Lung Surfactant Synthesis Research Articles

A novel LysoPhosphatidylcholine Acyl Transferase Activity is Expressed by Peroxiredoxin 6

The phospholipase A2 (PLA2) activity of peroxiredoxin 6 (Prdx6) has important physiological roles in the synthesis of lung surfactant and in the repair of peroxidized cell membranes. These functions require the activity of a lysopholipid acyl transferase as a critical component of the phospholipid remodeling pathway. We now describe a lysophosphatidylcholine acyl transferase (LPCAT) activity for Prdx6 that showed a strong preference for choline as the lysophospholipid head group and for palmitoyl CoA as the fatty acyl substrate. The calculated kinetic constants for acylation were Km 18 mM and Vmax 30 nmol/min/mg protein; the Vmax was increased 25 fold by phosphorylation of the protein while Km was unchanged. Study of recombinant protein in vitro and in mouse pulmonary microvascular endothelial cells infected with a lentiviral vector construct indicated that amino acid D31 is crucial for LPCAT activity. A linear incorporation of labeled fatty acyl CoA into dipalmitoyl PC indicated that lysoPC generated by Prdx6 PLA2 activity remains bound to the enzyme for the reacylation reaction. Prdx6 is the first LPCAT enzyme with demonstrated cytoplasmic localization. Thus, Prdx6 is a complete enzyme comprising both PLA2 and LPCAT activities for the remodeling pathway of phosphatidylcholine synthesis or for repair of membrane lipid peroxidation.

A novel lysophosphatidylcholine acyl transferase activity is expressed by peroxiredoxin 6

The phospholipase A2(PLA2) activity of peroxiredoxin (Prdx)6 has important physiological roles in the synthesis of lung surfactant and in the repair of peroxidized cell membranes. These functions require the activity of a lysophospholipid acyl transferase as a critical component of the phospholipid remodeling pathway. We now describe a lysophosphatidylcholine acyl transferase (LPCAT) activity for Prdx6 that showed a strong preference for lysophosphatidylcholine (LPC) as the head group and for palmitoyl CoA in the acylation reaction. The calculated kinetic constants for acylation wereKm18 μM andVmax30 nmol/min/mg protein; theVmaxwas increased 25-fold by phosphorylation of the protein whileKmwas unchanged. Study of recombinant protein in vitro and in mouse pulmonary microvascular endothelial cells infected with a lentiviral vector construct indicated that amino acid D31 is crucial for LPCAT activity. A linear incorporation of labeled fatty acyl CoA into dipalmitoyl phosphatidylcholine (PC) indicated that LPC generated by Prdx6 PLA2activity remained bound to the enzyme for the reacylation reaction. Prdx6 is the first LPCAT enzyme with demonstrated cytoplasmic localization. Thus, Prdx6 is a complete enzyme comprising both PLA2and LPCAT activities for the remodeling pathway of PC synthesis or for repair of membrane lipid peroxidation.

Acyl-CoA:Lysophosphatidylcholine Acyltransferase I (Lpcat1) Catalyzes Histone Protein O-Palmitoylation to Regulate mRNA Synthesis

The enzyme acyl-CoA:lysophosphatidylcholine acyltransferase (Lpcat1) is a critical cytosolic enzyme needed for lung surfactant synthesis that catalyzes an acyltransferase reaction by adding a palmitate to the sn-2 position of lysophospholipids. Here we report that histone H4 protein is subject to palmitoylation catalyzed by Lpcat1 in a calcium-regulated manner. Cytosolic Lpcat1 was observed to shift into the nucleus in lung epithelia in response to exogenous Ca(2+). Nuclear Lpcat1 colocalizes with and binds to histone H4, where it catalyzes histone H4 palmitoylation. Mutagenesis studies demonstrated that Ser(47) within histone H4 serves as a putative acceptor site, indicative of Lpcat1-mediated O-palmitoylation. Lpcat1 knockdown or expression of a histone H4 Ser(47A) mutant protein in cells decreased cellular mRNA synthesis. These findings provide the first evidence of a protein substrate for Lpcat1 and reveal that histone lipidation may occur through its O-palmitoylation as a novel post-translational modification. This epigenetic modification regulates global gene transcriptional activity.

Mild stretch activates cPLA 2 in alveolar type II epithelial cells independently through the MEK/ERK and PI3K pathways

Alveolar epithelial type II cells (AT II) in which lung surfactant synthesis and secretion take place, are subjected to low magnitude stretch during normal breathing. The aim of the study was to explore the effect of mild stretch on phospholipase A 2 (PLA 2) activation, an enzyme known to be involved in surfactant secretion. In A549 cells (a model of AT II cells), we showed, using a fluorometric assay, that stretch triggers an increase of total PLA 2 activity. Western blot experiments revealed that the cytosolic isoform cPLA 2 is rapidly phosphorylated under stretch, in addition to a modest increase in cPLA 2 mRNA levels. Treatment of A549 cells with selective inhibitors of the MEK/ERK pathway significantly attenuated the stretch-induced cPLA 2 phosphorylation. A strong interaction of cPLA 2 and pERK enzymes was demonstrated by immunoprecipitation. We also found that inhibition of PI3K pathway attenuated cPLA 2 activation after stretch, without affecting pERK levels. Our results suggest that low magnitude stretch can induce cPLA 2 phosphorylation through the MEK/ERK and PI3K–Akt pathways, independently.

Effects of terbutaline on lung surfactant components in fetal lung tissue, fetal lung washing and amniotic fluid in pregnant rabbit.

Synopsis Terbutaline (0.1 mg/kg) was given intramuscularly once or 3 times at intervals of 12 hours to pregnant rabbits. Fetuses were delivered at 28 days of gestation by cesarean section 4 hours after the last administration. In the 3-dose group, there were no changes in concentration of disaturated phosphatidylcholine in fetal lung tissue or of total phospholipid in the lung washing. Nor was there an increased number of lamellar bodies in type-II pneumocytes, but total phosphatidylcholine levels in amniotic fluid showed a significant increase (2.4 times the control group). In the single-dose group, levels of both total phospholipids and total phosphatidylcholine increased significantly, while lamellar bodies in type-II pneumocytes showed a significant decrease. This suggests that terbutaline is not involved in the synthesis of lung surfactant but enhances secretion and exerts a beneficial action upon fetal lung adaptability to the outside uterine environment through alveolar stabilization.

Leptin stimulatesXenopuslung development: evolution in a dish

The transition from uni- to multicellular organisms required metabolic cooperativity through cell-cell interactions mediated by soluble growth factors. We have empirically demonstrated such an integrating mechanism by which the metabolic hormone leptin stimulates lung development, causing the thinning of the gas exchange surface and the obligate increase in lung surfactant synthesis. All of these processes have occurred both phylogenetically and developmentally during the course of vertebrate lung evolution from fish to man. Here we show the integrating effects of the environmentally sensitive, pleiotropic hormone leptin on the development of the Xenopus laevis tadpole lung. The process described in this study provides a mechanistically integrated link between the metabolic regulatory hormone leptin and its manifold downstream effects on a wide variety of physiologic structures and functions, including locomotion and respiration, the cornerstones of land vertebrate evolution. It provides physiologic selection pressure at multiple levels to progressively generate Gene Regulatory Networks both within and between organs, from cells to systems. This model provides a cipher for understanding the evolution of complex physiology.

Effects of epidermal growth factor (EGF) on the development of EGF‐receptor (EGF‐R) binding in fetal rabbit lung organ culture

Epidermal growth factor (EGF) causes gender- and development-specific changes in fetal lung surfactant synthesis. We hypothesized that the effects of EGF on development of surfactant synthesis are related to effects on EGF receptor (EGF-R) expression. We prepared sex-specific fetal rabbit lung organ cultures on gestational days 21 and 24 (term = 31 days) in Waymouth's medium + 10% charcoal-stripped fetal calf serum as control or with added EGF (10 ng/mL). After 3, 5, and 7 days of culture, we measured specific EGF-R binding in fetal lung plasma membrane preparations. Analysis of variance (ANOVA) revealed significant effects of fetal gender (P = 0.0003), time in culture (P = 0.01), and EGF treatment (P = 0.0003) on EGF specific binding. In control cultures from days 21 and 24 (both male and female), EGF specific binding tended to decrease with time in culture. Specific binding in EGF-treated female 21-day cultures was significantly higher than in controls, both after 5 days (184% of control, P = 0.007) and after 7 days (151% of control, P = 0.01; Bonferroni multiple comparisons) of treatment, whereas males exhibited no response to EGF treatment. As opposed to these effects in 21-day cultures, EGF had little effect on 24-day cultures. We conclude that EGF affects the expression of the EGF-R on EGF specific binding in the fetal lung. The development of surfactant synthesis in the fetal lung may be controlled by upregulation of the EGF-R. Pediatr Pulmonol. 2000; 29:27–33. © 2000 Wiley-Liss, Inc.

Effect of epidermal growth factor on enzymes of phospholipid biosynthesis in lung and liver of fetal rat in vivo and in vitro.

Epidermal growth factor (EGF), a mitogenic polypeptide that binds to cell surface receptors, is an important regulator of cell differentiation and fetal lung surfactant synthesis, and may be used as a potential novel therapeutic agent in prematurity. Nevertheless, the distinct role in lung development and its mechanisms of action are not well understood. We investigated in vivo the systemic effect of intrafetally administered EGF (200 ng/g fetal body weight) and maternally administered dexamethasone (DEXA; 0.2 and 2.0mg/kg maternal body weight) on the activity of important enzymes of the phospholipid synthesis in the fetal rat lung and liver: choline kinase (EC 2.7.1.32), cholinephosphate cytidyltransferase (EC 2.7.7.15), choline phosphotransferase (EC 2.7.8.2), lysolecithin acyltransferase (EC 2.3.1.23) and glycerolphosphate phosphatidyltransferase (EC 2.7.8.5). Additionally, in vivo and in vitro effects of DEXA on EGF receptor synthesis, and the effects of EGF on protein content and morphogenesis of the fetal rat lung organoid culture, were evaluated. Whereas DEXA induced the activity of all investigated enzymes of phospholipid synthesis and increased EGF receptor synthesis, EGF has no effects on the enzymes, either in vivo or in vitro. EGF enhanced protein synthesis and morphogenesis in vitro. With respect to our data and the literature, we hypothesize that DEXA and EGF may act on different cellular sides. Whereas glucocorticoids induce surfactant phospholipid synthesis, EGF should be more involved in cell proliferation and morphogenesis.

ANTIOXIDANT ENZYMES, LIPID PEROXIDATION AND RED BLOOD CELL DEFORMABILITY IN CORD AND MATERNAL BLOOD. EFFECT OF DEXAMETHASONE ON OXIDATIVE SENSITIVITY. 1386

Background: The antioxidant enzyme activities (AOEAs) increase in parallel with lung surfactant synthesis and these enzymes display lower activities during intrauterin life and in preterm than in full-term infants. The red blood cell (RBC) deformality is also decreased in newborns in comperison with adults. The aim of our study was to determine activities of superoxide dismutase (SOD), catalase (C-ase) glutathione peroxidase (GP-ase), lipid peroxidation (LP) and RBC deformability in maternal blood, in neonates and premature infants of various gestational ages and also to establish the influence of dexamethasone (Oradexon) applied antenatally upon the AOEAs and deformability.

A novel pharmacological approach for paraquat poisoning in rat and A549 cell line using ambroxol, a lung surfactant synthesis inducer

Paraquat (PQ) is a widely used herbicide that causes acute adult respiratory distress syndrome (ARDS) and chronic lung damage (diffuse fibrosis). One of the earliest biochemical effects induced by PQ is damage to type II pneumocytes with consequent depletion of surfactant. With the aim of counteracting the toxic effects of PQ, a series of investigations were performed into the possible protective effect of the drug ambroxol, which induces the synthesis of surfactant in lung alveolar type II cells. The number of survivors and survival time of rats treated ip with 35 mg PQ/kg was significantly increased by 3 days of ambroxol pretreatment and by ambroxol treatment 30 min or 2 hr after PQ. Total phospholipid content in lung and bronchoalveolar lavage fluid (BALF) was significantly reduced 30 hr after treatment with PQ alone. The association of ambroxol with PQ significantly antagonized this reduction. In BALF the ratio between palmitic acid and stearic acid concentrations was significantly lower in animals treated with PQ alone but was returned to normal by the association with ambroxol. The cell line A549, exposed in vitro to PQ concentrations from 0.5 × 10 −4 to 2 × 10 −3 m, showed a significant dose-dependent loss of viability. Cells pretreated with ambroxol (10 mg/ml) were more resistant to PQ and their viability started to decrease significantly only from a PQ concentration of 0.8 × 10 −3 m. Membrane microviscosity was measured on the same cells. Cells treated with PQ alone showed a reduction of membrane microviscosity, which was significantly counteracted by ambroxol pretreatment. The curves of modification of membrane microviscosity of cells treated with PQ and with ambroxol plus PQ paralleled those of cell viability, indicating that the stimulation of surfactant synthesis in vitro may be a prerequisite for counteracting some of the early effects of PQ.

Fetal Dexamethasone Exposure Affects Basal Ornithine Decarboxylase Activity in Developing Rat Brain Regions and Alters Acute Responses to Hypoxia and Maternal Separation

Although glucocorticoids are widely used to stimulate fetal/neonatal lung function, they also interfere with cellular development in the central nervous system. Dexamethasone was administered to pregnant rats in late gestation at a dose (0.8 mg/kg) that lies just above the threshold for stimulation of lung surfactant synthesis, and the impact on ornithine decarboxylase (ODC) was evaluated in three brain regions. Dexamethasone treatment produced an initial inhibition of basal ODC activity followed by postnatal elevations, a pattern known to be associated with delays in cell replication and differentiation. Dexamethasone also interfered with the ability of the 1-day-old neonate to turn off ODC acutely in response to a 2-h period of maternal separation; as this response conserves energy in the absence of the dam, the effect of dexamethasone is maladaptive. Additionally, dexamethasone sensitized the neonatal brain to hypoxia: the acute increase of ODC associated with a 2-h exposure to 7% O2 was exacerbated in 8-day-old rats exposed to dexamethasone prenatally. These results suggest that administration of dexamethasone, in doses that promote respiratory competence, delays cell development in the central nervous system and renders the brain more vulnerable to adverse neonatal conditions, such as maternal separation or hypoxia.

De novo sn-glycerol-3-phosphorylcholine synthetase activity in lung and muscle and its subcellular location.

The activity of glycerophosphorylcholine synthetase, a newly discovered enzyme involved in the synthesis of acyl-specific phosphatidylcholines, is reported in rat lung and muscle. Its subcellular location appears to be mitochondrial. The implication of these findings in the synthesis of lung surfactant and the pathology of muscular dystrophy are discussed.

The influence of long-term administration of terbutaline on the disaturated phosphatidyl-choline concentration of fetal lung tissue and amniotic fluid in pregnant rabbits.

AbstractTerbutaline (40 μg/kg) was given intramuscularly to pregnant rabbits three times daily from the 23rd day of gestation until the 26th to 30th days, when the fetuses were delivered by cesarean section. The wet lung weight/body weight ratio of fetuses significantly decreased in the terbutaline‐treated group on the 27th and 28th days as compared with the control. The concentration of disaturated phosphatidyl‐choline (DSPC) of fetal lung tissue significantly increased only on the 29th and 30th days of gestation, which corresponded to the 7th and 8th days after the administration of terbutaline. However, a significant increase of the DSPC concentration in amniotic fluid had already occurred from the 26th day. These findings indicate that terbutaline accelerates the lung surfactant release, but does not primarily stimulate the synthesis of lung surfactant. The acceleration can be achieved only by long‐term administration of terbutaline. As this drug is able to dehydrate the fetal lung tissue by reducing the lung fluid, it might contribute to the stabilization of pulmonary respiration after birth.

Synergistic effect of triiodothyronine and dexamethasone on male and female fetal rat lung surfactant synthesis.

It has previously been reported that morphologic and functional indices of fetal lung maturation are delayed in male rabbit fetuses, and these differences cannot be overcome by glucocorticoid treatment. Since thyroid hormone also stimulates surfactant production we have studied the effects of triiodothyronine (T3) and dexamethasone on saturated phosphatidylcholine (SPC) synthesis by day 20 male and female rat lung. Control male lung slices incorporated significantly less 3H-choline into SPC than females; surgery on day 17 markedly stimulated 3H-choline incorporation into SPC by both sexes, but the sex difference persisted. The optimal dose of T3 inhibited SPC synthesis by both sexes while dexamethasone was stimulatory. When T3 and dexamethasone were given together they had a greater effect than that of dexamethasone or T3 alone. Based on these results we conclude that the male deficit in pulmonary surfactant production may be overcome by the synergistic effects of T3 and dexamethasone.

Sequential effects of irradiation on the pulmonary surfactant system

This study examines the effect of irradiation on lung surfactant synthesis and secretion in mice. Animals were irradiated with 650, 1300, or 1950 rad and morphological and biochemical indices of surfactant system function were followed for 18 weeks. No changes were seen at 650 rad; the results at 1300 and 1950 rad were virtually identical. Increased amounts of alveolar surfactant phospholipid were measureable by 24 hours. This persisted for four weeks and returned to normal by 18 weeks. Tissue surfactant phospholipid was initially reduced, returned to normal by four weeks and was increased at 18 weeks. At 18 weeks there was increased incorporation of surfactant precursor and increased production of alveolar surfactant. These biochemical changes were reflected in morphologic alterations showing release of lamellar body contents into alveoli in the first week and an increase in Iamellar bodies in type 11 pneumocytes by 18 weeks. Elevated tissue protein levels and morphologic evidence of increased collagen formation were also found at 18 weeks. These findings indicate effects of irradiation on the pulmonary surfactant system and have important implications for the pathogenesis and potential therapy of radiation pneumonitis.

Lung Surfactant in an Era of Replacement Therapy

In this paper current knowledge of the pulmonary surfactant system with particular emphasis on aspects that relate to exogenous surfactant replacement therapy for the neonatal respiratory distress syndrome (RDS) is considered. The work provides an overview, but concentrates on several facets of lung surfactant research that help to elucidate and evaluate past and present attempts toward such therapy. Subjects addressed include the functional need for specific lung surfactant components to obtain optimal surface activity and some of the required surface property measurements to characterize such activity. Also discussed is current knowledge of lung surfactant synthesis and secretion and the potential effect of exogenous surfactant on the endogenous pulmonary surfactant system. A primary theme throughout is that an analysis of previous clinical trials involving surfactant replacement shows the necessity for strong interactions with related basic science investigations. It is suggested that future human infant surfactant replacement trials proceed deliberately, and include maximal correlations with basic in vitro research on lung surfactant biophysics and biochemistry and with experiments in animal models.

Effects of the herbicide 2,4-dichlorophenyl- p-nitrophenyl ether (nitrofen) on fetal lung developments in rats

We have examined whether the neonatal mortality accompanied by lung hypoplasia and atelectasis induced by prenatal exposure to the diphenyl ether herbicide 2,4-dichlorophenyl- p-nitrophenyl ether (nitrofen) could be due to alterations in pulmonary surfactant synthesis. The specific hypothesis we tested was whether in utero exposure to nitrofen accelerated the catabolic metabolsim of glucocorticoids by induction of mixed function oxidase enzymes, leading to a depression in glucocorticoid levels and delay in fetal lung surfactant synthesis. Hepatic aminopyrine- n-demethylase and aniline hydroxylase activities in the 9000 g supernatant were not elevated in rat dams exposed to 50 mg/kg/day on days 8–18 of pregnancy. Cortisone and corticosterone levels in fetal plasma were not altered by nitrofen exposure. Fetal lung surfactant synthesis was also not affected, as indicated by measurements of total lung phospholipids, [ 14C]choline uptake into lung lipids, and cholinephosphotransferase (CPT) activity. Surface tension values from saline extracts of fetal lungs and fetal plasma corticosterone levels were monotored after co-administration of nitrofen and dexamethasone. Nitrofen exposure alone had no influence on these parameters, while dexamethasone, with or without nitrofen, depressed corticosterone levels and minimum surface tension measurements. These results suggest that the level of glucocorticoids and capacity of the fetal lung to respond to glucocorticoids by synthesizing and releasing surfactant are not affected by prenatal exposure to nitrofen.

The lecithin/sphingomyelin ratio in Macaca arctoides.

The amniotic fluid lecithin/sphingomyelin ratio (L/S ratio) of stumptail macaque monkeys (Macaca arctoides) was investigated by means of serial amniocenteses during the second half of pregnancy. Ten dated pregnancies were studied in 9 individual monkeys. The mean number of successful amniocenteses per pregnancy was 4. A rapid increase in the L/S ratio suggesting maturation of lung surfactant synthesis occurred as early as 130--140 days of pregnancy in some animals, and as late as 150--160 days in others. The earliest and latest times at which the L/S ratio first exceeded 2.0 were 135 and 164 days, respectively. These observations indicate that the L/S ratio of M. arctoides follows a course similar to that observed in human pregnancy, with a rise at 0.7--0.8 of term gestation. M. arctoides thus appears to be a useful model for the study of factors affecting fetal lung maturity, on the basis of the amniotic fluid L/S ratio. In particular, a rapid rise in L/S ratio before 130 days of pregnancy would be highly suggestive of accelerated maturation of pulmonary surfactant production.

Altered metabolism of phospholipids in the lung of rats with peritonitis.

Pulmonary alterations after shock and sepsis, described clinically as shock lung or adult respiratory distress syndrome, are of great importance in intensive care. Pathogenetically an alteration of the surfactant system of the lung is often discussed. Since phospholipids are constituents of lung surfactants, phospholipid metabolism is investigated in experimental peritonitis in rats in our laboratory. 15 hours after inducing a peritonitis, the lung incorporates more oleic acid than that in animals of the reference group. 33 hours after inducing peritonitis, the capacity of the lung to incorporate choline and fatty acids is markedly reduced, histologically the lungs represent morphological equivalents of the so-called shock lung at this time. Therefore we conclude, that an alteration of phospholipid metabolism with a diminished and/or altered synthesis of lung surfactant plays, at least in part, an important role in the pathogenesis of respiratory distress in sepsis and peritonitis.

201 THE RELATIONSHIP OF AMNIOTIC FLUID DEHYDROISOANDROSTERONE SULFATE (DS) AND CORTISOL (F) CONCENTRATIONS WITH LECITHIN TO SPHINGOMYELIN (L/S) RATIOS DURING HUMAN CESTATION

We have measured L/S ratios, together with the concentrations of F and DS of 414 amniotic fluid specimens obtained from 13 to 44 weeks gestation. We found that the L/S ratios begin to increase after 33 weeks gestation, from mean values of 0.7 ± 0.2 (± SE) at 33 weeks to 1.8 ± 0.3 at 34 weeks and 3.5 ± 0.7 at 35 weeks. There are no significant differences in concentrations of amniotic fluid F from 31-32 weeks up to 35 weeks. F concentrations rise thereafter from 51 ± 6.3 ng/ml at 35 weeks to 63 ± 4.5 ng/ml at 36 weeks and 67.5 ± 6.3 ng/ml at 37 weeks. DS concentrations in amniotic fluid rise slowly from 31-32 weeks (80 ± 13 ng/ml) to the 35th week 110 ± 7.8 ng/ml) and this rise continues to term. While others have shown that the L/S ratios in amniotic fluid correlate well with fetal lung maturation, this does not appear to be the case with cortisol, since cortisol concentrations increase following the increases of L/S ratios. However, DS concentrations in amniotic fluid do begin to increase before the rise in the L/S ratio, likely reflecting increased availability of fetal placental estrogen precursor. Augmented estrogen production could serve a role in increasing fetal lung surfactant synthesis, perhaps by leading to increased fetal pituitary prolactin secretion.