Laurice I. Gobran

Development of glycogen and phospholipid metabolism in fetal and newborn rat lung

Glucose, a major metabolic substrate for the mammalian fetus, probably makes significant contributions to surface active phospholipid synthesis in adult lung. We examined the developmental patterns of glycogen content, glycogen synthase activity, glycogen phosphorylase activity and glucose oxidation in fetal and newborn rat lung. These patterns were correlated with the development of phosphatidylcholine synthesis, content and the activities of enzymes involved in phosphatidylcholine synthesis. Fetal lung glycogen concentration increased until day 20 of gestation (term is 22 days) after which it declined to low levels. Activity of both glycogen synthase I and total glycogen synthase (I + D) in fetal lung increased late in gestation. Increased lung glycogen concentration preceded changes in enzyme activity. Glycogen phosphorylase a and total glycogen phosphorylase (a + b) activity in fetal lung increased during the period of prenatal glycogen depletion. The activity of the pentose phosphate pathway, as measured by the ratio of CO2 derived from oxidation of C1 and C6 of glucose, declined after birth. Fetal lung total phospholipid, phosphatidycholine and disaturated phosphatidylcholine content increased by 60, 90 and 180%, respectively, between day 19 of gestation and the first postnatal day. Incorporation of choline into phosphatidylcholine and disaturated phosphatidylcholine increased 10-fold during this time. No changes in phosphatidylcholine enzyme activities were noted during gestation, but both choline phosphate cytidylyltransferase and phosphatidate phosphatase activity increased after birth. The possible contributions of carbohydrate derived from fetal lung glycogen to phospholipid synthesis are discussed.

Effects of betamethasone on phospholipid content, composition and biosynthesis in the fetal rabbit lung.

Administration of betamethasone (0.2 mg/kg, intramuscularly) to pregnant rabbits had the following effects on the fetal lung at 26--27 days gestation. It increased the amount of phosphatidylcholine in lung lavage by 70% and almost doubled the phosphatidylcholine/sphingomyelin ratio, it increased the rate of incorporation of choline into phosphatidylcholine in fetal lung slices by up to 90%, it increased the activities of pulmonary cholinephosphate cytidylyltransferase and phosphatidate phosphatase by 50% and it reduced the amount of lung glycogen to 60% of the amount in the controls. Betamethasone had no effect on the activities of pulmonary cholinephosphotransferase or lysolecithin: lysolecithin acyltransferase but it slightly decreased the activity of choline kinase. Betamethasone administration to the doe did not increase the amount of surfactant phospholipid in fetal lung lavage to as great an extent as did direct administration of cortisol to the fetuses. Neither did betamethasone stimulate the activity of pulmonary cholinephosphotransferase. These data suggest that agents other than glucocorticoids mediate the stress-induced acceleration of fetal lung maturation and surfactant production.

Phospholipid content, composition and biosynthesis during fetal lung development in the rabbit

The phospholipid content and composition of lung wash and lung tissue as well as the activities of the enzymes involved in the synthesis of phosphatidylcholine and phosphatidylglycerol (the major surface active components of pulmonary surfactant) were studied in the rabbit during fetal lung development. In lung wash the amount of phospholipid increased four-fold during the period 27-31 days gestation. There was a further ten-fold increase following the onset breathing. During the same period the amount of phosphatidylcholine in lung wash increased from 29% of the total phospholipid to 80% while the amount of sphingomyelin decreased from 38% to 2%. The amount of phosphatidylcholine in lung tissue also increased during development but to a much lesser extent. During fetal lung development the activities of choline kinase and cholinephosphate cytidyltransferase changed little, cholinephosphotranserase decreased while lysophosphatidic acid acyltransferase and lysolecithin acyltransferase increased. There was a postnatal increase in the activities of cholinephosphate cytidyltransferase, cholinephosphotransferase and both acyltransferases. The amount of phosphatidylglycerol, as a percentage of the total phospholipid, in lung wash and lung tissue as well as the activity of pulmonary glycerolphosphate phosphatidyltransferase did not change appreciably during development.

Stimulation of phosphatidylcholine synthesis by 17β-estradiol in fetal rabbit lung

Abstract We investigated the mechanism by which estrogen stimulates pulmonary surfactant production in the fetal rabbit. Maternal administration of 17β-estradiol (5–75 μg) on day 25 of gestation resulted in a greater than twofold increase in the rate of choline incoporation into phosphatidylcholine in fetal lung slices on day 26 (full term = 31 days). Estrogen administration increased the activity of fetal lung cholinephosphate cytidylyltransferase by 62%. It had no effect on the liver enzyme. When assayed in the presence of phosphatidylglycerol fetal lung cholinephosphate cytidylyltransferase activity was increased 4.6-fold but it was not influenced by estrogen under these conditions. These findings suggest that estrogen stimulates cholinephosphate cytidyltransferase by increasing the activity of existing enzyme (possibly by increasing the amount of phosphatidylglycerol or other acidic phospholipid in the tissue) rather than by increasing the amount of enzyme-protein. Stimulation of fetal lung cholinephosphate cytidylyltransferase by estrogen as well as by glucocorticoids (Rooney, S.A., Gobran, L.I., Marino, P.A., Maniscalco, W.M., and Gross, I. (1979) Biochim. Biophys, Acta 572, 64–76) suggest that this enzyme may be rate-regulatory in the de novo biosynthesis of phosphatidylcholine. Estrogen administration also resulted in a 26% increase in the activity of pulmonary lysolecithin acyltransferase, an enzyme involved in the synthesis of disaturated, surface-active phosphatidylcholine. Lung choline kinase was slightly decreased following estrogen treatment bu ethanolaminephosphate cytidylyltransferase, cholinephosphotransferase, phosphatidate phosphatase and lysolecithin : lysolecithin acyltransferase were unaffected.

Thyroid Hormone Opposes Some Glucocorticoid Effects on Glycogen Content and Lipid Synthesis in Developing Fetal Rat Lung

ABSTRACT. Because of current interest in use of a combination of glucocorticoid and thyroid hormones for prevention of respiratory distress syndrome we examined the effects of dexamethasone and triiodothyronine (T3), alone and in combination, on glycogen content and rates of fatty acid and phosphatidylcholine synthesis in fetal rat lung. The hormones were administered to the mothers on the 2 days before delivery on days 17-22 of gestation. Both hormones increased the rate of choline incorporation into phosphatidylcholine, an index of surfactant synthesis, on day 20 just prior to the normal developmental surge but had no effect on this parameter on days 19, 21, or 22. There is a developmental increase in lung glycogen on days 17-20 with a decrease thereafter and a developmental increase in the rate of fatty acid synthesis between days 20 and 21. The increases in glycogen content and fatty acid synthesis were accelerated by dexamethasone and prevented by T3 and when the hormones were administered together T3 antagonized the stimulatory effects of dexamethasone on these parameters. Both dexamethasone and T3 accelerated the normal developmental decrease in lung glycogen later in gestation and the effects of the two hormones on this parameter were additive. The combination of dexamethasone and T3 led to significantly smaller fetuses and increased mortality late in gestation. These data show that glucocorticoid and thyroid hormones have opposite as well as common effects on parameters of fetal lung maturation. Although the relationship between changes in lung glycogen or fatty acid synthesis and surfactant production are not known the combination of hormones may be beneficial at certain gestational ages but harmful at others.

Stimulation of Surfactant Production by Oxytocin-Induced Labor in the Rabbit

The respiratory distress syndrome is believed to be due to insufficient surfactant. It is known that there is a greater incidence of the respiratory distress syndrome among infants delivered by cesarean section before labor than among those delivered after labor at the same gestational age. The purpose of this study was to determine the effect of labor on the production of pulmonary surfactant. We measured the phospholipid content of lung lavage in newborn rabbits delivered by cesarean section before labor at 29, 30, and 31 (full-term) days gestation and after oxytocin-induced labor at 31 days. We also measured the activities of pulmonary cholinephosphate cytidylyltransferase and choline-phosphotransferase, enzymes involved in the de novo synthesis of phosphatidylcholine, the major component of surfactant. There was a two- to fourfold increase in the amount of lung lavage phospholipid during the first 6 h after birth. This was not dependent upon gestational age at delivery. There was a further two- to fourfold increase in the next 18 h which was, however, dependent upon gestational age. Labor increased the amount of lavage phospholipid from rabbits delivered at full term by 132%, 177%, and 50% at 3, 6, and 24 h after birth, respectively. There was a postnatal increase in the activity of cholinephosphate cytidylyltransferase. This was almost linear with time during the first 12 h, by which time essentially adult values were attained. Choline-phosphate cytidylyltransferase was not affected by labor. There was also a postnatal increase in the activity of cholinephosphotransferase but this was stimulated 86%, 59%, and 21% by labor at 0, 1, and 24 h after birth, respectively. These studies suggest that labor stimulates both the synthesis and secretion of surfactant in the immediate postnatal period and thus may be an important factor in the prevention of the respiratory distress syndrome of the newborn.

Thyrotropin-Releasing Hormone Increases the Amount of Surfactant in Lung Lavage from Fetal Rabbits

Summary: Administration of thyrotropin-releasing hormone (TRH) to pregnant rabbits at 25 and 26 days of gestation results in increased pulmonary surfactant production by the fetus at 27 days (full term is 31 days). There was 60% more total phospholipid and 150% more phosphatidylcholine (the major component of surfactant) in the lung lavage from the fetuses in the treated group than in that from the controls. Lung lavage from the fetuses in the treated litters contained 13.4 ± 1.6 μg of total phospholipid phosphorus/g lung dry wt and 5.6 ± 1.1 μg of phosphatidylcholine phosphorus while that from the fetuses in the control litters contained only 8.2 ± 1.1 μg and 2.2 ± 0.4 μg, respectively. The phosphatidylcholine/sphingomyelin ratio increased from 1.0 in the lavage from the controls to 2.2 in that from the treated group. These changes in lung lavage phospholipid content and composition are in the direction of increased lung maturation. TRH administration had no effect on the incorporation of choline into phosphatidylcholine in fetal lung slices. These data suggest that TRH stimulates surfactant release rather than synthesis.Speculation: TRH has a physiologic role in fetal lung maturation and surfactant production. It may potentially be used in the prevention of the respiratory distress syndrome in humans.

The phospholipids of rabbit type II alveolar epithelial cells: comparison with lung lavage, lung tissue, alveolar macrophages, and a human alveolar tumor cell line.

The phospholipid composition of type II alveolar epithelial cells from the rabbit was compared with that of alveolar macrophages, lung lavage and lung tissue. In addition, the phospholipid composition of a human alveolar tumor cell line, which is morphologically similar to type II cells, was examined. Phosphatidylcholine accounted for 48% of the total phospholipid in the type II cells, 41% in the tumor cells, and 30% in the macrophages. Phosphatidylcholine was 51% disaturated in the type II cells, 54% in lung lavage, 39% in whole lung, 29% in lavaged lung and macrophages, and 16% in the tumor cells. Palmitic acid was the major fatty acid in phosphatidylcholine from all samples with the exception of the tumor cells in which almost half of the fatty acids were accounted for by oleic acid. The phospholipids of the type II cells were more similar to those of lung lavage, and thus surfactant, than to lung tissue and macrophages. This is consistent with their supposed role in surfactant production. The tumor cells, although morphologically similar to type II cells, were quite different with respect to phospholipid composition.

Alveolar lavage and lavaged lung tissue phosphatidylcholine composition during fetal rabbit development.

Pulmonary surfactant, the major surface-active component of which in the adult is dipalmitoylglycerophosphocholine, can be obtained by lavaging lungs with physiological saline. We have previously shown that there is an increase in the amount of phosphatidylcholine in fetal rabbit lung lavage during the latter part of gestation. We have now measured the amount of disaturated phosphatidylcholine as well as the fatty acid composition of phosphatidylcholine in lung lavage from fetal rabbits during the period 27 days’ gestation to full term (31 days). There was no developmental change in the amount of disaturated phosphatidylcholine during the period examined. About 50% of the total phosphatidylcholine was disaturated which is approximately the same as in adult rabbit lung lavage. The fatty acid composition, however, did change. There was an increase in the amount of 16∶0 from about 20% of the total fatty acids in phosphatidylcholine at 27–28 days to about 60% at full term, after birth, and in the adults. There was a corresponding decrease in the amounts of 14∶0, 18∶0, and longer chain fatty acids, most of which were saturated. In the lavaged lung tissue, there was a 2.6-fold increase in the percentage of phosphatidylcholine which was disaturated during the period 27–31 days’ gestation. It had not decrease to the adult value 24 hr after birth.

PKC isoforms and other signaling proteins involved in surfactant secretion in developing rat type II cells

We previously reported that there is a developmental increase in surfactant secretion in response to P2Y2 purinoceptor agonists. UTP does not stimulate secretion in type II cells from 1- or 2-day-old rats; there is a small response to UTP in cells from 4-day-old animals, and the response increases with increasing age thereafter. Second messenger formation in response to P2Y2 agonists has a similar developmental pattern. We have investigated whether the failure to respond to P2Y2 agonists is due to a deficiency in the P2Y2 receptor or in downstream signaling factors. We compared type II cells from adult and 1- to 2-day-old rats with respect to expression of the P2Y2 receptor gene and the levels of phospholipase C-β (PLC-β) and protein kinase C (PKC) isomers and of the α-subunit of the GTP-binding protein Gq. We measured gene expression by reverse transcriptase-polymerase chain reaction and protein levels by immunoblotting. We identified PKC-α, -βI, -βII, -δ, -η, -ζ, -θ, and -μ, PLC-β3, and Gqα in adult and newborn type II cells. PKC-ε, -γ, and -λ and PLC-β1, -β2, and -β4 were not present in adult or newborn type II cells. Expression of the P2Y2 receptor gene was essentially the same in newborn and adult cells. However, the levels of PKC-α, -βI, -βII, and -ζ in newborn type II cells were only 43-57% those of adult cells. The level of PKC-θ also tended to be lower in the newborn cells. There was little difference between newborn and adult type II cells in the levels of PKC-δ, -η, and -μ, PLC-β3, and Gqα. These data suggest that the lack of response of early newborn type II cells to P2Y2 agonists is not due to a lack of expression of the receptor gene but possibly to insufficient amounts of one or more of the α, βI, βII, or ζ PKC isoforms.