Seamus A. Rooney

Molecular and cellular processing of lung surfactant.

Pulmonary surfactant, a complex material that lines the alveolar surface of the lung, is synthesized in the type II pneumocyte. Surfactant consists largely of phospholipids, of which phosphatidylcholine∗∗∗ is by far the most abundant component, and is mainly responsible for surface activity. Surfactant also contains four unique proteins, surfactant protein (SP)‐A, SP‐B, SP‐C, and SP‐D, which are synthesized in a lung‐specific manner. SP‐A and SP‐D are glycoproteins (Mr ã 30,000‐40,000) whereas SP‐B and SP‐C are small (Mr ã 5,000‐18,000), extremely hydrophobic proteolipids released from large precursors by proteolysis. Synthesis of surfactant lipids and proteins is developmentally regulated in fetal lung and can be accelerated by glucocorticoids and other hormones. Developing fetal lung in vivo and in organ culture has been used extensively to study regulation of surfactant synthesis and gene expression. Glucocorticoids stimulate the rate of fetal lung phosphatidylcholine biosynthesis and the activity of the rate‐regulatory enzyme, choline‐phosphate cytidylyltransferase (CYT). The hormone, however, does not increase the amount of CYT; there is evidence that the increase in activity is mediated by increased fatty biosynthesis due to enhanced expression of the fatty acid synthase gene. Glucocorticoids also regulate expression of the SP‐A, SP‐B, and SP‐C genes in the late gestation fetal lung. Hormone response elements and other cis‐acting regulatory elements have been identified in the 5‐flanking regions of the SP‐A, SP‐B, and SP‐C genes. Surfactant phospholipids are stored in lamellar bodies, secretory granules in the type II cell, and secreted by exocytosis. Lamellar bodies are also rich in SP‐B and SP‐C but there are conflicting data on the cellular distribution of SP‐A. Secretion of SP‐A may be constitutive and occur independently of lamellar bodies. Phosphatidylcholine secretion is a regulated process, and in isolated type II cells it can be stimulated by physiological and other agents that act via at least three signal‐transduction mechanisms. After secretion, surfactant is transformed into tubular myelin, and the lipid and protein components are separated as the lipid is inserted into a monolayer at the air‐liquid interface. The majority of surfactant is removed from the alveolar space by reuptake into the type II cell by mechanisms that may include receptor‐mediated endocytosis. Some components of surfactant are directly recycled into new surfactant whereas other components are degraded.— ‐Rooney, S. A., Young, S. L., Mendelson, C. R. Molecular and cellular processing of lung surfactant. FASEB J. 8: 957‐967; 1994.

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.

Regulation of surfactant secretion

Lung surfactant is synthesized in the alveolar type II cell. Its lipids and hydrophobic proteins (SP-B and SP-C) are stored in lamellar bodies and secreted by regulated exocytosis. In contrast, the hydrophilic proteins (SP-A and SP-D) appear to be secreted independently of lamellar bodies. Regulation of surfactant secretion is mediated by at least three distinct signaling mechanisms: activation of adenylate cyclase with formation of cAMP and activation of cAMP-dependent protein kinase; activation of protein kinase C; and a Ca(2+)-regulated mechanism that likely results in the activation of Ca(2+)-calmodulin-dependent protein kinase. These signaling mechanisms are activated by a variety of agonists, some of which may have a physiological role. ATP is one such agent and it activates all three signaling mechanisms. There is increasing information on the identity of several of the signaling proteins involved in surfactant secretion although others remain to be established. In particular the identity of the phospholipase C, protein kinase C and phospholipase D isomers expressed in the type II cell and/or involved in surfactant secretion has been established. Distal steps in the secretory pathway beyond protein kinase activation as well as the physiological regulation of surfactant secretion, are major issues that need to be addressed.

The identification of phosphatidylglycerol in the rat, rabbit, monkey and human lung

Abstract There is some controversy in the literature as to whether phosphatidylglycerol, phosphatidyldimethylethanolamine or both are present in the lung. In order to clarify the confusion, we have studied the lung lipid composition of the adult rabbit, rat and rhesus monkey. We have also examined the lipid composition of fetal lung fluid and adult tracheobrorichial aspirates and lung wash from humans. 1. 1. In the rabbit, neutral lipid accounts for 50% of the lipid in whole lung but only 5% of that in lung wash. Phosphatidylcholine accounts for over 50% of the phospholipid in whole lung and 80–90% of that in lung wash. In addition, phosphatidylethanolamine and sphingomyelin are major components of the whole lung phospholipid but are present in only trace amounts in lung wash. Phosphatidylglycerol is the second most abundant phospholipid in lung wash where it accounts for 2–9% of the phospholipid. The phospholipid composition of rat and monkey whole lung is similar to that of rabbit. 2. 2. Phosphatidylglycerol was identified by comparison of its Chromatographic properties with those of standard Phosphatidylglycerol, by its glycerol/ phosphate ratio and by identification of its deacylation product. 3. 3. Phosphatidylglycerol is also present in the human lung material examined, where it accounts for up to 14% of the total phospholipid. 4. 4. A highly surface active fraction of phosphatidylglycerol from rabbit lung wash was obtained by mercuric acetate adduction of the unsaturated species. 5. 5. Phosphatidyldimethylethanolamine was not detectable in any sample studied.

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.

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

Abstract 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- 3 H]choline incorporation into phosphatidylcholine, disaturated phosphatidylcholine and sphingomyelin. Incorporation of [ 3 H]acetate into total phospholipid was increased by 63% ( P P 3 H] acetate in the various phospholipid fractions, it was found that there was a 29% increase in the phos-phatidylglycerol fraction ( P P P 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.

Influence of epidermal growth factor on fetal rat lung development in vitro.

ABSTRACT. Epidermal growth factor (EGF) has been shown to enhance cell multiplication or differentiation in a number of developing tissues. We have examined the effects of this growth factor on the biochemical development of explants of fetal rat lung, cultured in serum-free medium for 48 h. EGF enhanced the rate of choline incorporation into phosphatidylcholine and disaturated phosphatidylcholine in a dose dependent fashion. Half maximal stimulation occurred at a concentration of 1.0 nM, similar to the Kd for EGF binding to rat lung cell membranes. There was also significant stimulation of acetate incorporation into all phospholipids, particularly phosphatidylglycerol (539%), and increased distribution of radioactivity from acetate in this phospholipid fraction. Exposure to EGF stimulated PC synthesis in 18- and 19-day explants (term is 22 days) whereas maximal enhancement of DNA synthesis occurred after this time. This sequence differs from that observed during early embryonic development when EGF initially enhances cell multiplication. An additive interaction with regard to enhancement of PC synthesis was observed with EGF and thyroid hormone, but not EGF and dexamethasone. EGF had no effect on the activity of the enzymes of the choline incorporation pathway of phosphatidylcholine synthesis or on the activity of enzymes involved with acidic phospholipid synthesis. Fetal lung EGF content and EGF binding capacity were not increased by glucocorticoid treatment and similarly glucocorticoid binding capacity was not increased by EGF. These data indicate that EGF enhances fetal rat lung phospholipid synthesis in a dose-dependent manner and suggest that this is a direct effect on the lung tissue mediated by specific receptors.

Phospholipid biosynthesis and secretion by a cell line (A549) which resembles type II alveolar epithelial cells

The A549 cell line is a continuous cell line derived from a human adenocarcinoma of the lung. At low cell population density the cells contain relatively few lamellar bodies, but in mature cells in very confluent cultures lamellar bodies are abundant. The lamellar bodies from these cells are enriched for phosphatidylcholine and disaturated phosphatidylcholine. In mature cells, 45% of newly synthesized phosphatidylcholine is disaturated. Stimulation with the calcium ionophore A23187 produces exocytosis of phosphatidylcholine (46% disaturated). The A549 cell synthesizes, stores in lamellar bodies, and secretes phosphatidylcholine, and thus has many important biological properties of the alveolar epithelial type II cell.

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.