Surfactant Biosynthesis Research Articles

Altered morphology, growth, and biosynthetic activity of a type 2 pneumocyte-related cell strain induced by lymphokine-enriched supernatants of mitogen-stimulated spleen cells.

Hypertrophy and hyperplasia of type 2 pneumocytes during the evolution of subacute and chronic pulmonary injury cannot always be satisfactorily explained in terms of reparative responses to type 1 pneumocyte injury. We hypothesized that immunocompetent cells, evoked as part of the interstitial inflammatory response, may be secreting factors which affect proliferation and surfactant biosynthesis by type 2 pneumocytes. To evaluate this hypothesis in vitro, we tested the effects of lymphokine-enriched supernatants, from serum-free cultures of concanavalin A-stimulated spleen cells, upon the type 2 pneumocyte-related cell strain NAL 1A. Addition of these supernatants in culture induced irreversible morphologic and ultrastructural alterations in the NAL 1A cells, inhibited cell replication, and evoked increased and apparently abnormal surfactant phospholipid biosynthesis. Supernatants from unstimulated spleen cells had no effect. There was no evidence of toxic injury to the cells in culture, and an immunologically specific cytoplasmic protein continued to be expressed. The active factor(s) appeared to be a protein or peptide of greater than 10,000 molecular weight. A specific soluble factor such as is present in the mitogen-stimulated lymphoid cell supernatants may be capable of mediating a similar interaction with type 2 pneumocytes in vivo.

Influence of the major histocompatibility complex (H-2) on glucocorticoid-stimulated pulmonary surfactant synthesis in two congenic mouse strains.

Mice with different histocompatibility alleles in otherwise identical backgrounds (congenic inbred strains of H-2a and H-2b haplotypes), were used to study the possible influence of the major histocompatibility complex (MHC) upon glucocorticoid-stimulated pulmonary surfactant synthesis during embryonic and fetal stages of mouse lung development. Pregnant C57BL/10SnJ (H-2b) and congenic B10.A/SnSgJ (H-2a) mice were administered single or consecutive intraperitoneal injections of betamethasone during the period of 14 through 17 days gestation. Assays for incorporation of [3H]choline into total lipid and saturated phosphatidylcholine (an index for pulmonary surfactant synthesis) were conducted, 24 hr following the last treatment, on Days 17 or 18 of gestation. Control mice received injections of phosphate-buffered saline. The H-2 genotype appears to influence the relative responsiveness of glucocorticoid-stimulated surfactant synthesis during fetal lung development. The B10.A strain (H-2a haplotype) was more responsive to glucocorticoid-stimulated pulmonary surfactant synthesis than the H-2b haplotype. Whereas both strains were responsive to glucocorticoid-stimulated [3H]choline incorporation into lung surfactant, the differences in responsiveness at specific fetal stages suggest that genes within or closely linked to the H-2 complex might have a role in pulmonary surfactant biosynthesis during mouse lung development. The results also suggest the possibility that relative susceptibility to congenital pulmonary disorders may be influenced by the H-2 complex.

Lung Maturation in Fetuses of Diabetic Rats

Pulmonary maturation in diabetic pregnancy has been studied in the 20-day-old fetuses of manifest diabetic rats. The animals were either untreated or treated with insulin. The diabetic state was induced by a single IV injection of streptozotocin given about 2 wk before the onset of pregnancy. The biosynthesis of lung surfactant was estimated by monitoring the rate of incorporation of [methyl-3H]choline into phosphatidylcholine and lysophosphatidylcholine in fetal lung slices. In the untreated group, the biosynthesis of both phosphatidylcholine and lysophosphatidylcholine were decreased in the fetal lung. Insulin treatment abolished the decrease in the phosphatidylcholine biosynthesis, whereas the lysophosphatidylcholine biosynthesis reamined depressed. Light and transmission electron microscopical studies indicated a delayed pulmonary maturation in the untreated offspring accompanied by a decreased cytoplasmic content of glycogen in the alveolar epithelial cells.

PHOSPHATIDIC ACID PHOSPHOHYDROLASE AND PHOSPHOLIPIDS IN TRACHEAL AND AMNIOTIC FLUIDS DURING NORMAL OVINE PREGNANCY

Summary: Samples of amniotic fluid and fetal tracheal fluid were obtained from 36 ovine mancies which were studied either acutely or chronically during the last two-thirds of ation (65 to 149 days). The specific activity of phosphatidic acid phosphohydrolase (Pase), disaturated lecithin (DSL), total phospholipids (TPL), and the L/S ratio were Eyed in the amniotic and tracheal fluids. There was a significant and progressive ease in the specific activity of PAPase in trache3 fluid beginniqaher 110 days, easing from 66 t 7 nmoles phosphate released x mg-1 protein x hour-1 (Mean ± S.E.M.) I days, to 107 ± 6 at 111 to 120 days, and to 277 ± 70 at 131 to 144 days. The rise in case specific activity was followed by a parallel rise in the DSL fraction of the TPL DS-L/TPL ratio), increasing from a DSL/TPL ratio of 0.06 ± 0.01 in fetuses ≤ 120 days, 0.29 ± 0.06 at 121 to 130 days, and to 0.50 ± 0.18 at 131 ± 135 days. The PAPase specific activity and the L/S ratio in amniotic fluid did not change during pregnancy. Speculation: Neonatal respiratory distress syndrome is the result of inadequate production of race active material by the fetal and neonatal lung. The enzyme PAPase occupies a tral role in the biosynthesis of the glycerophospholipids, and increases in the specificity of PAPase in human amniotic fluid and in fetal rabbit lung tissue precede or are parallel with the increase in the L/S ratio and pulmonary surfactant synthesis, reactively. We have shown that this sequence is also demonstrable in ovine fetal heal fluid, thus providing an animal preparation in which the formation and speculation of surfactant biosynthesis during fetal lung maturation can be investigated in vivo.

PHOSPHATIDIC ACID PHOSPHOHYDROLASE AND PHOSPHOLIPIDS IN TRACHEAL AND AMNIOTIC FLUIDS DURING NORMAL OVINE PREGNANCY

Samples of amniotic fluid and fetal tracheal fluid were obtained from 36 ovine pregnancies which were studied either acutely or chronically during the last two-thirds of gestation (65 to 149 days). The specific activity of phosphatidic acid phosphohydrolase (PAPase), disaturated lecithin (DS-L), total phospholipids (TLP) and the L/S ratio were assayed in the amniotic and tracheal fluids. There was a significant and progressive increase in the specific activity of PAPase in tracheal fluid beginning after 110 days, increasing from 66+/- 7 nmoles phosphate released X mg-1 protein x hour-1 (Mean +/- S.E.M.) less than or equal to 110 days, to 107 +/- 6 at 111 to 120 days, and to 277 +/- 70 at 131 to 144 days. The rise in PAPase specific activity was followed by a parallel rise in the DS-L fraction of the TPL (DS-L/TPL ratio), increasing from a DS-L/TPL ratio of 0.06 +/- 0.-1 in fetuses less than or equal to 120 days, to 0.29 +/- 0.06 at 121 to 130 days, and to 0.50 +/- 0.18 at 131 +/- 135 days. The PAPase specific activity and the L/S ratio in amniotic fluid did not change during pregnancy. Neonatal respiratory distress syndrome is the result of inadequate production of surface active material by the fetal and neonatal lung. The enzyme PAPase occupies a central role in the biosynthesis of the glycerophospholipids, and increases in the specific activity of PAPase in human amniotic fluid and in fetal rabbit lung tissue precede or are parallel with the increase in the L/S ratio and pulmonary surfactant synthesis, respectively. We have shown that this sequence is also demonstrable in ovine fetal tracheal fluid, thus providing an animal preparation in which the formation and regulation of surfactant biosynthesis during fetal lung maturation can be investigated in vivo.

Biosynthesis of pulmonary surfactant: Comparison of 1-palmitoyl- sn-glycero-3-phosphocholine and palmitate as precursors of dipalmitoyl- sn-glycero-3-phosphocholine in adenoma alveolar type II cells

Urethan-induced pulmonary adenomas of mice are composed of cells that appear to be morphologically identical to alveolar type II cells and synthesize disaturated diacyl- sn-glycero-3-phosphocholine, the major component of pulmonary surfactant. 1-[1- 14C]Palmitoyl- sn-glycero-3-phosphocholine and [1- 14C]palmitic acid were compared as precursors of disaturated diacyl- sn-glycero-3-phosphocholine in the adenoma type II cells by incubating both substrates with whole adenomas. When the precursors were compared at equal concentrations (100 μ m) in the presence of albumin (1 mg/ml), the rates of incorporation of 1-[1- 14C]palmitoyl- sn-glycero-3-phosphocholine and [1- 14C]palmitic acid into diacyl- sn-glycero-3-phosphocholine were 5.2 and 2.9 nmol/min · g tissue, respectively. The concentration of monoacyl- sn-glycero-3-phosphocholine (lysolecithin) in the blood plasma of BALB/c mice was 150 μ m. In short-term labeling experiments, the label in disaturated diacyl- sn-glycero-3-phosphocholine was equally distributed between the sn-1 and sn-2 positions when 1-[1- 14C]palmitoyl- sn-glycero-3-phosphocholine was the precursor, whereas 75 to 80% was in the sn-2 position when [1- 14C]palmitic acid was the precursor. The ratios are consistent with incorporation of 1-palmitoyl- sn-glycero-3-phosphocholine via the lysolecithin:lysolecithin transacylase reaction and incorporation of palmitate via acylation of 1-palmitoyl- sn-glycero-3-phosphocholine by acyl-CoA:lysolecithin acyltransferase. 1-[1- 14C]Palmitoyl- sn-glycero-3-phospho-[ 3H- methyl]choline was incorporated into total cellular diacyl- sn-glycero-3-phosphocholine with an isotope ratio similar to that of the precursor; the disaturated species was more enriched in 14C. These findings indicate the cells take up intact monoacyl- sn-glycero-3-phosphocholine and incorporate it into diacyl- sn-glycero-3-phosphocholine. The ability of the cells to utilize intact lysophosphoglycerides for synthesis of cellular lipids was further demonstrated by showing that ether analogs, 1-alkyl- sn-glycero-3-phosphocholine and 1-alkyl- sn-glycero-3-phosphoethanolamine, are taken up and acylated by the cells. Activities of lysolecithin:lysolecithin transacylase and acyl-CoA:lysolecithin acyltransferase were measured in subcellular fractions of the adenoma type II cells; the specific activities of the enzymes were 2.1 nmol/min · mg soluble protein and 21 nmol/min · mg microsomal protein, respectively. The total activity of the acyltransferase in the cell fractions was about four-fold higher than the activity of the transacylase. Characteristics of the two enzymes were studied and are discussed. The findings indicate that exogenous 1-palmitoyl- sn-glycero-3-phosphocholine and palmitic acid both serve as efficient precursors of disaturated diacyl- sn-glycero-3-phosphocholine in the adenoma alveolar type II cells.

Biosynthesis of phosphatidic acid by liver and lung of maternal and fetal rabbits

1. 1. The synthesis of phosphatidic acid from labelled precursors, [ 3H]palmitic acid and [ 14C]glycerol 3-phosphate. was studied in microsomes of liver and lung from maternal and fetal rabbits. 2. 2. The rate of in vitro synthesis of phosphatidic acid by liver microsomes of pregnant rabbits was in good agreement with the levels of plasma glycerolipids. 3. 3. It agrees with previous data on the biosynthesis of phosphoglycerides and pulmonary surfactant during the perinatal development of the lung. 4. 4. On the other hand. metabolic maturity of liver in order to synthesize phosphatidic acid was achieved during the postnatal period.

Autoperfused heart-lung preparation: a structurally appropriate experimental model for the metabolic study of the isolated lung.

This study was carried out to confirm the hypothesis that an autoperfused heart-lung preparation provides a favorable situation to analyze the dynamics of the biosynthesis of alveolar surface-active material, because the model did not produce any significant changes in the structure of the lung. Light and electron microscopy confirmed the absence of pulmonary lesions throughout the whole time of perfusion. A relevant finding was the periodic delamellation of the type II pneumocytes observed ultrastructurally, which coincided with the data obtained from the study on 14C-glycerol-3-phosphate and 3H-palmitic acid incorporation into total lipids and phosphoglycerides from lung tissue. This confirmed that the biosynthesis of pulmonary surfactant is rhythmic and periodic. From a structural point of view, the results suggest that the autoperfused heart-lung preparation constitutes a useful method for the in vivo metabolic study of the isolated lung.

Glycerol kinase activity in adenoma alveolar type II cells

Although several in vivo [l-3] and whole-cell [4-121 studies have demonstrated lung tissue can incorporate glycerol into the glycerol backbone of lipids, the presence of glycerol kinase has not been conclusively demonstrated in the tissue [2,13-151. We have been particularly interested in the biosynthesis of dipahnitoyl-sn-glycero-3-phosphocholine, which is the major component of pulmonary surfactant and is synthesized by the alveolar type II cells. In previous studies [ 12,16-l 81 , pulmonary adenomas induced by urethan were demonstrated to be a useful model for studies of lipid metabolism in alveolar type II cells. These benign adenomas consist almost exclusively of type II cells that contain lamellar bodies [ 19,201 and synthesize surfactant as normal type II cells [12,16] . However, surfactant biosynthesis is more active in adenoma type II cells than in whole lung [12,18] where type II cells comprise only 10% of the cells [21] . We found that adenomas incubated in Krebs-Ringer phosphate buffer incorporated [2-3H, 1 ,3-14C]gly cerol into the glycerol moiety of phosphatidylcholine and other lipids [ 121. This finding implied glycerol kinase is present and led us to examine the type II cells for activity. In the studies reported here, we demonstrate the conversion of labeled glycerol to glycerophosphate by glycerol kinase in cell-free preparations from adenoma type II cells.

Biosynthesis of dipalmitoyl- sn-glycero-3-phosphocholine by adenoma alveolar type II cells

Adenomas induced in lungs of BALB/c mice by urethane treatment consist almost exclusively of pulmonary type II cells and, like normal type II cells, produce dipalmitoylphosphatidylcholine, the major lipid component of lung surfactant [Snyder, C., Malone, B., Nettesheim, P., and Snyder, F. (1973) Cancer Res. 33, 2437–2443]. In the present investigation, these adenomas were used for studying the biosynthesis of dipalmitoylphosphatidylcholine in the intact cells. Whole adenomas were incubated in Krebs-Ringer phosphate buffer or Medium 199 with [1- 14C]palmitate, [1- 14C]acetate, [U- 14Clglycerol, or [1,3- 14C,2- 3H]glycerol and the synthesis of [ 14C]dipalmitoylphosphatidylcholine was subsequently determined after its isolation by cryochromatography. The [1- 14C]acetate was converted to [ 14C]palmitate, which was incorporated into disaturated phosphatidylcholine in the same manner as exogenous [1- 14C]palmitate. After a 1-h incubation of either [1- 14C]palmitate or [1- 14C]acetate, approximately 60% of the [ 14C]phosphatidylcholine was disaturated while 75–80% of the label in the disaturated species was in the acyl group at the 2 position. The same results were obtained when minced lung was used instead of adenomas, except the incorporation rate was higher in the adenomas. It was concluded from these studies that palmitate, whether synthesized de novo or provided exogenously, enters a common pool before being used by the type II cells for the synthesis of dipalmitoylphosphatidylcholine. When [1,3- 14C,2- 3H]glycerol was used to label phosphatidylcholine in the system, all molecular species, based on the degree of unsaturation, retained the same 3 H 14 C ratio. This indicated that the dihydroxyacetone phosphate pathway is of no more significance for the synthesis of disaturated phosphatidylcholine than for the other molecular species. The study demonstrates the usefulness of the adenoma system as a model for studying surfactant biosynthesis in pulmonary type II cells and indicates that the major pathway by which palmitate enters dipalmitoylphosphatidylcholine is by the deacylation and reacylation of phosphatidylcholine.

Studies on the biosynthesis of pulmonary surfactant. The role of the methylation pathway of phosphatidylcholine biosynthesis in primate and non-primate lung

The relative importance of the choline incorporation and methylation pathways of phosphatidylcholine biosynthesis was studied in the rat, rabbit, rhesus monkey and human lung. In vitro studies showed that phosphatidylethanolamine methyltransferase activity in the lungs of all species was only a fraction of that in rat liver. In vivo experiments were carried out to study the incorporation of an intravenously administered equimolar mixture of l-[ methyl- 14C]-methionine and [ methyl- 3H] choline into lipid in the lung and liver of the rabbit and monkey. Over 90% of the total radioactivity incorporated into lipid was found m phosphatidylcholine. In the liver, methionine incorporation was 40–71% of that of choline in the rabbit and 99–120% in the monkey while in the lungs of both species it was less than 3%. On a weight basis, choline incorporation in the lung was 2–4 times that in the liver. These findings suggest that the methylation pathway is of little quantitative significance in the biosynthesis of phosphatidylcholine in primate and non-primate lung.

Phospholipid Biosynthesis: The Activity of Phosphatidic Acid Phosphohydrolase in the Developing Lung and Amnionic Fluid

The presented data suggest that PAPase may, indeed, be important in the biosynthesis of lung phospholipids. The observed increase in the specific activity of rabbit lung precedes the surge of phosphatidylcholine concentration and surfactant biosynthesis by at least 24 hours. In human amnionic fluid, PAPase specific activity was markedly increased in pregnancies of 35 weeks of gestation or more. The increase in the specific activity of PAPase preceded the surge of acetone precipitated phosphatidylcholine. The reported studies may provide an additional procedure to examine the problems associated with surfactant biosynthesis.

Phospholipid biosynthesis: the activity of phosphatidic acid phosphohydrolase in the developing lung and amnionic fluid

The presented data suggest that PAPase may, indeed, be important in the biosynthesis of lung phospholipids. The observed increase in the specific activity of rabbit lung precedes the "surge" of phosphatidylcholine concentration and surfactant biosynthesis by at least 24 hours. In human amnionic fluid, PAPase specific activity was markedly increased in pregnancies of 35 weeks of gestation or more. The increase in the specific activity of PAPase preceded the "surge" of acetone precipitated phosphatidylcholine. The reported studies may provide an additional procedure to examine the problems associated with surfactant biosynthesis.

The enzymes of lecithin biosynthesis in human newborn lungs. 3. Phosphorylcholine glyceride transferase.

Extract: Phosphorylcholine glyceride transferase (EC. 2.7.8.2), the enzyme responsible for the final step of lecithin biosynthesis via the cytidine diphosphorylcholine (CDP-choline) pathway, is active in human neonatal lung tissue. A crude homogenate enzyme preparation in phosphate buffer was made from lung samples obtained at autopsy. The synthesis of product was linearly dependent upon protein concentration and linear with time for 30 min. The Michaelis constant for CDP-choline was 1.4 × 10-5 m. Optimal activity was attained at pH 7.5–8.0, at 35–39°, in the presence of Mg. Whole homogenate was 20–50 times as active as supernatant. No effect of oxygen or cysteine could be demonstrated. Triton and calcium inhibited the enzyme. The difference in enzyme activity between premature infants (3.53 × 10-7 mM lecithin/mg protein) and infants of longer (>32 weeks) gestation periods (1.76 × 10-7 mM lecithin/mg protein) is statistically significant at the 95% confidence limit. Speculation: Phosphorylcholine glyceride transferase could play a significant role in the relation between lung lecithin biosynthesis and respiratory distress syndrome of the human neonate. Characterization and study of the enzyme in human lung may lead to definition of its role as the possible regulator of pulmonary surfactant biosynthesis. There may be a relation between the level of enzyme activity and gestational age.

Intra- and extracellular compartmentalization of the surface-active fraction in dog lung

Adult mongrel dogs were killed at various times after injection of (3)H-labeled palmitate. The lungs were removed and subjected to an extensive saline lavage. The surface-active fraction was isolated from the lavage and from homogenized residual lung by a procedure based upon differential centrifugation in sucrose solutions. The material isolated from the lavage was designated extracellular surfactant; material from the residual lung was designated intracellular surfactant. Both had similar chemical composition and surface activity. The results of the isotopic labeling studies demonstrate that the two fractions have distinctly different specific activity curves. Label was incorporated into the intracellular surfactant rapidly and reached a peak at 1 hr. No radioactivity was found in the extracellular surfactant for the first 15 min, and the specific activity increased much more slowly than in the intracellular surfactant. These results demonstrate at least two anatomically distinct metabolic "pools" of pulmonary surfactant in the lung. While our data are not conclusive, one possible interpretation is that the biosynthesis of pulmonary surfactant takes place intracellularly with a subsequent secretion onto the alveolar surface.

Acyl transferase activities in dog lung microsomes

Mammalian lung has a high concentration of dipalmitoyl phosphatidylcholine and other phospholipids in which both fatty acid ester chains are saturated, as opposed to the usual asymmetric phospholipid (one saturated fatty acid and one unsaturated fatty acid). The acyl transferase system in dog lung microsomes was studied by determining the reactivities of various acyl CoA derivatives with 1-lyso-2-acyl- and 1-acyl-2-lyso-phosphatidylcholine. The 16:0 derivative had equal reactivity for both the 1- and 2-lyso positions. The 18:0 derivative also exhibited marked reactivity toward both positions, although the specific activity of the enzyme when palmitoyl CoA was used was approximately twice that compared to when stearoyl CoA was used. The 16:1 derivative showed approximately the same reactivity toward the 1-lyso position as did 16:0 but both 16:1 and 18:1 were more active with the 2-lyso position. These results suggest that acyl transferases may be important in the lung to insure that sufficient amounts of dipalmitoyl phosphatidylcholine will always be present for use in pulmonary surfactant biosynthesis. It is also conceivable that the acyl transferase system described acts on 1- and 2-lyso-palmitoyl phosphatidylcholine (produced by phospholipase hydrolysis of dipalmitoyl phosphatidylcholine) in order to produce phosphatidylcholine species needed for cellular purposes other than surfactant function.