Surfactant chemical composition and biophysical activity in acute respiratory distress syndrome.

Acute Respiratory Distress Syndrome (ARDS) is characterized by lung injury and damage to the alveolar type II cells. This study sought to determine if endogenous surfactant is altered in ARDS. Bronchoalveolar lavage was performed in patients at-risk to develop ARDS (AR, n = 20), with ARDS (A, n = 66) and in normal subjects (N, n = 29). The crude surfactant pellet was analyzed for total phospholipids (PL), individual phospholipids, SP-A, SP-B, and minimum surface tension (STmin). PL was decreased in both AR and A (3.48 +/- 0.61 and 2.47 +/- 0.40 mumol/ml, respectively) compared to N (7.99 +/- 0.60 mumol/ml). Phosphatidylcholine was decreased in A (62.64 +/- 2.20% PL) compared to N (76.27 +/- 2.05% PL). Phosphatidylglycerol was 11.58 +/- 1.21% PL in N and was decreased to 6.48 +/- 1.43% PL in A. SP-A was 123.64 +/- 20.66 micrograms/ml in N and was decreased to 49.28 +/- 21.68 micrograms/ml in AR and to 29.88 +/- 8.49 micrograms/ml in A. SP-B was 1.28 +/- 0.33 micrograms/ml in N and was decreased to 0.57 +/- 0.24 micrograms/ml in A. STmin was increased in AR (15.1 +/- 2.53 dyn/cm) and A (29.04 +/- 2.05 dyn/cm) compared to N (7.44 +/- 1.61 dyn/cm). These data demonstrate that the chemical composition and functional activity of surfactant is altered in ARDS. Several of these alterations also occur in AR, suggesting that these abnormalities occur early in the disease process.

Surfactant cholesterol metabolism of the isolated perfused rat lung.

1. The isolated perfused rat lung was used as the principal model to study surfactant cholesterol metabolism. 2. Cholesterol was found to represent over 50% of the neutral lipid of both the total surfactant and the lamellar body fractions. 3. De novo synthesis of cholesterol from [1-14C]acetate accounted for only 1% of the surfactant cholesterol, the remainder being derived from exogenous cholesterol supplied as serum lipoproteins. 4. Lipoprotein [1,2-3H2]cholesterol was incorporated into the lamellar body and extracellular surfactant fractions. The increase in the cholesterol specific activities of these fractions with time was consistent with a precursor-product relationship between the lamellar body cholesterol and that of the extracellular surfactant. 5. Incorporation of [methyl-14C]choline and [1,2-3H2]cholesterol indicated that the metabolism and secretion of lamellar body and extracellular surfactant cholesterol parallels that of phosphatidylcholine and suggests that most if not all extracellular surfactant cholesterol is derived from the lamellar body. 6. Comparison of the relative specific activities of incorporated [1,2-3H2]-cholesterol indicate that 59% of the total surfactant cholesterol is located extracellularly.

Altered phospholipid secretion in type II pneumocytes isolated from streptozotocin-diabetic rats.

To study the effect of diabetes on pulmonary surfactant secretion, type II pneumocytes from adult streptozotocin-induced diabetic rats were placed in short-term culture. As opposed to a linear secretory rate by control type II cells, the secretory rate of type II cells from diabetic animals was biphasic reaching a minimum at 1.5 h. When exogenous surfactant containing radioactive phosphatidylcholine was added to the incubation media for 1.5 h, the cells from diabetic animals incorporated more exogenous phosphatidylcholine into lamellar bodies than control cells. This suggests that in the type II cell from diabetic animals, the rate of reutilization is greater than the rate of secretion until 1.5 h, at which time the rate of secretion becomes greater. The altered secretory pattern was reversed by in vivo insulin treatment 30 min prior to killing but not by the addition of insulin to the incubation media. When challenged by isoproterenol, a beta-adrenergic agonist, the secretory pattern of cells from diabetic animals was biphasic as observed with basal secretion; however, secretion was stimulated 30% as opposed to 100% increase in control cells. These data suggest that basal and stimulated secretion are altered in the cultured type II cell from diabetic animals and restored by in vivo but not in vitro insulin treatment.

Altered phospholipid biosynthesis in type II pneumocytes isolated from streptozotocin-diabetic rats.

To determine whether type II pneumocytes isolated from diabetic animals could serve as a useful model for the study of surfactant phospholipid biosynthesis and its regulation, type II pneumocytes were isolated from adult streptozotocin-diabetic rats and placed in short-term primary culture. On a DNA basis, total cellular disaturated phosphatidylcholine (disaturated PC) and phosphatidylglycerol (PG) were decreased 36 and 66%, respectively, in type II cells from diabetic animals. 7 days of insulin treatment of diabetic rats returned the cellular disaturated PC and PG content to control values and increased the total cellular phosphatidylethanolamine (PE) content by 51%. The rates of glucose and acetate incorporation into disaturated PC per unit DNA were reduced 32 and 38%, respectively, in cells isolated from diabetic rats, while glycerol incorporation was increased by 143%. Insulin treatment of diabetic rats returned the glucose and glycerol incorporation rates to control values and increased acetate incorporation into disaturated PC by 66%. These data suggest that the biosynthesis of surfactant is altered by both diabetes mellitus and in vivo insulin treatment.

Phosphatidic acid phosphatase activity in subcellular fractions derived from adult rat type II pneumocytes in primary culture.

The development of a method for the subcellular isolation of lamellar body, microsomal, mitochondrial and cytosolic enriched fractions from the adult rat type II pneumocyte is presented. These fractions were used to investigate the distribution and specificity of phosphatidic acid phosphatase (EC 3.1.3.4). The cytosolic enriched fraction possessed the greatest proportion of total phosphatidic acid phosphatase activity and the highest relative specific activity. The small amounts of phosphatase activity in the lamellar body, microsomal and mitochondrial enriched fractions correlated best with a nonspecific phosphatase activity. 1,2-Disaturated phosphatidic acid served as a slightly better substrate than did 1-saturated, 2-unsaturated phosphatidic acid in the cytosolic enriched fraction. This observation indicates that the de novo pathway could contribute to dipalmitoylphosphatidylcholine synthesis in the rat type II pneumocyte.

Effect of experimental diabetes and insulin on lipid metabolism in the isolated perfused rat lung.

The isolated perfused rat lung was used as a model to study the possible hormonal regulation of lipid metabolism in the mammalian adult lung. Experimental diabetes, whether induced by alloxan or streptozotocin, decreased the incorporation of [U-14C]glucose into neutral lipids and phospholipids of both the surfactant fraction and the residual fraction of the lung by 60-80%. Glucose incorporation into phosphatidylcholine and phosphatidylglycerol is decreased in experimental diabetes in both the surfactant and residual fractions to a comparable degree. Glucose incorporation is decreased in both the fatty acid and the glycerophosphocholine moieties of phosphatidylcholine isolated from the surfactant and residual fractions. Insulin treatment of normal animals 30 or 15 min prior to perfusion resulted in an approximate doubling of the incorporation of glucose into the phosphatidylcholine and phosphatidylglycerol isolated from the surfactant and residual fractions of the lung. The incorporation of glucose into palmitic acid isolated from phosphatidylcholine was also shown to increase similarly. The results of these investigations indicate that insulin may play a role in regulating the synthesis of the important lipid components of the mammalian pulmonary surfactant complex.

Glycerol metabolism in type II pneumocytes isolated from streptozotocin-diabetic rats.

Glycerol utilization for phospholipid biosynthesis was examined in type II pneumocytes isolated from normal and streptozocinin-diabetic rats. With glucose in the incubation medium, incorporation of exogenous [1,3-14C]glycerol into disaturated phosphatidylcholine, total phosphatidylcholine (PC), phosphatidylglycerol (PG) and phosphatidylethanolamine (PE) was increased 4-fold in cells from diabetic rats. In the absence of glucose, glycerol incorporation was 5-fold greater than in its presence in cells from normal animals, but was further increased 2.2-fold in cells from diabetic rats. Insulin treatment of diabetic rats returned all incorporation rates to control values. The increased glycerol incorporation rates were not due to differences in either phospholipid turnover or the size of the glycerol 3-phosphate precursor pool. Kinetic analysis of glycerol entry into the acid-soluble cell fraction indicated that glycerol transport occurred largely by simple diffusion, and was not rate limiting for its entry into lipids. Glycerol entry into the total lipid fraction was saturable, reaching a Vmax of 48 pmol/micrograms DNA per h in normal cells and 120 pmol/micrograms DNA per h in cells from diabetic rats, with no change in the Km (0.31 mM). While glycerol oxidation was reduced 23% in cells from diabetic rats in the presence of glucose and by 44% in the absence of glucose, glycerol kinase activity in sonicates of cells from diabetic animals was increased 210% and was reversed by in vivo insulin treatment. These results suggest that glycerol utilization in type II pneumocytes is a hormonally regulated function of both glycerol oxidation and glycerol phosphorylation.

Glycerol as a substrate for phospholipid biosynthesis in type II pneumocytes isolated from streptozotocin-diabetic rats.

Glycerol and glucose utilization for phospholipid biosynthesis was examined in type II pneumocytes isolated from normal and streptozotocin-diabetic rats. In cells from diabetic rats, incorporation of [1,3-14C]glycerol into total phosphatidylcholine (PC), disaturated phosphatidylcholine (DSPC), phosphatidylglycerol (PG) and phosphatidylethanolamine (PE) occurred to a greater degree by the glycerol 3-phosphate pathway as opposed to the dihydroxyacetone phosphate pathway. Total incorporation of glycerol into each of the major cellular phospholipids was increased up to 6-fold in cells from diabetic rats, while the total incorporation of glucose into the same lipids was decreased 2-fold. While the percentage of both glucose and glycerol carbons incorporated into the backbone of DSPC was increased in cells from diabetic rats, the percentage of carbons from both substrates incorporated into the fatty acid moieties was decreased. As a measure of DSPC synthesis, choline incorporation into DSPC was significantly decreased in type II cells from diabetic animals if the cells were incubated in the presence of glucose, palmitate and choline but not glycerol. Addition of 0.1 or 0.3 mM glycerol to the incubation medium restored choline incorporation to the control value in cells from diabetic rats, but did not affect the rate of choline incorporation into DSPC in cells from normal rats. These results suggest that exogenous glycerol can compensate for reduced glucose metabolism in type II cells of diabetic animals to maintain a constant rate of DSPC synthesis.

Acyltransferase activities in adult rat type II pneumocyte-derived subcellular fractions.

Acyl-CoA: lysophosphatidylcholine, acyl-CoA: lysophosphatidylethanolamine, and lysophosphatidylcholine:lysophosphatidylcholine acyltransferases were investigated using subcellular fractions derived from adult rat type II pneumocytes in primary culture. Acyl-CoA:lysophospholipid acyltransferase activities were determined to be microsomal, while lysophosphatidylcholine:lysophosphatidylcholine acyltransferase activity was found to be cytosolic. Total palmitoyl CoA:lysophosphatidylcholine acyltransferase activity was 30-fold greater than lysophosphatidylcholine:lysophosphatidylcholine acyltransferase activity, indicating that the former enzyme is more important in the synthesis of dipalmitoyl phosphatidylcholine. Palmitoyl-CoA and oleoyl-CoA lysophosphatidylcholine acyltransferase activities were approximately equal under optimal substrate conditions. Specific activities of the enzyme using arachidoyl-CoA and arachidonoyl-CoA were 46% and 18%, respectively, of those with palmitoyl-CoA. Acyl-CoA:lysophosphatidylethanolamine acyltransferase showed a preference for palmitoyl-CoA as opposed to oleoyl-CoA under optimal conditions. However, when equimolar concentrations of either palmitoyl-CoA and oleoyl-CoA or palmitoyl-CoA and arachidoyl-CoA were assayed together, the relative utilization of the two substrates was found to be dependent on total acyl-CoA concentration. At higher concentrations, the incorporation of palmitoyl-CoA into phosphatidylcholine was less than other acyl-CoAs. However, at lower concentrations palmitoyl-CoA was utilized quite selectively. Whole lung microsomes did not show as marked a preference for palmitoyl-CoA as did type II pneumocyte microsomes under these same conditions. In similar experiments, low total acyl-CoA concentrations produced greater incorporation of oleoyl-CoA into phosphatidylethanolamine. For both enzymes total activity at the lowest concentrations used was at least 45% that at optimal conditions. This demonstrates that the type II pneumocyte acyltransferase system(s) can selectively utilize palmitoyl-CoA. No evidence for direct exchange of palmitoyl-CoA with 1-saturated-2-unsaturated phosphatidylcholine in subcellular fractions from type II pneumocytes was found.

A study of the molecular species of diacylglycerol, phosphatidylcholine and phosphatidylethanolamine and of cholinephosphotransferase and ethanolaminephosphotransferase activities in the type II pneumocyte.

The percent distributions of the molecular species of diacylglycerol, phosphatidylcholine and phosphatidylethanolamine in rat whole lung and type II pneumocytes were found to differ significantly. Diacylglycerol from the type II pneumocyte is enriched in the disaturated species and diminished in the polyenoic species compared to whole lung. Type II pneumocyte phosphatidylcholine is enriched in the disaturated species and diminished in all other species compared to whole lung. Relative to whole lung, type II pneumocyte phosphatidylethanolamine is greatly enriched in monoenoic and depleted in polyenoic fatty acid species. Analysis of the fatty acid composition of the molecular species in general indicated differences in relative amounts of fatty acids which were most pronounced in palmitic, palmitoleic, stearic and oleic acids, both within and between type II pneumocyte and whole lung glycerolipids. Significant differences between molecular species also existed within type II pneumocyte glycerolipids. In this cell type, phosphatidylcholine is enriched in disaturated and diminished in monoenoic species compared to diacylglycerol. Phosphatidylethanolamine is enriched in monoenoic and polyenoic species relative to diacylglycerol. In order to determine whether differences observed in type II pneumocyte glycerolipid molecular species were attributable to differences in the specificities of cholinephosphotransferase and ethanolaminephosphotransferase, the selectivity of these enzymes was examined. While cholinephosphotransferase showed diminished activity towards 1-stearoyl-2-linoleoyl-sn-glycerol, neither enzyme showed selectivity towards other tested diacylglycerols under a variety of conditions. Therefore, while in the type II pneumocyte significant amounts of phosphatidylcholine (particularly the disaturated species) and phosphatidylethanolamine may be synthesized de novo, enzymes responsible for remodeling (phospholipase A2 and acyltransferases) may play an important role in establishing the final molecular species composition of both phosphatidylcholine and phosphatidylethanolamine.

Lysolecithin acyltransferase and lysolecithin: lysolecithin acyltransferase in adult rat lung alveolar type II epithelial cells.

1. The specific activity of lysolecithin acyltransferase (EC 2.3.1.23) in sonicated adult rat lung alveolar type II epithelial cells, measured either alone or in combination with acyl-CoA synthetase (EC 6.2.1.3), was found to be an order of magnitude greater than that of lysolecithin:lysolecithin acyltransferase. 2. Lysolecithin acyltransferase in type II cells was found to prefer palmitoyl-CoA over oleoyl-CoA as substrate. The combination of lysolecithin acyltransferase and acyl-CoA synthetase was found to prefer palmitate over oleate for incorporation into phosphatidylcholine. 3. Compared to whole lung homogenate, sonicated adult rat type II cells are highly enriched in lysolecithin acyltransferase but not in lysolecithin:lysolecithin acyltransferase. 4. These observations indicate that in normal adult rat type II cells the deacylation-reacylation cycle is more important for the formation of dipalmitoyl phosphatidylcholine than the deacylation-transacylation process.

The synthesis of phosphatidylcholine by adult rat lung alveolar type II epithelial cells in primary culture.

1. The formation of phosphatidylcholine from radioactive precursors was studied in adult rat lung alveolar type II epithelial cells in primary culture. 2. The incorporation of [Me-14C]choline into total lipids and phosphatidylcholine was stimulated by addition of palmitate, whereas the incorporation of [U-14C]glucose into phosphatidylcholine and disaturated phosphatidylcholine was stimulated by addition of choline. Addition of glucose decreased the absolute rate of incorporation of [1(3)-3H]glycerol into total lipids, phosphatidylcholine and disaturated phosphatidylcholine, decreased the percentage [1(3)-3H]glycerol recovered in phosphatidylcholine, but increased the percentage phosphatidylcholine label in the disaturated species. 3. At saturating substrate concentrations, the percentages of phosphatidylcholine radioactivity found in disaturated phosphatidylcholine after incubation with [1-(14)C]acetate (in the presence of glucose) [1-(14)C]palmitate (in the presence of glucose), [Me-14C]choline (in the presence of glucose and palmitate) and [U-14C]glucose (in the presence of choline and palmitate) were 78, 75, 74 and 90%, respectively. 4. Fatty acids stimulated the incorporation of [U-14C]glucose into the glycerol moiety of phosphatidylcholine. The degree of unsaturation of the added fatty acids was reflected in the distribution of [U-14C]glucose label among the different molecular species of phosphatidylcholine. It is suggested that the glucose concentration in the blood as related to the amount of available fatty acids and their degree of unsaturation may be factors governing the synthesis of surfactant lipids.

Partial deficiency of surfactant protein B in an infant with chronic lung disease.

OBJECTIVE: To evaluate components of pulmonary surfactant and identify mutations in the surfactant protein B gene (SP-B) of a term infant with severe respiratory distress and chronic lung disease. PATIENT AND TESTING: Respiratory distress developed in an infant delivered at term, and he required extracorporeal bypass support for 2 weeks. Until his unexpected death at 9.5 months, he was ventilator and oxygen dependent and required continual dexamethasone therapy. Tracheobronchial lavage samples were analyzed for content of surfactant proteins (SPs), and DNA from blood samples were sequenced and analyzed by polymerase chain reaction restriction analysis for the presence of SP-B gene mutations. Surfactant lipid composition and function, the contents of SPs and their messenger RNAs (mRNAs), and the immunostaining pattern for SPs were determined in postmortem lung tissue. RESULTS: The lavage sample contained SP-A but not SP-B, and DNA restriction analysis indicated that the patient and his mother were heterozygous for the previously described 121ins2 mutation of SP-B. Postmortem lung tissue contained normal levels of SP-A and its mRNA, a low but detectable level of SP-B, and near normal content of SP-B mRNA. SP-C was abundant on staining, and some 6-kd precursor was present in tissue. A surfactant fraction was deficient in phosphatidylglycerol and was not surface active. On DNA sequencing, a point mutation was found in exon 7 of the patient's SP-B gene allele without the 121ins2 mutation, resulting in a cysteine for arginine substitution, and the father was a carrier for the same mutation. CONCLUSIONS: We describe a patient who is a compound heterozygote with a new mutation and only a partial deficiency of SP-B. Some forms of inherited SP-B deficiency may have low expression of immunoreactive and possibly functional SP-B with milder lung disease and longer survival. These infants may benefit from glucocorticoid therapy and may not develop antibodies to SP-B after either lung transplant or gene therapy.

Role of microtubules in surfactant secretion.

In the isolated perfused rat lung and cultured type II cells, surfactant secretion and cellular adenosine 3',5'-cyclic monophosphate (cAMP) content was stimulated by beta-adrenergic agonists. Isoproterenol-induced surfactant secretion was inhibited by the antimicrotubule agents colchicine and vinblastine. Incorporation of [3H]glycerol into disaturated phosphatidylcholine was augmented by beta-adrenergic agents but was not significantly different from the enhanced incorporation rate when colchicine was present. This suggests that the augmented incorporation of [3H]glycerol into disaturated phosphatidylcholine was a secondary response to storage depletion rather than direct cAMP stimulation. beta-Adrenergic agents shifted the equilibrium in the isolated perfused rat lung and cultured type II cells to favor microtubules. The stimulatory effect of 1.0 microM isoproterenol on tubulin polymerization was observed as early as 1 min and was augmented 2.8-fold at a half-maximal stimulation of 4 nM in cultured type II cells. Cytochalasin B, an antimicrofilament agent, potentiated the isoproterenol-induced secretion. These results suggest that an intact microtubule-microfilament system may be obligatory for enhanced surfactant secretion and that beta-adrenergic agents not only induce surfactant release but also tubulin polymerization.

Effect of exogenous surfactant instillation on experimental acute lung injury.

Pulmonary surfactant replacement has previously been shown to be effective in the human neonatal respiratory distress syndrome. The value of surfactant replacement in models of acute lung injury other than quantitative surfactant deficiency states is, however, uncertain. In this study an acute lung injury model using rats with chronic indwelling arterial catheters, injured with N-nitroso-N-methylurethane (NNNMU), has been developed. The NNNMU injury was found to produce hypoxia, increased mortality, an alveolitis, and alterations in the pulmonary surfactant system. Alterations of surfactant obtained by bronchoalveolar lavage included a reduction in the phospholipid-to-protein ratio, reduced surface activity, and alterations in the relative percentages of the individual phospholipids compared with controls. Treatment of the NNNMU-injured rats with instilled exogenous surfactant (Survanta) improved oxygenation; reduced mortality to control values; and returned the surfactant phospholipid-to-protein ratio, surface activity, and, with the exception of phosphatidylglycerol, the relative percentages of individual surfactant phospholipids to control values.

Regulation of lung surfactant cholesterol metabolism by serum lipopoteins.

The isolated perfused rat lung was used as an experimental model in the study of the lipoprotein regulation of surfactant cholesterol metabolism. Addition of low density lipoproteins (LDL) to the perfusion medium at a cholesterol concentration of 0.5 micrometer had no inhibitory effect on [1-14C]-acetate incorporation into cholesterol of either the surfactant or residual fractions. Increasing the concentration of cholesterol in the medium to 2.5 micrometer resulted in significant inhibition of incorporation into cholesterol of both fractions. A similar inhibition resulted when lungs were perfused with 2.5 micrometer cholesterol in the form of high density lipoproteins (HDL). No inhibition of fatty acid synthesis, measured as incorporation into cholesteryl esters, was observed. The rate of uptake by perfused lung of cholesterol from both high and low density lipoproteins was similar. Competitive binding studies with 125I-labeled lipoproteins indicated the existence of lung receptors for both classes of lipoprotein. The rate of uptake of the apoprotein moiety of low density lipoproteins was significantly greater than that of high density lipoproteins. These data suggest that lung cholesterol metabolism may be subject to regulation by both low and high density serum lipoproteins.

Adrenergic and cholinergic regulation of lung surfactant secretion in the isolated perfused rat lung and in the alveolar type II cell in culture.

The purpose of this study is to define the hormonal regulation of pulmonary surfactant secretion in two models, the isolated perfused rat lung and the isolated alveolar type II cell in culture. In the perfused lung, both cholinergic and adrenergic stimulation independently increased labeled disaturated phosphatidylcholine secretion, the major phospholipid component of surfactant, each by 2.3-fold. A concomitant increase in lung cGMP and cAMP concentration of 275- and 25-fold, respectively, was observed. The effect of each agonist was inhibited only by its appropriate antagonist. In alveolar type II cells in culture, both adenylate and guanylate cyclase responded to their appropriate agonists and antagonists. The release of 3H-labeled disaturated phosphatidylcholine was enhanced by beta-adrenergic but not alpha-adrenergic or cholinergic agonists. The effect of isoproterenol (10 microM) on surfactant release was seen by 2.5 min, and secretion was stimulated 2.9-fold at a half-maximal concentration of 1 nM. Cyclic AMP levels were increased by 4.9-fold by isoproterenol at a half-maximal concentration of 40 nM. These results indicate that while in the perfused lung, secretion is stimulated by both adrenergic and cholinergic effectors, in the type II cell model, surfactant secretion is under only beta-adrenergic control.

Sublethal hydrogen peroxide inhibits alveolar type II cell surfactant phospholipid biosynthetic enzymes.

Alveolar type II cell injury by phagocytic cell-derived reactive oxygen metabolites represents a potential mechanism for the altered surfactant metabolism found in patients with the adult respiratory distress syndrome (ARDS). Previous studies demonstrated altered surfactant phospholipid metabolism after sublethal oxidant exposure. In this study, we measured intracellular ATP levels and the activities of several enzymes involved in surfactant phospholipid biosynthesis after sublethal H2O2 exposure of cultured rat alveolar type II cells. Intracellular ATP levels were reduced by 46.6% after exposure to 75 microM H2O2. The activity of CTP:phosphorylcholine cytidyltransferase was unchanged after H2O2 exposure when measured in whole cell homogenates. However, when measured in the microsomal fraction, cytidyltransferase activity significantly fell after exposure of type II cells to 75 microM H2O2. Activity in the cytosolic fractions remained unchanged. Similarly, microsomal cholinephosphotransferase was reduced after H2O2 exposure. We conclude that H2O2 decreases surfactant phosphatidylcholine biosynthesis independently of its ability to deplete intracellular ATP content. These deleterious effects may partially explain the diminished alveolar surfactant observed in patients with ARDS.