Testosterone regulation of sex differences in fetal lung development.

Fetal lung development, in particular surfactant synthesis, exhibits a sexual dimorphism. Dihydrotestosterone (DHT) has been shown to delay fetal pulmonary surfactant production, but the potential role for testosterone is unknown. Both testosterone and DHT are potent masculinizing hormones, yet in some instances, an end organ specificity for DHT is present. We hypothesized that the delay in fetal lung surfactant production is dependent upon DHT such that inhibition of the synthesis of DHT from the precursor hormone testosterone would eliminate the sex difference by allowing the male fetus to produce surfactant at the female level. We tested this hypothesis using 17 beta-N,N-diethylcarbamoyl-4-aza-4-methyl-5-alpha-androstane-3-one (4-MA), a potent inhibitor of the enzyme 5 alpha-reductase, which converts testosterone into DHT. First, studies were performed in vivo. 4-MA (20 mg/kg/day) or an equivalent volume of vehicle was injected into pregnant rabbits from Day 12 through Day 26 of gestation. On Day 26, the fetuses were delivered, the lungs were lavaged, and fetal sex was noted. Treatment with 4-MA resulted in a lack of any male-female difference in the anogenital distance and no DHT was detected in the serum of any treated fetus. Phosphatidylcholine (PC), saturated phosphatidylcholine (SPC), and sphingomyelin (S) were measured in the lung lavage, and were expressed as the ratios of PC to sphingomyelin (PC:S) and SPC to sphingomyelin (SPC:S). Sex differences in the PC to sphingomyelin ratio of 4-MA-treated fetuses (female PC:S ratio, 1.43 +/- 0.14; male PC:S ratio, 1.00 +/- 0.13 [mean +/- SE]; P = 0.04) and in the SPC:S ratio of the 4-MA-treated group (female SPC:S ratio, 0.68 +/- 0.10; male SPC:S ratio, 0.35 +/- 0.10; P = 0.03) were present after treatment with 4-MA. The effect of testosterone and of 4-MA on fibroblast pneumonocyte factor (FPF) production was studied in vitro. Fetal rat lung fibroblasts were cultured to confluence with either no added androgen, DHT, testosterone, or testosterone plus 4-MA, and conditioned media for FPF were prepared. Conditioned media were added to fetal Type II cell cultures and FPF activity was measured as the degree of stimulation of the incorporation of [3H] choline into SPC. The conversion of radiolabeled testosterone to DHT by the fibroblasts was inhibited by 4-MA (10(-5) M). Conditioned media from untreated female fibroblasts stimulated with cortisol exhibited significant FPF activity ([3H]choline incorporation into SPC, 140 +/- 17% of control).(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Dihydrotestosterone blocks fetal lung fibroblast-pneumonocyte factor at a pretranslational level

Fibroblast pneumonocyte factor (FPF) synthesis by fetal rat lung fibroblasts is augmented during gestation in the presence of cortisol. The control and cortisol-augmented levels of FPF production, as determined by FPF ability to stimulate saturated phosphotidylcholine synthesis by lung epithelial Type II cells, is delayed during development in fibroblasts derived from male fetuses as compared to those derived from female fetuses. The mechanism by which this delay occurs has been addressed. Pregnant rats treated in vivo with dihydrotestosterone (DHT) showed decreased FPF activity from control or cortisol-treated fibroblasts derived from 20-day-old male or female fetuses. In vitro translated proteins of size-fractionated lung RNA from 19-day-old fibroblasts that were pretreated with DHT in vitro showed decreased FPF activity compared to nontreated samples. This decreased FPF activity was present even if the DHT-pretreated cells were stimulated with cortisol prior to RNA preparation. Using a mouse model of testicular feminization that contains no receptors for androgens showed no change in the cortisol augmented FPF activity when the fibroblasts were pretreated with DHT. These data taken together suggest that the delayed FPF production of male-derived lung fibroblasts is a physiologic process which requires androgen receptors, and the mechanism by which androgens inhibit FPF production appears to affect events occurring mainly at a pretranslational level.     

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.     

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.     

Epidermal growth factor influences the developmental clock regulating maturation of the fetal lung fibroblast

Growth factors may play a significant role in regulating the orderly progression of organ growth and differentiation during fetal development. We hypothesized that epidermal growth factor (EGF) would help regulate the development of surfactant synthesis in the fetal lung by influencing fibroblast-epithelial cell interactions. The effect of EGF (10 ng per ml) on the ability of the fetal lung fibroblast to produce fibroblast pneumonocyte factor (FPF) was studied in sex-specific fibroblasts cultured from day 16, day 17 or day 18 fetal mouse lungs. FPF which is normally not produced by day 16 fibroblasts, is found only in female fibroblasts on day 17, and then in both males and females on day 18. EGF advanced this pattern such that female fibroblasts produced activity on day 16 and fibroblasts from both sexes produced FPF activity on day 17 and day 18. Fibroblasts from an androgen receptor-deficient mouse model confirmed that the effect of EGF was sex-specific and related to the state of development of the fetal lung. We conclude that EGF advances the fetal lung fibroblast through specific stages of development. It appears, therefore, to help control the timing of the clock regulating fetal lung maturation.     

Fatty acid synthesis in the fetal lung: relationship to surfactant lipids

The aims of this study were to investigate the control of fatty acid synthesis and its relationship to surfactant production in the fetal lung during alteration of hormonal and substrate conditions. Lung explants from 18 day fetuses (term = 22 days) which were cultured 2 days in the presence of 10 mM lactate showed parallel acceleration of de novo fatty acid synthesis (3H2O incorporation) and [14C]choline incorporation into disaturated phosphatidylcholine (DSPC) compared to culture of explants in glucose. Both the cultured and fresh explants were resistant to the classical short term (4 h) cAMP inhibition of fatty acid synthesis with 3 mM dibutyryl cAMP or 0.5 mM aminophylline. In the cultured explants short term cAMP elevation increased DSPC production, and long term (2 day) cAMP elevation caused a further increase in DSPC synthesis and also stimulated fatty acid synthesis. In cultured explants from 17 day fetuses, dexamethasone (0.1 microM) caused a synergistic increase with aminophylline in both fatty acid synthesis and DSPC production whereas, in explants from 18 day fetuses, dexamethasone inhibited both processes and reduced the level of stimulation of DSPC and fatty acid synthesis seen with aminophylline alone. Dexamethasone also reduced the stimulation of both DSPC and fatty acid synthesis produced in the culture of 18 day explants with bacitracin (0.5 mg/ml), whereas the combination of bacitracin and aminophylline resulted in a synergistic increase in DSPC production. Culture with glucagon (0.1 microM) also stimulated DSPC synthesis but at physiological levels insulin had no effect on either DSPC or fatty acid synthesis. These data show that lung fatty acid synthesis exhibits unique features of fatty acid synthesis regulation compared to other lipogenic tissues and also suggest a link between de novo fatty acid synthesis and surfactant production during the critical period of accelerated lung maturation.     

Carbon tetrachloride inhibits synthesis of pulmonary surfactant disaturated phosphatidylcholines and ATP production in alveolar type II cells

Other studies have shown that inhalation of carbon tetrachloride (CCl4) decreases the amount of pulmonary surfactant lining the alveolar surface. Therefore, we studied the effects of CCl4 on the synthesis of surfactant phosphatidylcholines (PCs) in rat alveolar type II cells in vitro. The rate of incorporation of choline, palmitate or glycerol into disaturated PC (DSPC) is decreased in a concentration-dependent manner. The CCl4 concentrations which cause maximal inhibition and 50% inhibition are similar for each substrate. The rate of incorporation of choline or glycerol into total PC is diminished to the same extent as their incorporation into DSPC. In addition, the rate of incorporation of glycerol into phosphatidylglycerol is decreased by the same extent as its incorporation into PC. All of these data suggest that there is a common site(s) at which CCl4 inhibits PC synthesis and that the inhibition occurs early in the biosynthetic pathway. However, individual enzymes involved in phospholipid synthesis do not seem to be affected by the solvent. Exposure of alveolar type II cells to CCl4 does cause a rapid and dramatic loss in cellular ATP, a cofactor required by some enzymes involved in PC synthesis. Studies with isolated lung mitochondria suggest that CCl4 inhibits the enzyme complex which catalyzes the synthesis of ATP from ADP. In addition, CCl4 causes a decrease in the amount of 3-O-methylglucose associated with type II cells, suggesting that glucose influx is impaired. This may also contribute to lower cellular ATP levels. The results of this study suggest that inhalation of CCl4 may impair surfactant phospholipid synthesis by decreasing ATP levels in alveolar type II cells.     

Coordination of growth and differentiation in the fetal lung

The male fetal lung begins to synthesize surfactant later in gestation than the female. This delay appears to be caused by androgens. We hypothesized that male fetal lung differentiation is delayed as a consequence of an extended phase of growth which is elicited by androgens. We observed that in vivo fetal lung protein synthesis relative to DNA synthesis peaked earlier in gestation in the female fetal lung and that this event was synchronous with the onset of differentiation. Pregnant rats were treated with dihydrotestosterone (DHT) during pregnancy, and fetal lung growth parameters were measured. Lung wet weight, dry weight, and DNA and protein concentrations were significantly elevated by DHT treatment. Type II cells and fibroblasts were isolated from lungs of DHT-treated fetuses. The number of total cells recovered was increased by 30%; the number of type II cells recovered was increased by 87%; and the number of fibroblasts recovered was increased by 42%. The type II cells which were recovered exhibited increased incorporation of [3H]thymidine into DNA and a reduced ratio of radiolabeled protein to radiolabeled DNA compared to that of cells from control lungs. Further studies were done in vitro with fibroblasts and type II cells isolated from untreated fetal rat lungs. Treatment of the fibroblasts with DHT during culture caused an increase in thymidine incorporation into DNA. This effect was not blocked by simultaneous treatment with cortisol, which normally causes reduced DNA synthesis and induces fibroblast differentiation. Treatment of the type II cells with DHT in culture caused a dose-dependent increase in cell number but a decrease in synthesis of disaturated phosphatidylcholine. These studies provide more direct evidence of the interrelationships between the control of growth and the control of differentiation in the fetal lung. DHT, a signal which delays the onset of expression of differentiation, also induces growth. We conclude that the controls of growth and of differentiation of the fetal lung are reciprocally linked.     

Effect of cortisol on the synthesis of lamellar body glycerophospholipids in fetal rabbit lung tissue in vitro

The effect of cortisol on the rate of choline incorporation into tissue phosphatidylcholine was investigated in lung explants from fetal rabbits of 19-28 days gestational age. The explants were incubated in medium with or without fetal calf serum for up to 7 days. When lung tissues were incubated in serum-free medium, a stimulatory effect of cortisol on tissue phosphatidylcholine synthesis was found in explants from 21-, 24-, 26- and 28-day fetal rabbits; a stimulatory effect of cortisol was observed in 19-day fetal lung explants only if fetal calf serum was present in the culture medium. To assess directly the effect of cortisol on the synthesis of lamellar body phosphatidylcholine, choline incorporation into phosphatidylcholine associated with a purified lamellar body fraction isolated from lung explants of 21- and 28-day fetal rabbits was also investigated. Cortisol caused a marked stimulation of synthesis and accumulation of lamellar body phosphatidylcholine in lung explants from both 21- and 28-day fetal rabbits. The magnitude of the stimulatory effect of cortisol on the rate of synthesis of lamellar body phosphatidylcholine was always greater than the effect of cortisol on the rate of choline incorporation into lipids of tissue homogenates. The relative rates of synthesis of lamellar body phosphatidylinositol and phosphatidylglycerol were also significantly altered in lung explants from 21- and 28-day fetal rabbits by cortisol treatment. Lamellar bodies that were formed initially in the fetal lung explants were enriched in phosphatidylcholine and phosphatidylinositol and had a relatively low phosphatidylglycerol content. With cortisol treatment there was a decrease in the relative rate of synthesis of lamellar body phosphatidylinositol and an increase in the relative rate of synthesis of phosphatidylglycerol. The stimulatory effect of cortisol on the synthesis of lamellar body phosphatidylcholine was observed at an earlier time-point of incubation than was the effect of cortisol on the relative rates of synthesis of lamellar body phosphatidylinositol and phosphatidylglycerol. The temporal sequence of the cortisol-induced changes in the synthesis of lamellar body glycerophospholipids, therefore, reflects that which occurs with maturation in vivo.     

Glucocorticoids affect the synthesis of pulmonary fibroblast-pneumonocyte factor at a pretranslational level

Glucocorticoids accelerate fetal lung maturation by acting on the fetal lung fibroblast to induce the synthesis of fibroblast-pneumonocyte factor which in turn stimulates pulmonary surfactant synthesis by the alveolar type II cell. We have studied the site of glucocorticoid regulation of fibroblast-pneumonocyte factor synthesis in primary cultures of fetal rat lung fibroblasts. Conditioned media from fetal rat lung fibroblasts exposed to cortisol stimulate [Me-3H]choline incorporation into saturated phosphatidylcholine by primary cultures of fetal rat lung alveolar type II cells. This effect is blocked by the presence of actinomycin D during the first, but not the second, 24 h of incubation of the fibroblasts with cortisol. Cycloheximide blocks this effect if present during either the first or second 24 h of incubation. We fractionated mRNA from fetal rat lung fibroblasts incubated in the presence or absence of dexamethasone and observed that cell-free translation products from a fraction of approximately 500 bases possess biological activity in the bioassay. Such activity is only present in cell-free translation products of mRNA isolated from fibroblasts treated with dexamethasone. These results suggest that glucocorticoids act at a pretranslational level to induce production of fibroblast-pneumonocyte factor and that the primary translation products are biologically active.     

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.     

Accelerated fetal lung maturation by estrogen is associated with an epithelial-fibroblast interaction

Summary The role of epithelial-mesenchymal interactions in the stimulation of lung development by estrogen is now investigated using organ cultures of lung from male and female fetal rats taken from Days 17 to 21 of gestation. Estradiol at 1 μg/ml was found to reduce cell proliferation in explants taken during a rapid growth phase (Day 18) and to stimulate surfactant synthesis in both males and females only in Day 20 explants when cell division is much slower. At this time more epithelial cells from estrogen-treated explants contained lamellar bodies, which were also secreted to fill the air sacs. These cultures also showed a significant increase in the frequency of cell-to-cell contacts between epithelial cells and fibroblasts. Uptake of tritiated estradiol by explants increased from Day 18 onward, and by autoradiography, labeling was located predominantly over fibroblasts. Using pure cultures of fetal and adult cells, uptake of labeled estradiol was significantly higher in fibroblasts than in corresponding epithelial cells, and estradiol did not directly enhance palmitate incorporation into epithelial cells. The results suggest that the earlier maturation and increased surfactant synthesis in female fetal lung is related at least in part to enhanced binding of estrogen by the fibroblast with subsequent transfer of a maturation factor to the fetal epithelium. This research project was supported by grants from the Medical Research Council of Canada and the Council for Tobacco Research, U.S.A., Inc.     

Glycerol as a substrate for phospholipid biosynthesis in type II pneumocytes isolated from streptozotocin-diabetic rats

Glycerol and glucose utilization for phospholipid biosynthesis was examined in type II pneumocytes isolated from normal and streptozotocin-diabetic rats. In cells from diabetic rats, incorporation of [1,3-14C]glycerol into total phosphatidylcholine (PC), disaturated phosphatidylcholine (DSPC), phosphatidylglycerol (PG) and phosphatidylethanolamine (PE) occurred to a greater degree by the glycerol 3-phosphate pathway as opposed to the dihydroxyacetone phosphate pathway. Total incorporation of glycerol into each of the major cellular phospholipids was increased up to 6-fold in cells from diabetic rats, while the total incorporation of glucose into the same lipids was decreased 2-fold. While the percentage of both glucose and glycerol carbons incorporated into the backbone of DSPC was increased in cells from diabetic rats, the percentage of carbons from both substrates incorporated into the fatty acid moieties was decreased. As a measure of DSPC synthesis, choline incorporation into DSPC was significantly decreased in type II cells from diabetic animals if the cells were incubated in the presence of glucose, palmitate and choline but not glycerol. Addition of 0.1 or 0.3 mM glycerol to the incubation medium restored choline incorporation to the control value in cells from diabetic rats, but did not affect the rate of choline incorporation into DSPC in cells from normal rats. These results suggest that exogenous glycerol can compensate for reduced glucose metabolism in type II cells of diabetic animals to maintain a constant rate of DSPC 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.     

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.     

Altered lung phospholipid metabolism in mice with targeted deletion of lysosomal-type phospholipase A2

Lung surfactant dipalmitoylphosphatidylcholine (DPPC) is endocytosed by alveolar epithelial cells and degraded by lysosomal-type phospholipase A2 (aiPLA2). This enzyme is identical to peroxiredoxin 6 (Prdx6), a bifunctional protein with PLA2 and GSH peroxidase activities. Lung phospholipid was studied in Prdx6 knockout (Prdx6-/-) mice. The normalized content of total phospholipid, phosphatidylcholine (PC), and disaturated phosphatidylcholine (DSPC) in bronchoalveolar lavage fluid, lung lamellar bodies, and lung homogenate was unchanged with age in wild-type mice but increased progressively in Prdx6-/- animals. Degradation of internalized [3H]DPPC in isolated mouse lungs after endotracheal instillation of unilamellar liposomes labeled with [3H]DPPC was significantly decreased at 2 h in Prdx6-/- mice (13.6 +/- 0.3% vs. 26.8 +/- 0.8% in the wild type), reflected by decreased dpm in the lysophosphatidylcholine and the unsaturated PC fractions. Incorporation of [14C]palmitate into DSPC at 24 h after intravenous injection was decreased by 73% in lamellar bodies and by 54% in alveolar lavage surfactant in Prdx6-/- mice, whereas incorporation of [3H]choline was decreased only slightly. Phospholipid metabolism in Prdx6-/- lungs was similar to that in wild-type lungs treated with MJ33, an inhibitor of aiPLA2 activity. These results confirm an important role for Prdx6 in lung surfactant DPPC degradation and synthesis by the reacylation pathway.     

An apolipoprotein preferentially enriched in cholesteryl ester-rich very low density lipoproteins

Phosphatidyl glycerol is present in lamellar bodies and in the material obtained by alveolar wash representing 12.3 and 11.5%, respectively, of the total phospholipid phosphorus. Lung microsomes catalyze the formation of phosphatidyl glycerol from the known precursors, L-glycerol 3-phosphate and CDP-diglyceride. The rate of [¹⁴C]L-glycerol 3-phosphate incorporation into phosphatidyl glycerol was 30% higher in microsomes as compared to mitochondria. The addition of mercuric chloride inhibited the synthesis of phosphatidyl glycerol, and stimulated the incorporation into another as yet incompletely identified lipid. After pulse labeling of microsomal phosphatidyl glycerol $\text{in vitro}$, further incubation of microsomes with lamellar bodies or alveolar wash resulted in nearly quantitative appearance of label in surfactant.     

Adrenalectomy and surfactant in adult rats

We examined the effect of adrenalectomy (ADX) on aspects of the surfactant system of adult rats. Five days after bilateral ADX, ADX rats had about 20% less disaturated phosphatidylcholine (DSPC) in lung lavage returns (airway DSPC) than sham-operated rats, but the amount of tissue DSPC was not different between the groups; airway DSPC formed 12.8 +/- 0.5% of total DSPC (airway + tissue) in ADX and 15.9 +/- 0.7% in sham-ADX rats. An ultrastructural morphometric analysis of alveolar type 2 cells did not reveal an effect of ADX on lamellar body volume density or surface-to-volume ratio. ADX rats had heavier lungs (not as a result of edema) than sham-ADX rats. Treatment of ADX rats with hydrocortisone returned the amount of DSPC toward normal and eliminated the increase of lung weight. ADX did not alter the recoil of saline-filled lungs but did slightly increase the recoil of air-filled lungs. We conclude that corticosteroid hormones influence the in vivo functioning of the surfactant system of adult rats, but this effect seems to be slight.