated phosphatidylcholine was increased when compared to that in control cultures, suggesting that unsaturated phosphatidylcholine synthesis was reduced to a greater extent than that of the disaturated species. Synthesis of sphingomyelin and phosphatidylethanolamine from labeled palmitate was also reduced, whereas that of phosphatidylinositol and phosphatidylglycerol was significantly increased. Addition of phospholipase C resulted in increased synthesis of phosphatidylcholine from both labeled precursors; no significant changes were found in synthesis of most of the other 3H-labeled lipids. Added phospholipase A2 did not lead to any changes in either choline or palmitate incorporation. However, when melittin (a phospholipase A2 activator) was added to the cultures, greater incorporation of both palmitate and choline was observed, along with a significant increase in the percentage of total cellular radioactivity in 14C-labeled lipids, indicating also stimulation of phosphatidylcholine synthesis. A marked increase in CTP: phosphorylcholine cytidylyltransferase activity was found after treatment of the cultures with phospholipase C. Exposure to quinacrine also increased the activity of this enzyme. Addition of phospholipase C and melittin to prelabeled pneumocyte cultures accelerated degradation of cell phospholipids and the release of free fatty acids as the main degradation products. These findings suggest that intracellular phospholipases are regulators of synthesis of surfactant phospholipids in fetal type II pneumocytes, and that activation or inhibition of these phospholipases could represent a mechanism through which hormones and pharmacological agents modify surfactant and other phospholipid synthesis.     

Role of lamellar inclusions in surfactant production: studies on phospholipid composition and biosynthesis in rat and rabbit lung subcellular fractions.

Lamellar inclusion bodies in the type II alveolar epithelial cell are believed to be involved in pulmonary surfactant production. However, it is not clear whether their role is that of synthesis, storage, or secretion. We have examined the phospholipid composition and fatty acid content of rabbit lung wash, lamellar bodies, mitochondria, and microsomes. Phosphatidylcholine and phosphatidylglycerol, the surface-active components of pulmonary surfactant, accounted for over 80% of the total phospholipid in lung wash and lamellar bodies but for only about 50% in mitochondria and microsomes. Phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, and sphingomyelin accounted for over 40% of the total in mitochondria and microsomes but for only 6% in lung wash and 15% in lamellar bodies. The fatty acid composition of lamellar body phosphatidylcholine was similar to that of lung wash, but different from that of mitochondria and microsomes, in containing palmitic acid as a major component with little stearic acid and few fatty acids of chain length greater than 18 carbon atoms. The biosynthesis of phosphatidylcholine and phosphatidylglycerol was examined in the mitochondrial, microsomal, and lamellar body fractions from rat lung. Cholinephosphotransferase was largely microsomal. The activity in the lamellar body fraction could be attributed to microsomal contamination. The activity of glycerolphosphate phosphatidyltransferase, however, was high in the lamellar body fraction, although it was highest in the mitochondria and was also active in the microsomes. These data suggest that the lamellar bodies are involved both in the storage of the lipid components of surfactant and in the synthesis of at least one of those components, phosphatidylglycerol.     

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.     

Studies on pulmonary surfactant. Effects of cortisol administration to fetal rabbits on lung phospholipid content, composition and biosynthesis.

Corticosteroids are known to accelerate maturation of the fetal lung and production of surfactant. We examined the effect of cortisol administration to fetal rabbits on the phospholipid content and composition of lung lavage and lung tissue, as well as on the activities of enzymes involved in the synthesis of phosphatidylcholine and phosphatidylglycerol, the major surface-active components of surfactant. Cortisol was administered by intrauterine injection at 25 days' gestation and the fetuses were delivered at 27 days (full term, 31 days). Saline-injected fetuses, littermates of the cortisol-treated as well as non-littermates, were used as controls. The amount of phospholipid in lung lavage from the hormone-treated fetuses was almost double that of the saline-injected controls and was similar to that of an untreated fetus of more than 30 days' gestation. Similarly, the phospholipid composition of lung lavage from the hormone-treated fetuses was similar to that of an untreated fetus at a greater gestational age. These data, therefore, suggest that cortisol acts by accelerating physiological development. Cortisol administratration stimulated the activity of cholinephosphate cytidylyltransferase and lysolecithin acyltransferase to a small, but statistically significant extent. This is also consistent with an acceleration of normal development. The stimulation of lysolecithin acyltransferase is of interest, since this enzyme is believed to be involved in the synthesis of dipalmitoylglycerophosphocholine, the major surface-active species of phosphatidylcholine. Cortisol administration had no effect on the activities of pulmonary choline kinase, cholinephosphotransferase, lysophosphatidic acid acyltransferase and glycerolphosphate phosphatidyltranferase, although we have previously shown the latter enzyme to be stimulated following a longer period of exposure to the hormone. Saline injection produced some maturational effects presumably as a result of stress, which may be mediated by corticosteroids or other hormones.     

Phospholipid composition of lamellar bodies formed by fetal rabbit lung type II cells in organ culture

Lung tissue obtained from fetal rabbits of 23 days gestational age was maintained in organ culture to study the in vitro formation of lamellar body phospholipids. During the culture period, the epithelium of the prealveolar ducts of the explants differentiated to form type II pneumonocytes. After 8 days in culture, the explants were harvested, homogenized, and two lamellar body fractions were isolated by sucrose density gradient centrifugation. The lamellar body fraction which best retained the distinct multilamellar structure was recovered at the interface between a solution of buffer without sucrose and buffer containing 0.41 m sucrose. The phospholipid compositions of both lamellar body fractions were similar to those reported for lamellar bodies and surfactant isolated from fetal rabbit lung, with the exception of a slightly higher phosphatidylethanolamine content. The disaturated phosphatidylcholine content of the lamellar body fractions, expressed as a percentage of total lipid phosphorus, was not influenced by the presence of palmitate in the medium.     

Regulation of fetal lung disaturated phosphatidylcholine synthesis by de novo palmitate supply

Lung surfactant disaturated phosphatidylcholine (PC) is highly dependent on the supply of palmitate as a source of fatty acid. The purpose of this study was to investigate the importance of de novo fatty acid synthesis in the regulation of disaturated PC production during late prenatal lung development. Choline incorporation into disaturated PC and the rate of de novo fatty acid synthesis was determined by the relative incorporation of [14C]choline and 3H2O, respectively, in 20-day-old fetal rat lung explants and in 18-day-old explants which were cultured 2 days. Addition of exogenous palmitate (0.15 mM) increased (26%) choline incorporation into disaturated PC but did not inhibit de novo fatty acid synthesis, as classically seen in other lipogenic tissue. Even in the presence of exogenous palmitate, de novo synthesis accounted for 87% of the acyl groups for disaturated PC. Inhibition of fatty acid synthesis by agaric acid or levo-hydroxycitrate decreased the rate of choline incorporation into disaturated PC. When explants were subjected to both exogenous palmitate and 60% inhibition of de novo synthesis, disaturated PC synthesis was below control values and 75% of disaturated PC acyl moieties were still provided by de novo synthesis. These data show that surfactant disaturated PC synthesis is highly dependent on the supply of palmitate from de novo fatty acid synthesis.     

Influence of dexamethasone on the lipid distribution of newly synthesized fatty acids in fetal rat lung

There is a developmental increase in fatty acid biosynthesis and surfactant production in late-gestation fetal lung and both are accelerated by glucocorticoids. We have examined the distribution of the newly synthesized fatty acids to determine whether they are preferentially incorporated into surfactant. Explants of 18 day fetal rat lung were cultured with and without dexamethasone for 48 h and then with [3H]acetate for 4 h after which labeled fatty acids were measured. Incorporation of radioactivity from acetate was considered a measure of newly synthesized fatty acids. Phospholipids contained 86% of the newly synthesized fatty acids of which approx. 80% were in phosphatidylcholine. Phosphatidylcholine and disaturated phosphatidylcholine contained a much greater percentage of the labeled fatty acids than of the phospholipid mass determined by phosphorus assay while phosphatidylethanolamine, phosphatidylserine and sphingomyelin contained less. Dexamethasone increased the rate of acetate incorporation into total lipid fatty acids but it had little effect on fatty acid distribution, except that it increased the percentages in phosphatidylglycerol and disaturated phosphatidylcholine. The hormone also increased the mass of these two phospholipids to a greater extent than that of the total. These data suggested that the newly synthesized fatty acids are preferentially incorporated into surfactant phospholipids and that this process is accelerated by dexamethasone. However, since phosphatidylcholine and phosphatidylglycerol are not exclusive to surfactant, we compared isolated lamellar bodies with a residual fraction not enriched in surfactant. The rate of acetate incorporation into fatty acids in lamellar body phosphatidylcholine as well as its specific activity (radioactivity per unit phosphorus) were both increased by dexamethasone. Specific activity, however, was no greater in the lamellar bodies than in the residual fraction in both control and dexamethasone-treated cultures. Therefore, there is no preferential incorporation of newly synthesized fatty acids into phospholipids in surfactant as opposed to those in other components of the lung.     

Phosphatidylglycerol stimulates cholinephosphate cytidylyltransferase activity and phosphatidylcholine synthesis in type II pneumocytes

Phosphatidylcholine synthesis in type II pneumocytes is stimulated by inclusion of phosphatidylglycerol and other phospholipids in the culture medium (Gilfillan, A.M., Chu, A.J. and Rooney, S.A. (1984) Biochim. Biophys. Acta 794, 269-273). We have now examined the effect of phosphatidylglycerol in the medium on enzymes of de novo phosphatidylcholine synthesis in adult rat type II cells. Activities of choline kinase, cholinephosphate cytidylyltransferase and cholinephosphotransferase in homogenates of whole lung and type II cells were generally similar. Phosphatidate phosphatase activity in type II cells, however, was only 16% that in whole lung. Addition of phosphatidylglycerol (10 microM) to the culture medium had no effect on choline kinase, cholinephosphotransferase or phosphatidate phosphatase activities in type II cells but it increased the activity of cholinephosphate cytidylyltransferase by 56%. Since it is known that cholinephosphate cytidylyltransferase is stimulated in vitro by addition of phospholipids to the assay mixture, we also measured its activity in the presence of sufficient phosphatidylglycerol (1.1 mM) to maximally stimulate in vitro. Even under these conditions cholinephosphate cytidylyltransferase activity in type II cells cultured in the presence of phosphatidylglycerol was 32% greater than in control cells. These data show that the stimulatory effect of phospholipid in the culture medium on phosphatidylcholine synthesis in type II cells is mediated by increased cholinephosphate cytidylyltransferase activity. The mechanism of increased cytidylyltransferase activity remains to be elucidated but it is not due to direct in vitro activation by the phospholipid.