Developing Rat Lung Research Articles

Metabolic Activity of Developing Rat Lung

The uptake and metabolism of <sup>3</sup>H-ρalmitate, <sup>14</sup>C-choline, <sup>14</sup>C-proline and <sup>3</sup>H-glucosamine were studied in lung slices prepared from rats aged 2–12 days. Palmitate incorporation increased from 18.2 ± 2.8 nmol/mg protein/h 2 days before birth to 40.0 ± 4.0 nmol/mg protein/h 3 days after birth, and decreased to 24.0 ± 2.3 nmol/mg protein/h at 12 days of age, it remained at the same level in the adult lung. At all ages tested, more than 75% of the palmitic acid taken up by the lung was in esterified form. Incorporation of palmitic acid into lecithin was highest 1 day after birth. Choline incorporation into lecithin increased eightfold between 2 days before and 1 day after birth (from 0.45 ± 0.12 to 3.75 ± 0.25 nmol/mg protein/h, respectively) and decreased 60% by 12 days of age. There was no difference between the incorporation rate at 12 days and that in the adult lung (1.52 ± 0.15 nmol/mg protein/h). Total lung phospholipid increased 220% between 2 days before birth and the day of birth, and changed little thereafter. Phosphatidyl choline made up 50–60% of lung phospholipids at all ages. Proline incorporation into lung protein was highest in the perinatal period (0.57–0.58 nmol/mg protein/h 2 days before and 1 day after birth) and decreased rapidly thereafter. Glucosamine incorporation was highest in the fetal lung (0.84 ± 0.05 nmol/mg protein/h) and decreased 67% 1 day after birth, the incorporation remained between 0.1 and 0.2 nmol/mg protein/h thereafter (from 3 days to adult age). The data presented show that in the rat lung the biosynthesis of structural components and of surfactant is highest during the immediate perinatal period.

Development of fetal rat lung during prolonged gestation

Pregnancies were prolonged by daily subcutaneous injection of progesterone to the maternal rats from day 20 through 24 fo gestation (Term is 22 days). The fetus were harvested by ceasarean section on gestation days (GD) 21 through 25. In spite of reduction in placental weight the fetal body weight increased during prolonged gestation. Both lung wet and dry weights decreased resulting in smaller lung per body weight. Compared to term fetuses, post term fetuses (GD 25) exhibited a 10% reduction in lung cell numbers, and a 90% reduction in lung glycogen. Lung disaturated phosphatidyl choline expressed per lung increased up to GD 23 and subsequently decreased by GD 25. Surface tension of lung extracts and pulmonary lavage fluids increased with prolongation of gestation. Histological studies revealed a decrease in the number of lamellar bodies in the Type II cells and a gradual reduction in the size of terminal scas leading to areas of complete atelectasis at GD 25. It is suggested that lung hypoceullularity, depletion of lung glycogen, reduction in intracellular reserve of phospholipids, loss of pulmonary fluid leading to atelectasis, and loss of surface activity of pulmonary fluid may be predisposing factors for the development of respiratory distress in postmaturity.

Coordinate increases in the enzyme activities responsible for phosphatidylglycerol synthesis and CTP:cholinephosphate cytidylyltransferase activity in developing rat lung

The enzymes responsible for the biosynthesis of phosphatidylglycerol, CTP:phosphatidate cytidylyltransferase, CDP-diacylglycerol: glycerophosphate phosphatidyltransferase and phosphatidylglycerophosphate phosphatase demonstrated a coordinate increase in activity in fetal rat lung at term when the demand for pulmonary surfactant increases. The activity of CTP:cholinephosphate cytidylyltransferase, the enzyme responsible for CDP-choline production also increased in the perinatal period. The activity of cholinephosphate cytidylyltransferase in fetal and neonatal cytosol was stimulated by the addition of phosphatidylglycerol but no effect was noted with cytosol from adult lung. These results are consistent with the suggestion that the activity of cholinephosphate cytidylyltransferase, a potential rate-determining enzyme in pulmonary phosphatidylcholine synthesis, may be regulated in the perinatal period both through an activation by phosphatidylglycerol and by an increase in total enzyme units.

The influence of hormones on the biochemical development of fetal rat lung in organ culture. II. Insulin.

Summary: The influence of insulin on the biochemical and morphologic maturation of 19-day fetal rat lung (term is 22 days) was studied in an organ culture system. Exposure to insulin for 24 hr resulted in a significant increase in the glycogen content and the rate of glucose oxidation to CO2. In association with this, there was morphologic evidence of delayed lung maturation as evidenced by a significant decrease in the number of lamellar bodies and type II cells in the explants.Insulin treatment had no effect on the rate of choline incorporation into phosphatidylcholine (PC) or disaturated PC and did not antagonize the dexamethasone-induced stimulation of choline incorporation into PC. When the incorporation of [3H]acetate into the various phospholipid fractions was examined, however, it was found that exposure to insulin resulted in a significant decrease in the percentage of phospholipid radioactivity in the disaturated PC fraction and an increase in the percentage of radioactivity in the “membrane” phospholipids, phosphatidylethanolamine, phosphatidylinositol plus phosphatidylserine, and sphingomyelin. There was no significant change in the unsaturated PC and phosphatidylglycerol fractions. Treatment with dexamethasone generally had the opposite effect to insulin with regard to acetate incorporation into the various phospholipid fractions, and when the two hormones were combined, this effect was diminished or abolished. The effects of insulin could not be accounted for on the basis of a change in activity of any of the enzymes of phospholipid synthesis that were examined.These findings suggest that insulin stimulates the synthesis of the cell membrane phospholipids while decreasing that of the surfactant phospholipid, disaturated PC.Speculation: Insulin stimulates lung cell growth while delaying lung cell differentiation.

Postnatal development of rat lung. Changes in lung lectin, elastin, acetylcholinesterase and other enzymes.

The development of rat lung from a primitive gas-exchange organ to the mature respiratory organ is in large part a postnatal phenomenon that has been well characterized by morphological and morphometric methods. The alveolarization of the lung is achieved during the first 3 weeks of life. Cholinergic innervation of rat lung also appears postnatally. We have monitored the presence or activity of several proteins during postnatal rat lung development. Newborn-rat lung contains negligible amounts of acetylcholinesterase, but the specific activity of acetylcholinesterase reaches adult values by postnatal day 10-11. Neonatal-rat lung does not contain significant amounts of beta-galactoside-binding protein [Powell (1980) Biochem. J.187, 123-129]. The activity of this endogenous lung lectin was apparent at about day 6, was maximal between days 10 and 13 before declining 8-10-fold to reach adult values. Elastin has been implicated from morphological evidence as critical to lung restructuring. We have quantified the amount of desmosine and isodesmosine per g wet wt. of lung. The concentration of elastin, by this criterion, was low and stationary until postnatal day 7; a dramatic increase in elastin concentration occurred between days 10 and 20, when adult values were reached. The peak of lung-lectin activity was coincident with the maturation of acetylcholinesterase and the beginning of rapid elastin cross-linking. The specific activities of angiotensin-converting enzyme, carbonic anhydrase, choline kinase and glucose 6-phosphate dehydrogenase were also monitored.

The influence of hormones on the biochemical development of fetal rat lung in organ culture: I. Estrogen

It was recently demonstrated that 17β-estradiol enhances phosphatidylcholine synthesis in fetal lung in vivo. In order to determine whether estrogen acts directly on the lung, we examined the influence of 17β-estradiol on phospholipid and glycogen metabolism in explants of 18 day fetal rat lung in organ culture. Exposure of the explants to 17β-estradiol resulted in significant stimulation of [Me- 3H]choline incorporation into phosphatidylcholine, disaturated phosphatidylcholine and sphingomyelin. Incorporation of [ 3H]acetate into total phospholipid was increased by 63% ( P < 0.001). Incorporation into individual phospholipids was similarly increased, except for that into phosphatidyl-glycerol which was enhanced by 111% ( P < 0.005). When the data were expressed as the percentage of radioactivity from [ 3H] acetate in the various phospholipid fractions, it was found that there was a 29% increase in the phos-phatidylglycerol fraction ( P < 0.005) and a 12% reduction in phosphatidyl-inositol plus phosphatidylserine combined ( P < 0.005). There was no significant change in the percentage of radioactivity in any of the other phospholipid fractions, or in the activity of any of the enzymes of phospholipid synthesis examined. Estrogen treatment also resulted in a decrease in the glycogen content ( P < 0.05), but no change in the protein/DNA ratio. These data indicate that estrogen acts directly on the fetal lung and stimulates the incorporation of choline and acetate into phospholipids. It may specifically enhance incorporation into phosphatidylglycerol, the second most abundant phospholipid in pulmonary surfactant.

Toxic effects of cadmium on the developing rat lung. I. Altered pulmonary surfactant and the induction of respiratory distress syndrome

The effects of Cd on the growth of the fetal rat lung and the maturation of the pulmonary surfactant system were studied. Pregnant rats received sc injections of cadmium chloride on d 12-15 of gestation. Animals were sacrificed throughout late gestation. Fetal lungs were assayed for pulmonary surfactant lecithin and spingomyelin. Some animals were allowed to give birth and the neonates were observed for symptoms of respiratory distress. The treatment resulted in high fetal mortality and growth retardation. Lung-body weight ratios were reduced by 20-30% in treated fetuses. Pulmonary spingomyelin content was not affected by the Cd absolute quantity but not in lecithin-lung weight ratio on the last days of gestation. Parturition was delayed almost a full day by the Dd treatment, and birth weights were reduced. Of the treated neonates, 11% developed respiratory distress syndrome. All but one of these individuals died and had lungs with hyaline membranes. Prenatal exposure to Cd can (1) cause lung hypoplasia, (2) affect pulmonary surfactant, and (3) induce respiratory distress syndrome in term pups.

Role of cyclic AMP and related enzymes in rat lung growth and development

Cyclic AMP levels in rat lungs showed phasic elevations which peaked during fetal, neonatal and late postnatal periods of development. Lung phospholipids showed major alterations in their levels during fetal and early neonatal life. Alterations in glycogen levels were accompanied by parallel changes in phosphorylase a/total phosphorylase activity which may be related to changes in cyclic AMP during development. Cyclic AMP levels were dependent on the relative activities of adenylate cyclase and cyclic AMP phosphodiesterase which also changed with age. Activation of adenylate cyclase by norepinephrine and NaF, and of cyclic AMP phosphodiesterase by calcium, was maximum neonatally and declined variably thereafter. These data suggest a relationship between cyclic AMP, glycogen and phospholipids during rat lung development.

Hormones and the lung. I. Thyroid hormones and glucocorticoids in lung development

AbstractIt has been proposed that thyroid hormones may have a role in the regulation of lung development and the maturation of the surfactant system. The purpose of this study was to examine the effects of exogenous thyroxine (T4) on rat lung development and to investigate possible interactions of this hormone with the glucocorticoids, other hormones implicated in the regulation of lung development.A number of different experimental protocols were utilized: T4 was administered to day 17 and 18 rat fetuses from untreated animals as well as from animals which had been adrenalectomized or treated with the 11‐β‐hydroxylase inhibitor, Metopirone; replacement hydrocortisone was administered to some groups. Sham‐operated and untreated animals were included as controls. Animals were sacrificed from days 19‐22 (term, day 22‐23) of gestation and the fetal lungs were examined. Accelerated lung development was observed in all experimental groups receiving T4. Alveolar epithelial cells were more differentiated in comparison to controls. Glycogen deposits were diminished, cell flattening was more advanced, mitochondria, Golgi complexes and rough endoplasmic reticulum were more prominent, and lamellar bodies, storage sites for pulmonary surfactant, were greatly increased in number. Secretion of lamellar bodies into the alveolar space was also stimulated. The results also indicated that T4 and glucocorticoids act together in the acceleration of lung development. Under conditions of decreased glucocorticoids (Metopirone treatment and adrenalectomy), stimulatory effects of T4 were still evident but less pronounced; hydrocortisone replacement increased the observed stimulation. Maximal acceleration of lung development was observed subsequent to T4 and uncontrolled surgical stress. These results demonstrate that T4 can accelerate rat lung development and that this acceleration is maximal in the presence of glucocorticoids.

Acetylcholinesterase- and norepinephrine-containing nerves in developing rat lung

The lungs of rat fetuses at various stages of gestation and lungs of infant rats were examined histochemically for acetylcholinesterase (AChE) and norepinephrine (NE). No AChE is present in the fetal lungs until 15 days of gestation. At this stage a number of large round cells appear which stain heavily for AChE. These cells disappear by the 18th day of development and at 18 days no AChE-positive structures are demonstrable within the lung. The large AChE-positive cells are of similar size and distribution to fluorescent cells which become apparent after treatment of the mothers with L-DOPA. At 20 days, the day before delivery, a diffuse AChE reaction appears in the walls of large branches of intrapulmonary bronchi. At 20 days, also, sparse NE-containing nerves are present near the hilum and extend along bronchial arteries into the lung. Not until birth do AChE-containing nerves appear in intrathoracic structures. These are vagal preganglionic and postganglionic fibers near the trachea, bronchi, and esophagus. AChE-positive ganglion cells are present in the walls of extrapulmonary bronchi at birth, and perimuscular nerve plexuses containing AChE are also present in the bronchial walls. NE-containing nerves are visible in several divisions of the bronchial artery at birth. Three days postnatally, AChE-containing nerves have not yet invaded intrapulmonary structures, but at this stage the adult pattern of adrenergic innervation is present. By the fifth postnatal day, sparse AChE-positive nerves are associated with intrapulmonary bronchi, and rats 9 days old present the adult pattern of cholinesterase-containing nerves.

An organ culture model for study of biochemical development of fetal rat lung

We have developed a short-term organ culture model for the study of the biochemical and morphological development of late gestation fetal rat lung. Explants (1 mm3) of 19-day lung were cultured in an oxygen enriched environment in the presence of synthetic serum-free medium for 3 days. Morphological maturation continued in culture. The rate of incorporation of choline into disaturated phosphatidylcholine and the content of this phospholipid in the explants increased in vitro in a pattern very similar to that which occurs in vivo. The activities of choline kinase and cholinephosphotransferase were also similar in cultured lung and in vivo. Studies of glucose oxidation to CO2 provided additional evidence that the explants remained viable in culture. The explants retained the sensitivity of fetal lung to hormonal action. This was demonstrated by the stimulation of choline incorporation into phospholipid by cyclic AMP and an increase in the glycogen content after exposure to insulin.

908 FETAL LUNG DEVELOPMENT AFTER AMNIOCENTESIS

The effects of amniocentesis (Ax) on the growth and development of fetal rat lung were studied in littermate fetuses subjected to minimum (Ax-Min; 0.1 ml) and maximum volume (Ax-Max; 0.5 ml+) amniocentesis at 17 days gestation. Non-operated littermates served as controls. Lungs of 12-14 fetuses/group were analyzed at term (22 days) for weight, composition (DNA, glycogen, lipid, phospholipid, phosphatidyl-choline) and histology (light and EM). Only Ax-Max reduced the weight of the lung (p>.1). DNA content was not influenced by Ax. Both Ax-Max (p>.02) and Ax-Min (p>.1) reduced lung glycogen. Ax did not alter the quantity of lung lipid at birth. The phospholipid fraction/lung was reduced (p>.05) after Ax-Max but not Ax-Min. The lungs of Ax-Max fetuses showed reduced alveolar space size and type II pneumocytes with few lamellar bodies and little glycogen. Histologic changes were not observed after Ax-Min. These studies indicated that large volume Ax reduces lung size by obliterating the fetal lung space rather than inhibiting lung cell proliferation. Ax-Max also reduces the phospholipid and surfactant pool size. Ax-min results in few detectable changes in lung growth or development. These studies explain certain aspects of the pulmonary dysfunction of infants with prolonged oligohydramnios.

Changes in the structural and metabolic heterogeneity of phosphatidylcholines in the developing rat lung

In the present study the developmental profiles of the structural and metabolic heterogeneity of rat lung phosphatidylcholine are presented. The individual molecular species of phosphatidylcholine at different stages of the developing rat lung were analyzed as diacylglycerol derivatives. The metabolic heterogeneity of rat lung phosphatidylcholine was also studied by incubation using lung slices with radioactive precursors. The results obtained were as follows: 1. 1. A significant increase of lung phosphatidylcholine during perinatal development (up to 1 day after birth) was found to be largely due to the increase of dipalmitoyl species. The percentage of palmitoyl-palmitoleoyl species also increased from −4 to −1 day of gestation, while palmitoyl-oleoyl species were found to decrease during development. Other molecular species showed no significant changes. 2. 2. The incorporation of [1- 14C]palmitoyl lysophosphatidylcholine into saturated phosphatidylcholine was relatively low at the earlier stage of fetal development, but it increased significantly in the last stage and reached its maximum at one day prior to birth and one day prior to the marked accumulation of dipalmitoylphosphatidylcholine occurring in the lung. In contrast, the incorporation of [ 3H]glycerol into saturated phosphatidylcholine remained, without showing a marked alteration during development. These results suggest that the lysophosphatidylcholine pathway which is mainly attributed to transacylation mechanism between 2 molecules of lysophosphatidylcholine may contribute to the marked production of dipalmitoylphosphatidylcholine in the lung in the last stage of gestation.

INSULIN DELAYS THE MORPHOLOGIC MATURATION OF FETAL RAT LUNG IN VITRO

Infants of diabetic mothers, who have increased circulating levels of insulin, are prone to an increased incidence of RDS. In order to evaluate the relationship between hyperinsulinism and lung maturation we have examined the influence of insulin on the morphologic development of fetal rat lung in organ culture. Ex-plants of 19 day gestation fetal lung were cultured in F12 medium, to which 1.0 u/ml insulin had been added, for 24 hours. Explants grown in F12 medium alone served as controls. After 24 hours the control cultures demonstrated evidence of continued maturation as evidenced by: larger alveolar spaces, less interstitial mesenchyme, decreased glycogen content, and increased numbers of lamellar bodies. Explants cultuted in medium containing insulin had the following features: very tall alveolar lining cells which were filled with glycogen and extended into the alveolar spaces, frequently obliterating them, more interstitial tissue and markedly fewer lamellar bodies than in the control cultures. The control cultures had 8.3±2.1 lamellar bodies per 10 alveolar lining cells, whereas the insulin treated cultures had 1.1±0.4 lamellar bodies per 10 lining cells.These observations indicate that insulin delays the appearance of lamellar bodies and increases the glycogen content of the alveolar lining cells in this culture system. Insulin may act by stimulating glycogen synthesis from glucose thereby diverting substrate away from phospholipid synthesis. Supported by USPHS grant no. HL 19752.

The influence of postnatal nutritional deprivation on the phospholipid content of developing rat lung

It has been previously reported that fasting may result in decreased lung surfactant production. In order to investigate this relationship and the role of nutrition in lung phospholipid synthesis, 21-day-old rats were exposed for 60 h to one of five dietary regimens: standard rat chow (controls), fasting, pure glucose, pure fat, or pure protein. After the period of fasting there was a 33% decrease in lung protein content, but there was no change in DNA content. Exposure to any of the experimental diets resulted in a decrease in tissue total phospholipid and phosphatidylcholine content per lung, but not per unit lung protein. Similarly lung lavage phospholipid and phosphatidylcholine content was decreased by 25% after fasting when expressed per lung or per unit DNA, but not per unit protein. Pulmonary cholinephosphotransferase (EC 2.7.8.2) activity was decreased in the fasted animals and those fed the protein diet, but not in the glucose or fatfed animals. The activities of acetyl-CoA carboxylase (EC 6.4.1.2) and microsomal fatty acid elongation were decreased in all the experimental groups except for the glucose-fed group. It is concluded that fasting results in a decrease in lung cell size but not in lung cell number. Total phospholipid and phosphatidylcholine content in lung tissue and lung lavage is decreased per cell but not per unit cell mass.

Mechanism of premature development of embryonic rat lung after unilateral pneumonectomy on the mother during pregnancy

The relationship between proliferative (mitosis) and destructive (karyolysis and karyopyknosis) processes during development of the lung was investigated in rat embryos after unilateral pneumonectomy on the mother during pregnancy. A lowering of the level of destructive processes and a sharp increase in the number of mitoses in the experimental animals compared with the controls (normal development) were found.

Fine structure of myogenesis and elastogenesis in the developing rat lung.

AbstractThe processes of myogenesis and elastogenesis are studied under the electron microscope in developing rat lungs, throughout the 15th to the 21st days of the gestation period. Myogenesis follows bronchial development and stops at the beginning of the alveolar zone, at the primitive respiratory bronchiole level. Elastogenesis appears at the periphery of the myoblasts during their differentiation. Thin myofilaments only are observed within myoblasts and their formation precedes that of dense bodies.Primitive respiratory bronchioles are visible on the 19th day and are characterized by an early elastogenesis carried out by fibroblasts. At this stage there are no elastic fibers around the alveolar tubules. Then (20th and 21st days) elastogenesis spreads throughout the alveolar zone, accompanying the alveolization process. Peculiar morphological characteristics of the pulmonary fibroblast are underlined. In relation to both muscular cells and fibroblasts the fine structural features of the rat pulmonary elastogenesis are identical to those previously described in other organs. Myoblasts and fibroblasts probably originate from the same primitive mesenchymal cell. Their differentiation depends on the zone where they are located. The relations between connective tissue and epithelial cell differentiation suggest a control of lung development by means of reciprocal induction processes.

Fine structural observations of ruthenium red binding in developing and adult rat lung.

AbstractThe ruthenium red staining technique of Luft ('71b) was utilized in an electron microscopic investigation of developing and adult rat lung. Electron‐dense deposits of ruthenium red‐positive material were observed on all exposed surfaces of the tissue block, regardless of the stage of development. In the more central areas of the block, sites of ruthenium red binding changed with age. In early prenatal lung (days 16 to 20) dense accumulations of ruthenium red‐positive material were found in association with the basement membranes of endodermal epithelial cells. Ruthenium red binding was also observed between adjacent epithelial cells; however, their luminal surfaces were negative. The main intracellular site of ruthenium red binding in intact cells was the lamellar body of the developing type II pulmonary epithelial cells. By day 21 of development, accumulations of granular product were observed in association with most lamellar bodies, as well as on epithelial cell luminal surfaces. Ruthenium red binding in postnatal tissue decreases with increasing age. By the second postnatal week, the predominant site of binding is the luminal surface of the type I and type II pulmonary epithelial cells. When compared to fetal and early neonatal stages, adult rat lung has a still more limited distribution of ruthenium red‐positive material. Changes in the distribution of ruthenium red‐positive material correlate with numerous morphologic and biochemical events in rat lung development.

Choline phosphokinase, phosphorylcholine cytidyltransferase and CDP-choline: 1,2-diglyceride cholinephosphotransferase activity in developing rat lung.

CHIDA, N., HIRONO, H., NISHIMURA, Y. and ARAKAWA, Ts., Choline Phosphokinase, Phosphorylcholine Cytidyltransferase and CDP-choline-1, 2-diglyceride Cholinephosphotransferase Activity in Developing Rat Lung. Tohoku J. exp. Med., 1973, 110 (3), 273-282-Incorporation of choline-methyl-14C into phosphorylcholine, CDP-choline, and phosphatidylcholine by lung tissue of developing rats was studied. Choline phosphokinase, phosphorylcholine cytidyltransferase, and CDP-choline: 1, 2-diglyceride choline phosphotransferase were determined in the lung tissue of developing rats. Results indicated that phosphorylcholine cytidyltransferase was the rate-limiting enzyme in the reactions-choline→phosphoryl-choline→CDP-choline→phosphatidylcholine

Fetal rat lung development: lipids and surface tension properties after decapitation in utero.

SummaryDecapitation in utero produces hormonal deficiencies which do not influence lung organogenesis but retards pneumocyte differentiation at the organelle and molecular levels. Such lungs contain decreased quantities of phospholipid and functionally are impaired in their ability to reduce surface tension to normal levels. The mechanisms by which these alterations are produced are most likely related to hormonal deficiencies during fetal development. These deficiencies may specifically alter lung phospholipid metabolism. Retardation of phospholipid metabolism may, however, be secondary to a more generalized retardation of pneumocyte differentiation. We favor the latter hypothesis in so far as pituitary failure occurring in man and animals with fully mature lungs does not lead to clinical respiratory distress (33, 38). Further inquiry into the manner in which hormones influence lung metabolism will require evaluation of the effect of specific hormonal replacements on in utero decapitated or hypophysectom...