Maturation of fetal rat lung in diabetic pregnancies of graduated severity.

Biochemical and morphologic maturation of fetal rat lung was studied in pregnant, diabetic rats with different levels of glucose intolerance (sub-, mildly, and severely diabetic). Fetuses were almost normoglycemic and hyperinsulinemic in subdiabetic rats, both hyperglycemic and hyperinsulinemic in mildly diabetic rats, and hyperglycemic but hypoinsulinemic in severely diabetic rats. A similar delay in type II pneumocyte differentiation and a similar decrease of disaturated phosphatidylcholine (DSPC) content in lung tissue and broncho-alveolar material recovered by lung lavage occurred in the fetuses of the three diabetic groups, independently of the severity of diabetes. Phosphatidylglycerol (PG) was decreased in fetuses from severely diabetic rats only. DSPC appeared specifically affected in fetuses of sub- and mildly diabetic groups, whereas in those of severely diabetic groups, DSPC alterations accompanied a variety of abnormalities including whole lung hypoplasia and hypotrophy and decreases of sphingomyelin, unsaturated PC, and lysoPC. The mechanisms leading to abnormal lung development could therefore be different in fetuses of sub- and mildly diabetic rats on one hand and fetuses of severely diabetic rats on the other. Since hyperinsulinemia was the prominent feature of fetal "milieu intérieur" in subdiabetic rats, this study presents arguments gained from in vivo experiments for an implication of hyperinsulinemia in lung developmental retardation due to maternal diabetes. However, the decrease of PG seems to depend on increased blood glucose level in itself. Diminished lung glycogen breakdown and decreased lung triglyceride content, more pronounced in fetuses of sub- and mildly diabetic rats than in those of severely diabetic rats, suggest that in the former, the decrease of DSPC biosynthesis could be due to decreased availability of substrates because of abnormal glycogen utilization. Fetuses from sub- and mildly diabetic rats constitute experimental models most closely resembling the human fetus of the diabetic mother with respect to circulating glucose and insulin. They appear therefore more adequate for elucidating the mechanisms of abnormal lung development in the diabetic pregnancy. In contrast, fetuses from severely diabetic rats associate very high blood glucose levels and hypoinsulinemia, which are features closer to those of adult diabetic subjects than to those of the human fetus of the diabetic mother.

1,25-dihydroxyvitamin D3 receptors in rat lung during the perinatal period: regulation and immunohistochemical localization.

We have previously reported that in contrast to what has been described in adult lung, 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] has specific binding sites in rat fetal lung at the end of gestation, and it stimulates in vitro the phospholipid biosynthesis and surfactant release from fetal rat type II pneumocytes. In the present study an immunohistochemical technique using a rat monoclonal antibody (9A7 gamma) and binding studies were carried out on fresh lung tissues from fetal and newborn rats during the perinatal period to identify the cell(s) directly responsive to 1,25-(OH)2D3 in fetal lung and to look for a down-regulation of the 1,25-(OH)2D3 receptors in the perinatal period. We also searched for a regulation of 1,25-(OH)2D3 binding to fetal lung by 1,25-(OH)2D3 itself and by factors known to affect lung maturation or be involved in parturition. Our results suggest that 1) fetal type II pneumocytes are target cells for 1,25-(OH)2D3; 2) a physiological down-regulation of the 1,25-(OH)2D3 receptors in rat lung occurs in the perinatal period, starting a few hours before birth and lasting at least up to the fifth day of life; and 3) the capacity of rat fetal lung to bind 1,25-(OH)2D3 can be modulated in vitro by different hormones; a small inhibitory effect is observed with oxytocin (100 microU/ml), while PRL (10(-8) M), T4 (10(-6)-10(-10) M), 1,25-(OH)2D3 (10(-9)-10(-10) M), and, to a lesser extent, dexamethasone (10(-7) M) induce a 2- to 4-fold increase in the number of 1,25-(OH)2D3 receptors without altering the binding affinity of receptor for 1,25-(OH)2D3.

The endocrine control of embryonic lung maturation in the chicken. I. Morphological and biochemical differentiation of lungs after "in ovo" decapitation.

The effects of "in ovo" hypophysectomy on lung maturation of the chick embryo were investigated. Both biochemical and morphological aspects of differentiation were markedly delayed in experimental embryos: the phospholipid content of lungs was lower than in controls at all stages, whereas the water content remained very high. The type II pneumocytes, which normally appear within the epithelium on day 16 of incubation, started to differentiate only between days 18 and 20 of incubation in the decapitated embryos. The differentiation of type I pneumocytes leading to the formation of air capillaries was also slowed down: they did not appear until the end of incubation in decapitated embryos, whereas they normally start to appear on day 19. The presence of an intact hypophysis is thus essential for normal lung maturation in the chicken.

Lipid and phospholipid content and fatty acid composition of the chick lung during embryonic development.

The evolution of total phospholipid, phosphatidylcholine and phosphatidylethanolamine content of chick lung during embryonic development is in good agreement with morphological data. Saturated fatty acids are predominant. A sex-linked difference is observed in the evolution of phosphatidylcholine.

Pulmonary di-and-triacylglycerols during the perinatal development of the rat.

Diacylglycerol (DG) and triacylglycerol (TG) levels in rat lung tissue were determined from day 17 of gestation to day 10 post partum and studied in parallel with ultrastructural differentiation. The DG level, although rather low at all measured stages, rose significantly between days 17 and 19 and at birth. TG level increased steadily during the whole studied period and especially between days 17 and 19 and at birth. In DG as well as in TG, saturated fatty acids were predominant. The rising of TG levels paralleled the appearance and accumulation of lipid vacuoles in mesodermal cells lying in contact with type II cells. The possible role of these cells is discussed.

[Phospholipid and fatty acid composition of lamellar bodies isolated from chicken lungs at different stages of development]. / Composition en phospholipides et en acides gras des inclusions lamellaires isolées à partir de poumons de poulets à différents stades du développement.

In order to elucidate whether the lamellar body content represents functional surfactant as soon as these bodies appear in lung epithelium during fetal development, lamellar bodies were isolated from lungs of Chicken at various developmental stages. Their content was analyzed and compared to extra-cellular functionnal surfactant. The results show that this content undergoes gradual changes in the phospholipid/protein ratio, as well as in the relative phosphatidylcholine amount. Thus, in the Chicken, nascent lamellar bodies do not represent the final form of extracellular surfactant.

1,25(OH)2D3 stimulates phospholipid biosynthesis and surfactant release in fetal rat lung explants.

Lung tissue from 18-day-old rat fetuses was cultured in the presence of 1,25-dihydroxyvitamin D3 - 1,25(OH)2D3 - (10(-9) M) and dexamethasone (10(-7) M) for 48 h. 1,25(OH)2D3 increased the lung content in phospholipids more specifically related to lung surfactant, phosphatidylcholine and phosphatidylglycerol. This increase was similar to that observed with dexamethasone. In addition, unlike dexamethasone, 1,25(OH)2D3 stimulated the surfactant release into luminal spaces, as evidenced by light and electron microscopy. Thus, vitamin D3 might represent an additional factor controlling fetal lung maturation by stimulating phospholipid synthesis and surfactant release from type II cells.

Effects of maternal protein-calorie malnutrition on the phospholipid composition of surfactant isolated from fetal and neonatal rat lungs. Compensation by inositol and lipid supplementation.

The effects of a maternal protein-calorie malnutrition during gestation and lactation were analyzed on fetal and postnatal lung growth and maturation, including a surfactant fraction isolated from lung tissue. There was a considerable reduction in body weight and in wet and dry lung weights of malnourished pups. Lung protein and DNA concentrations were similar in both groups except in late gestation (lung hyperplasia) and 2 and 15 d after delivery (hypocellularity). Lung glycogen breakdown was slowed down in malnourished newborns. Surfactant material was decreased the most perinatally and the reduction was more marked than for the nonsurfactant fraction of the lung. Disaturated phosphatidylcholine, the major surface active surfactant component, was decreased the most at birth (1.70 +/- 0.31 nmol/mg wet wt versus 3.68 +/- 0.17 nmol/mg in controls, n = 8) and on d 2 (5.04 +/- 0.53 nmol/mg versus 7.67 +/- 0.44 nmol/mg in controls, n = 8). There was an apparent recovery in the composition of surfactant in malnourished rats 5 d after delivery, due in fact to a decrease in controls, and an actual return to normal levels 15 to 20 d after birth. Postnatal lipid supplementation with Intralipid led to partial recovery on d 10. Inositol supplementation totally reverted the effects of malnutrition on surfactant phospholipids (8.36 +/- 0.94 nmol disaturated phosphatidylcholine/mg wet wt on d 2 versus 7.67 +/- 0.44 nmol/mg in controls and 5.55 +/- 0.62 nmol/mg in untreated malnourished rats, n = 10; 2.43 +/- 0.32 nmol disaturated phosphatidylcholine/mg wet wt on d 10 versus 3.26 +/- 0.32 nmol/mg in controls and 1.18 +/- 0.27 nmol/mg in untreated malnourished rats, n = 8).(ABSTRACT TRUNCATED AT 250 WORDS)

Maturational changes induced by 1 alpha,25-dihydroxyvitamin D3 in type II cells from fetal rat lung explants.

Specific binding sites for 1 alpha,25 dihydroxyvitamin D3 [1 alpha,25-(OH)2D3] localized to type II pneumocytes have been evidenced in fetal rat lung at the end of gestation, suggesting a role for vitamin D3 in the control of lung maturation. In this study, we describe the morphological changes that occur in lung explants from 18-day-old rat fetuses grown for 1 and 2 days in control conditions and in the presence of 1 alpha,25(OH)2D3 (10(-9) M) or dexamethasone (10(-7) M). Point counting and planimetric measurements on light and electron micrographs show that 1 alpha,25-(OH)2D3 1) dramatically decreases the mean glycogen content of type II cell profiles between days 1 and 2 of the culture, suggesting an acceleration of the glycogenolytic processes normally occurring at that stage and 2) does not change the intracellular osmiophilic lamellar body (OLB) content of cell profiles, but increases the amount of intraluminal surfactant by 126% when expressed as surfactant clusters surface area/section surface area and by 129% when expressed on a per cell basis, suggesting a stimulation of surfactant synthesis and secretion. By contrast, dexamethasone increases the mean intracellular OLB content of type II cell profiles by 306% and decreases the relative surface area of secreted material by 53 and 73%. In conclusion, 1 alpha,25(OH)2D3 accelerates the physiological maturation of fetal rat type II pneumocytes and could represent a key factor for the onset of normal lung function at birth.

Effects of the antiglucocorticoid RU 486 on the maturation of fetal rat lung surfactant.

The role of endogenous glucocorticoids in the control of surfactant system maturation was investigated in the fetal rat using an antiglucocorticoid molecule synthesized by Roussel-UCLAF, RU 486. The drug was administered to the mother from day 16 of gestation on. In a preliminary step, the transplacental transfer of RU 486 and its antiglucocorticoid effects on fetal target tissues were verified by evidencing RU 486-receptor complexes in fetal liver and lung, by measuring liver glycogen content, and by evaluating fetal blood corticosterone. The maturational state of fetal lungs was assessed biochemically on days 19, 20, and 21 of gestation by measuring their glycogen content, the phospholipid content of whole lung tissue and isolated surfactant fraction, and the incorporation of [methyl-3H]choline into DSPC. Morphological development was studied by analyzing electron micrographs of type II cells. The measured parameters clearly indicated a slowing of maturational processes in lungs of fetuses from RU 486-treated mothers, thereby demonstrating that endogenous glucocorticoids are actually involved in the control of lung maturation. In addition, the obtained results showed that endogenous corticosteroids specifically acted on the surfactant system of the fetal lung.

Optimization of fetal lung organ culture for surfactant biosynthesis.

Lung organ culture has been a widely used system for studying differentiation and maturation of alveolar epithelium through various culture conditions. The purpose of this work was to carefully characterize in vitro lung biochemical differentiation through isolation of surfactant fraction from tissue and to search for optimal culture conditions. Fetal rat lung was explanted on the 18th gestational day for studying glycogen storage, and on the 20th gestational day for studying surfactant accretion, and cultivated for 48 h. Morphologic differentiation was studied by electron microscopy on tissue explanted on the 17th or 18th gestational days and cultivated for various times. Glycogen storage was greater on fluid medium, although less than occurring in vivo. Cellular integrity and surfactant accumulation were maximal on a semisolid medium containing 0.5% agar. Use of O2-CO2 instead of air-CO2 for gassing the explants slightly decreased phospholipid accumulation. Among media used in previous lung culture studies, Waymouth MB 752/1 was the only one to allow net glycogen accumulation in vitro. The most favorable media for surfactant phospholipid accretion were Waymouth MB 752/1, Eagle's minimum essential and its Dulbecco's modification, CMRL 1066, and NCTC 109. They allowed a 12- to 14-fold increase of surfactant fraction phospholipids in vitro, which is similar to the increase occurring in vivo during the same period. Ham's F10 and F12 media allowed a six fold increase. RPMI 1640 and medium 199 (M199) allowed only a three fold increase. Phospholipid concentration in nonsurfactant fraction only doubled during culture, and differences between various media were much less marked. DNA concentration changed little during culture. Morphologic differentiation of epithelial cells was advanced as compared with in vivo timing in a medium allowing maximal surfactant accretion (Waymouth MB 752/1) but not in a medium allowing low surfactant increase (RPMI 1640). The possible role of compositional differences between media is discussed.

Role of myo-inositol in impairment of fetal lung phosphatidylglycerol biosynthesis in the diabetic pregnancy: physiological consequences of a phosphatidylglycerol-deficient surfactant in the newborn rat.

To test the hypothesis that the deficit of fetal lung surfactant phosphatidylglycerol (PG) in diabetic pregnancies was due to increased plasma myo-inositol, lung PG content and plasma myo-inositol level were compared in fetuses of streptozotocin-diabetic rats and in fetuses of rats injected with myo-inositol during the 4 last gestational days. An inverse linear correlation was established between circulating myo-inositol and lung phosphatidylglycerol content, including data from fetuses of diabetic rats, which is consistent with the hypothesis. Changes in phospholipid synthetic rates were estimated in fetuses of rats given myo-inositol by measuring incorporation of labelled glycerol on a six hour period on the 21st gestational day, after i.v. injection to the mother. Incorporation into PG was 2.5 times smaller but incorporation into PI or PC were not modified. Pulmonary function in PG-deficient newborns of rats given a high-dosage myo-inositol was assessed by pressure/volume measurements on the lung in situ and by measurement of oxygen tension in aortic blood. Opening pressure of alveoli for lung inflation was increased and blood oxygenation was reduced (30%) in newborns with PG-deficient surfactant as compared with controls, thus suggesting an important physiological role for surfactant PG at birth.

Lung maturation in the hyperinsulinemic rat fetus.

Hyperinsulinemic rat fetuses were obtained either by repeated in utero injections of long-acting insulin (resulting in fetal hypoglycemia) or by chronically infusing intravenous glucose to the mother (resulting in fetal hyperglycemia). Fetuses were examined at term. In insulin-injected fetuses (n = 15), surfactant (S) fraction phosphatidylcholine (PC) and disaturated phosphatidylcholine (DSPC) were significantly decreased (3.6 +/- 0.1 nmol Pi/mg tissue; p less than 0.001 and 2.8 +/- 0.1 nmol/mg; p less than 0.025, respectively) as compared with their saline-injected controls (4.8 +/- 0.2 and 3.3 +/- 0.1 nmol/mg, respectively, n = 19). However, residual (R) fraction was unchanged, and there was no difference in whole-lung phospholipids (combined S and R fractions). These results are consistent with morphological data showing a lower lamellar body area per type II cell profile in insulin-injected fetuses as compared with their controls [1.41 +/- 0.13 micron 2 (n = 72) versus 1.99 +/- 0.14 micron 2 (n = 129) p less than 0.01]. Glycogen content was slightly higher in insulin-injected fetuses (18.5 +/- 1.0 micrograms/mg, n = 17) than in their controls (15.1 +/- 0.8 micrograms/mg, n = 18; p less than 0.05). In the second model, changes in S fraction PC and DSPC were similar to those observed after insulin-injections: 4.3 +/- 0.25 and 3.4 +/- 0.2 nmol Pi/mg in fetuses of glucose-infused rats (n = 10) versus 5.7 +/- 0.45 and 4.3 +/- 0.3 nmol Pi/mg, respectively, in controls (n = 10, p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Persistent elastase/proteinase inhibitor imbalance during prolonged ventilation of infants with bronchopulmonary dysplasia: evidence for the role of nosocomial infections.

Acute imbalance between elastase and alpha-1-proteinase inhibitor (alpha 1Pi) may contribute to the development of bronchopulmonary dysplasia (BPD). The question of whether such an imbalance persists in BPD infants still requiring mechanical ventilation after 4 wk of life has not been previously addressed. We studied 14 infants still on mechanical ventilation at 4 wk of age: nine had BPD and five did not. Weekly (4 to 9 wk) serum and bronchoalveolar lavage (BAL) specimens were taken. alpha 1Pi and alpha-2-macroglobulin were measured in serum and BAL by immunoturbidimetric assay. BAL elastase activity was measured by cleavage of a synthetic substrate and expressed as ng of porcine pancreatic elastase equivalent. Infants with BPD had higher levels of serum alpha 1Pi and alpha-2-macroglobulin than those without BPD. In contrast, the corresponding BAL levels were either similar or even decreased (alpha 1Pi). Moreover, there was a 3-fold increase in elastase-1Pi imbalance expressed as the BAL ng of porcine pancreatic elastase equivalent/2 alpha 1Pi ratio. The role of nosocomial infections was evident in a subgroup of 11 infected BAL aspirates in BPD infants. In such cases we found a 3-fold increase in the BAL ng of porcine pancreatic elastase equivalent/alpha 1Pi ratio as compared to 35 noninfected BAL in BPD infants. These data suggest a persistent alveolitis with imbalance between elastase and proteinase inhibitors in prolonged severe BPD. Such an imbalance is, in part, explained by a local destruction and/or inactivation of alpha 1Pi. Our results also emphasize the increase in proteolysis with nosocomial pneumonia.

Evidence for a vitamin D paracrine system regulating maturation of developing rat lung epithelium.

Rat fetal lung is a target tissue for 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25 (OH)2 D3]. We have identified the cells that respond to the hormone and tested the hypothesis that the lung is also a source of 1 alpha,25(OH)2D3. We found that 1) at the end of pregnancy (days 20-21) alveolar type II cells (ATII) bore 1 alpha,25(OH)2D3 receptors and responded to the hormone. Incubating these cells with 10(-9) M 1 alpha,25(OH)2D3 for 48 h stimulated the synthesis (87.3 +/- 9.1%) and release (61.7 +/- 6.1%) of disaturated phosphatidylcholine; 2) EB-1213, a 1 alpha,25(OH)2D3 analogue with low calcemic activity, had similar effects on ATII; 3) neither fetal lung fibroblasts nor neonatal ATII (day 2 postpartum) expressed 1 alpha,25(OH)2D3 receptors; and 4) in contrast, fetal lung fibroblasts taken on days 19-22 of gestation converted [3H]25(OH)D3 to [3H]1 alpha,25(OH)2D3, whereas ATII and skin fibroblasts did not. These findings suggest that 1 alpha,25(OH)2D3 is a local mediator of epithelial-mesenchymal cell interactions in the developing rat lung and that 1 alpha,25(OH)2D3 or EB-1213 might be therapeutically useful in treating the respiratory distress syndrome of premature neonates.

Lung catecholamines and cyclic nucleotides during perinatal development in the rat. Possible relationships with biochemical and morphological differentiation.

In this study, we investigated in parallel the evolution of endogenous catecholamine and cyclic nucleotide levels in lung tissue in the course of normal fetal development of the rat from day 17 of gestation to day 10 postpartum; the findings were correlated with the successive stages of lung differentiation. These were assessed morphologically by electron microscopy, and biochemically by measuring the levels of the main lung tissue phospholipids and studying their fatty acid composition. Dopamine levels were low and always remained under 5 ng/g of wet tissue. Nevertheless, slight rises were observed between days 17 and 19 of gestation (1.8 and 2.78 ng/g, respectively) and between days 21 of gestation and birth (2.56 and 4.25 ng/g, respectively). These changes were significant (P less than 0.05). Norepinephrine (NE) levels first decreased by about 50% between days 17 and 19 of gestation (7.34 and 3.88 ng/g, respectively), (P less than 0.01) and then rose sharply about 4 times until birth (17.24 +/- 3.90 ng/g), and more slowly from then on. On day 10 postpartum, the mean NE level was 34.31 +/- 3.28 ng/g. The levels of the two cyclic nucleotides [cyclic adenosine 3':5'-monophosphate and cyclic guanosine 5'-monophosphate (cGMP)] were quite high on day 17 and remained so until day 19 (cAMP = 220 pmoles /g; cGMP = 20 pmoles/g). From day 19 until day 21, the levels of the two nucleotides decreased (cAMP = 140 pmoles/g; cGMP = 8 pmoles/g; P less than 0.05). During this period, the decrease in cGMP level was about twice that of cAMP. During the last day of gestation, the cAMP level was still decreasing, whereas cGMP was increasing. After birth, the levels of the two cyclic nucleotides rapidly increased to day 5. Between days 5 and 10 postpartum, no significant changes were observed in either cAMP or cGMP levels. The time course of the variations reported suggests that they are related to the maturation processes of lung assessed on the basis of morphological and biochemical criteria. On day 19 of gestation, when the first lamellar bodies appear, large changes occur in the evolution of NE and cyclic nucleotide levels, suggesting that these changes may be related to the onset of surfactant synthesis. On day 21 of gestation, the cAMP and cGMP ratio rises sharply; at the same time, the total phospholipid level is increased by 50%. At this stage, lamellar bodies are actively differentiating within type II pneumocytes and massively released into the fetal airways. The relationships between the variations of the studied biochemical parameters and the maturation processes of lung suggest that dopamine and NE could be involved in surfactant synthesis and release via their second messenger, the cyclic nucleotides. Further investigations are needed to elucidate the mechanisms regulating these changes.

Role of 1,2-sn diacylglycerol in airway smooth muscle stimulated by carbachol.

Activation of muscarinic receptors in airway smooth muscle leads to breakdown of membrane polyphosphoinositides. In agreement with others, we show here that muscarinic stimulation elicits inositol-1,4,5-triphosphate formation. In addition, we show that carbachol also elicits total diacylglycerol and 1,2-sn diacylglycerol accumulation in a specific and dose-dependent manner (EC50 values: 2.1 x 10(-8) M for 1,2-sn diacylglycerol). The time-course of inositol-1,4,5-triphosphate accumulation is compatible with that of the clonic phase of muscle contraction. Since this derivative can mobilize intracellular Ca2+ stores, it may play a second-messenger role in the initial phase of contraction. The time-course of diacylglycerol accumulation is compatible with the muscarinic-induced tonic phase of smooth-muscle contraction. Carbachol induces similar dose-dependent reductions in isoproterenol-induced muscle relaxation (EC50 values for relaxation concentration-response curves to isoproterenol: 3 x 10(-6) M and 2.1 x 10(-5) M, with carbachol at 10(-7) M and 10(-4) M, respectively), and increases in adenylate cyclase activity (EC50 values for the concentration-response to isoproterenol: 1.2 x 10(-6) M and 1.5 x 10(-5) M, with carbachol at 10(-7) M and 10(-4) M, respectively). Since it is known that carbachol-induced uncoupling of beta 2-adrenergic receptors is proportional to the breakdown of polyphosphoinositides, and that 1,2-sn diacylglycerol is a potent activator of protein kinase C, 1,2-sn diacylglycerol could be mediating the uncoupling of beta 2-adrenergic receptors, via activation of alpha-protein kinase C and subsequent phosphorylation of receptor, and/or cyclase, and/or G proteins.(ABSTRACT TRUNCATED AT 250 WORDS)