Surfactant apoprotein Mr = 26,000-36,000 enhances uptake of liposomes by type II cells

The alveolar type II cell which synthesizes and secretes surfactant also plays a major role in the reuptake of surfactant lipids. In a recent in vivo study we found that the subfractions of natural surfactant that contained the surfactant protein with molecular weights of 26,000-36,000 (SP-26-36) were preferentially taken up into lamellar bodies of type II cells to a greater extent than were fractions that did not contain SP-26-36. Because the subfractions of natural surfactant in that study differed in other properties than the presence or absence of SP-26-36, the current study was undertaken to determine whether purified SP-26-36 enhanced the uptake of surfactant-like lipids by freshly isolated type II cells. SP-26-36 increased the uptake of label in radioactive surfactant-like lipids by up to 10-fold, and the effect of SP-26-36 was dependent on time, protein concentration, and temperature. The enhancement was inhibited by heat-treating the protein, by a polyclonal antibody against SP-26-36, and by metabolic inhibitors. The distribution of radioactivity in cell-associated phospholipids differed if cells were incubated with or without SP-26-36. If SP-26-36 was present during the incubation, greater than 96% of the radioactivity remained associated with phosphatidylcholine. In the absence of SP-26-36, only 85% of the radioactivity remained associated with phosphatidylcholine and 7% of the label appeared in phosphatidylglycerol. We hypothesize that SP-26-36 may act as a ligand to direct surfactant lipids to type II cells, perhaps to different metabolic pathways, and to regulate recycling and surfactant homeostasis.     

Altered surfactant function and metabolism in rabbits with acute lung injury

Lung injury was induced in rabbits with N-nitroso-N-methylurethane (NNNMU), and saturated phosphatidylcholine (Sat PC) pool sizes and phospholipid compositions were measured in alveolar wash subfractions isolated by differential centrifugation (large and small surfactant aggregates). Surfactant metabolism also was studied using intravascular and intratracheal radiolabels. Protein permeability, gas exchange, and compliance were significantly abnormal as lung injury progressed. At peak injury, there was a decrease in the large aggregate Sat PC pool size in alveolar wash accompanied by increased uptake of Sat PC from the air space and increased specific activity of both intravascular and intratracheal radiolabels in lamellar bodies. This was followed by a marked rise in the small aggregate pool size in the alveolar wash and increased secretion of Sat PC into the air spaces. Phospholipid compositions, total phospholipid-to-protein ratios, and in vivo functional studies using a preterm ventilated rabbit model were abnormal for both large and small aggregate surfactant fractions from the lung-injured rabbits. These studies characterize quantitative, qualitative, and functional changes of alveolar wash surfactant subfractions in NNNMU-injured lungs.     

Changes in quantity, composition, and surface activity of alveolar surfactant at birth

We hypothesized that when the lung makes the transition from the fluid- to the air-filled state at birth, there are changes in physical and functional properties of the alveolar surfactant. To test this hypothesis, newborn rabbits were killed at different times in the first 24 h of life, their lungs lavaged with ice-cold saline, and the lavage fluid subfractionated by differential centrifugation. The phospholipid and protein content and composition and the kinetics of surface tension lowering of the subfractions were examined. We found that with the onset of breathing, shifts occur in the distribution of surfactant subfractions as a surfactant apoprotein-free phospholipid fraction is generated. The ratio of rapidly sedimentable apoprotein-rich to slowly sedimentable, apoprotein-free fractions decreases from 31 at birth to 4 at 24 h of life. Concurrently, rates of surface tension lowering by the subfractions increase with time. The results suggest that the adult pattern of pool sizes and surface activity of alveolar surfactant is not present at birth but evolves slowly over the 1st day of life.     

Binding of annexins to lung lamellar bodies and the PMA-stimulated secretion of annexin V from alveolar type II cells

To identify lung lamellar body (LB)-binding proteins, the fractions binding to LB-Sepharose 4B in a Ca(2+)-dependent manner from the lung soluble fractions were analyzed with Mono Q column. Four annexins (annexins III, IV, V, and VIII) were identified by partial amino acid sequence analyses as the LB-binding proteins in the lung soluble fractions. A control experiment using phospholipid (phosphatidylserine/phosphatidylglycerol/phosphtidylcholine) liposome-Sepharose 4B revealed that annexins III, IV and V were the Ca(2+)-dependent proteins binding to the column in the lung soluble fractions, while annexin VIII was not detected. Thus, annexin VIII might preferentially bind to LB. On the other hand, the only Ca(2+)-dependent LB-binding protein identified in the bronchoalveolar lavage fluids was annexin V. It was further demonstrated that annexin V was secreted by isolated alveolar type II cells from rats and that the secretion was stimulated by the addition of phorbol ester (PMA), a potent stimulator of surfactant secretion. The PMA-dependent stimulation of annexin V was attenuated by preincubation with surfactant protein-A (SP-A), a potent inhibitor of surfactant secretion. As LB is thought to be an intracellular store of pulmonary surfactant, which is secreted by alveolar type II cells, annexin V is likely to be secreted together with the lamellar body.     

Protein composition of rabbit alveolar surfactant subfractions

The goal of this investigation was to characterize the proteins in subfractions of alveolar surfactant obtained by lung lavage and separated by differential centrifugation. It was previously demonstrated that the material in the more sedimentable fraction, which was enriched in tubular-myelin and was surface-active may be a precursor to the less sedimentable, vesicular, inactive material [1]. Separation of the proteins by polyacrylamide gel electrophoresis showed that the more sedimentable subfractions and rabbit surfactant isolated by conventional methods contained proteins with molecular weights comparable to those previously reported for alveolar surface active material (approximately 36 000 and 10 000). The less sedimentable subfractions contained less of these proteins. Immunoblots with anti-dog surfactant apoprotein antibodies, which cross-react with rabbit proteins, supported these observations. Immunoblots also showed that all of the subfractions contained serum proteins and secretory IgA, with the less sedimentable subfractions containing more secretory IgA. These results suggested that changes in protein composition may accompany functional changes in surfactant in the alveoli.     

Reutilization of surfactant phosphatidylglycerol and lysophosphatidylcholine by adult rabbits

Adult rabbits reutilize the phosphatidylcholine (PC) of surfactant much less efficiently than developing rabbits (22% vs. 95%). Comparisons of reutilization efficiency of other components of surfactant in adult rabbits have not been determined. We injected adult rabbits intratracheally with [3H]dipalmitoylphosphatidylcholine (DPPG) mixed with [14C]lysophosphatidylcholine (lysoPC) and natural surfactant or [14C]DPPC mixed with [3H]dipalmitoylphosphatidylglycerol (DPPG) and natural surfactant. Recovery in the alveolar wash and lamellar bodies of labelled DPPC, lysoPC and DPPG was determined at different times after injection. By plotting the ratio of [3H]DPPG to [14C]DPPC in the alveolar wash versus time after injection we found that phosphatidylglycerol was reutilized with an efficiency of only 0-7% which was much less than the reutilization of PC in these animals. At early times after injection, adult rabbits injected with [14C]lysoPC had a ratio of [14C]PC in their alveolar wash to lamellar bodies that was larger than 1.0. By comparison, 3-day old rabbits injected intratracheally with [14C]lysoPC had a ratio of [14C]PC in alveolar wash to lamellar bodies less than 1.0 at the earliest times measurable. Thus adult rabbits demonstrate a pathway for accumulation of PC in their alveolar space prior to its appearance in lamellar bodies. This was not detected in developing rabbits. As in developing rabbits, adult rabbits reutilize the phosphatidylglycerol of surfactant less efficiently than the PC of surfactant.     

Characterization of rabbit lung lysosomes and their role in surfactant dipalmitoylphosphatidylcholine catabolism

Although alveolar surfactant is rapidly catabolized in adult rabbit lungs, the pathways have not been characterized. Pathways of surfactant secretion and recycling involve lamellar bodies and multivesicular bodies, organelles shown to be related to lysosomes by cytochemistry and autoradiography. Since lysosomes are central to intracellular catabolic events, it is possible that lysosomes are involved in intrapulmonary surfactant catabolism. Lysosomes relatively free of contaminating organelles (as determined morphologically and by marker enzymes for mitochondria, endoplasmic reticulum, peroxisomes, and plasma membranes) were obtained from post-lavage lung homogenates of 1-kg rabbits by differential centrifugation in buffered sucrose and gradient separation in percoll (density, 1.075-1.165). The role of lung lysosomes in catabolism of dipalmitoylphosphatidylcholine (DPC) was then studied in rabbits killed 4, 12, and 24 h following intratracheal injection of [3H]DPC and [14C] dihexadecyl phosphatidylcholine (DPC-ether). While equal amounts of label were in the lamellar body containing fractions at 4 h, nearly 6-fold more DPC-ether label than DPC label was recovered in the lysosomal fractions. By 24 h, there was 15-fold more DPC-ether in the lysosomes. This is the first report of successful isolation of lysosomes relatively free of other organelles from rabbit lungs. The tracer studies indicate DPC and DPC-ether follow similar intracellular processing after alveolar uptake. The subsequent accumulation of the ether analog in the lysosomal fractions supports a role for these organelles in surfactant DPC catabolism.     

Phosphatidylglycerol in lung surfactant. III. Possible modifier of surfactant function.

Lamellar bodies and alveolar lavage from adult mammalian lung contain unusually high concentrations of phosphatidylglycerol that could serve as a sensitive indicator of surfactant. Phosphatidylglycerol was absent and phosphatidylinositol was correspondingly prominent in surfactant from the preterm rabbit fetus. Phosphatidylglycerol rapidly appeared and phosphatidylinositol decreased following the delivery. Surfactant isolated from the prematurely born rabbit or from humans with respiratory distress syndrome never contained phosphatidylglycerol. Comparison between lamellar bodies from fetal and postnatal rabbits revealed remarkably similar composition except for the acidic phospholipids; however, the physico-chemical properties were different. The compressibility of the surface film (i.e. the ratio of the fractional decrease in surface area and the corresponding decrease in surface tension) at low surface tensions was higher with fetal than with postnatal surfactant, whereas the difference in minimum surface tensions was small. These data suggest that phosphatidylglycerol is not an essential component required for the formation of the complex, but it improves the properties of surfactant in stabilizing the alveoli.     

Quantification of surfactant pool sizes in rabbit lung during perinatal development

Methods are presented for the quantitative isolation of surfactants from fetal and newborn rabbit alveolar lavage returns and post-lavaged lung tissue homogenates. The phospholipid content of both fractions progressively increased between 27 days gestation and term (31 days). The tissue-stored fraction increased approximately 16-fold (from 0.48 +/- 0.13 to 7.83 +/- 0.86 mg/g dry lung) and the alveolar fraction more than 30-fold (from 0.08 +/- 0.02 to 2.69 +/- 0.52 mg/g dry lung). Developmental changes in phospholipid composition were also observed. Tissue-stored surfactant was prepared using differential and density gradient centrifugation. Alveolar surfactant was isolated during fetal development as a high-speed pellet following a one-step differential centrifugation. There was little change in the phospholipid content of fetal alveolar lavage supernatant (range 0.12 +/- 0.04 to 0.28 +/- 0.09 mg/g dry lung). By the first postnatal day the phospholipid content of both lavage fractions significantly increased (pellet, 7.51 +/- 1.79; supernatant, 4.01 +/- 1.36 mg/g dry lung) and both were identified as surfactant. This increase in alveolar surfactant was accompanied by an approximately twofold decrease (to 3.81 +/- 1.1 mg/g dry lung) in the tissue-stored fraction. These data provide a quantitative profile of surfactant accumulation and secretion in developing rabbit lung.     

Postnatal transformations of alveolar surfactant in the rabbit: changes in pool size, pool morphology and isoforms of the 32-38 kDa apolipoprotein

To clarify perinatal transformations of surfactant we performed lung lavage in term fetuses and in 0-24-h-old newborn rabbits. Lavage fluid was separated into three pools, namely lavage pellet, lavage supernatant and cells. We found that at birth the pellet contains 94.1 +/- 1.4% (S.E.) saturated phosphatidylcholine, while the supernatant and cells contain traces of it. At birth the pellet contains secreted lamellar bodies while the supernatant lacks any recognizable structure. After birth, the alveolar saturated phosphatidylcholine level increases 5.1-times in 24 h, the proportions between pools reaching adult values in 90 min (pellet = 75.9 + 4.8%, supernatant = 22.7 +/- 4.9%), and small vesicles appear in the supernatant, probably originating from the turnover of alveolar surfactant during breathing. The saturated phosphatidylcholine associated with cells remains unchanged. At birth, the 32-38 kDa surfactant apolipoprotein appears to be less extensively sialylated than in adult life.     

The in vivo labeling with acetate and palmitate of lung phospholipids from developing and adult rabbits

The labeling with radiolabeled acetate and palmitate of lung, microsomes isolated from lung, and surfactant phospholipids from adult, 3-day-old, and newborn rabbits was studied. The half-life of phosphatidylcholine from lung and microsomal fractions was shorter when labeled with acetate than when labeled with palmitate. Half-time values similarly measured for phosphatidylglycerol, phosphatidylinositol or phosphatidylethanolamine were not different for the two labels. Acetate and palmitate-labeled phospholipids appeared in the surfactant fraction with similar accumulation curves. The relative specific activities of acetate-labeled phosphatidylcholine from adult, 3-day-old, and newborn rabbits, respectively, were 1.30, 1.86 and 1.77 times those measured for those measured for the palmitate label. Surfactant phosphatidylinositol and phosphatidylethanolamine from 3-day-old animals similarly were labeled preferentially with acetate. However, phosphatidylglycerol purified from the surfactant fraction contained equivalent relative amounts of the acetate and palmitate labels in 3-day-old and adult rabbits. These results suggest that the type II pneumocyte may use acetate preferentially for the synthesis of palmitic acid which then is incorporated into surfactant phospholipids.     

Lysophosphatidylcholine uptake and metabolism in the adult rabbit lung

Tracer quantities of 3H-labeled lysoPC and 32P-labeled natural rabbit surfactant were given intratracheally via a bronchoscope and [14C]palmitate was given intravenously to 25 rabbits with labeled PC and lysoPC measured in the alveolar wash, lung homogenate, lamellar bodies and microsomes at five times from 10 min to 6 h after tracheal injection. Surprisingly, only 31% of the administered lysoPC remained in its original form in the total lungs (alveolar wash + lung homogenate) by 10 min, of which 77% was in the alveolar wash. Meanwhile, by 10 min an additional 37% was already converted to PC, of which more than 98% was in the lung homogenate. LysoPC continued to be rapidly and efficiently converted to PC, with 62% conversion measured at 3 h. The converted lysoPC initially appeared with high specific activity in microsomes, then in lamellar bodies, and finally in the alveolar wash. The intravascular palmitate labeled lung PC had similar specific activity-time profiles in the subcellular fractions, while intratracheally administered natural rabbit surfactant had a constantly low specific activity in microsomes and much higher specific activities in lamellar bodies and alveolar wash. Another 25 rabbits received intratracheal lysoPC labeled in both the choline and palmitate moieties and then were studied from 1 to 24 h after tracheal injection. The ratio of the palmitate to choline labels indicated uptake and conversion to PC primarily by direct acylation rather than transacylation and by intact reuptake and conversion rather than breakdown and resynthesis. LysoPC is an attractive 'metabolic probe' of surfactant metabolism which undergoes very rapid and efficient intracellular conversion to PC via a subcellular pathway that parallels the remodeling and de novo synthetic pathways.     

Organotypic cultures of diploid type II alveolar pneumonocytes: surfactant associated esterase activity

Organotypic cultures, established from enzymatically dispersed day 19 fetal rat lung, are comprised primarily of cells which are morphologically similar to type II alveolar pneumonocytes, the cells involved in surfactant synthesis. To further characterize these cultures, the nonspecific esterase pool was examined to determine if these cultures contained certain nonspecific esterases previously shown to be enzyme markers for the surfactant system. The results of biochemical, electrophoretic and cytochemical studies indicate that these organotypic cultures contain the same nonspecific esterases already demonstrated in surface active fractions derived from rat and mouse lung homogenates and pulmonary lavage fluid. As in whole lung, the major site of esterase activity in the organotypic cultures is the type II cell lamellar body, the putative site of surfactant synthesis and storage. These findings support the concept that the organotypic cultures derived from fetal rat lung are comprised predominantly of type II cells which retain surfactant associated functions in vitro.     

Postnatal transformations of alveolar surfactant in the rabbit: changes in pool size,pool morphology and isoforms of the 32–38 kDa apolipoprotein

To clarify perinatal transformations of surfactant we performed lung lavage in term fetuses and in 0–24-h-old newborn rabbits. Lavage fluid was separated into three pools, namely lavage pellet, lavage supernatant and cells. We found that at birth the pellet contains 94.1 ± 1.4% (S.E.) saturated phosphatidylcholine, while the supernatant and cells contain traces of it. At birth the pellet contains secreted lamellar bodies while the supernatant lacks any recognizable structure. After birth, the alveolar saturated phosphatidylcholine level increases 5.1-times in 24 h, the proportions between pools reaching adult values in 90 min (pellet = 75.9 + 4.8%, supernatant = 22.7 ± 4.9%), and small vesicles appear in the supernatant, probably originating from the turnover of alveolar surfactant during breathing. The saturated phosphatidylcholine associated with cells remains unchanged. At birth, the 32–38 kDa surfactant apolipoprotein appears to be less extensively sialylated than in adult life.     

Surfactant subtypes in mice: characterization and quantitation

Surfactant obtained by bronchoalveolar lavage of normal adult mice was separated into subtypes by a one-step centrifugation to equilibrium on continuous sucrose gradients. Mouse surfactant resolved in this way exists in three subtypes with similar phospholipid compositions. A "light" subtype of buoyant density 1.027 +/- 0.012 (SD) g/ml comprises 43 +/- 18% of the total alveolar lavage phospholipid, has little surface activity, and consists exclusively of small unilamellar vesicles. A "heavy" subtype of buoyant density 1.055 +/- 0.016 g/ml comprises 48 +/- 11% of the total, is surface active, and consists of small amounts of tubular myelin among large empty vesicles. A third component, called "ultraheavy," comprises 9 +/- 4% of the total alveolar lavage phospholipid, has a density of 1.072 +/- 0.020 g/ml, is surface active, and consists of large aggregates of tubular myelin associated with lamellar bodylike structures. Labeling studies suggested that the ultraheavy material was labeled first and was of the same density as purified lamellar bodies. These results are consistent with the view that, in mice, surfactant is secreted into the alveolar compartment in an ultraheavy form, which evolves into the heavy and light forms.     

Alterations in rat alveolar surfactant phospholipids and proteins induced by administration of chlorphentermine

Chlorphentermine is a cationic amphiphilic drug which produces a phospholipid storage disorder in rat lungs. Experiments were carried out to characterize changes in the composition of acellular alveolar lavage materials and to study possible mechanisms by which pulmonary surfactant phospholipidosis is produced by administration of the drug. Following ten daily injections of chlorphentermine (25 mg/kg body weight), there are 12.2- and 13.6-fold increases of pulmonary lavage total phospholipids and disaturated phosphatidylcholines (disaturated PC), respectively. In addition, there is a 2.8-fold increase in total protein and a 12.7-fold increase in the surfactant apoprotein group with molecular weights from 28,000 to 32,000. We measured incorporation of labeled palmitate, choline and glycerol into disaturated PC in type II cells and alveolar macrophages isolated from control and chlorphentermine-treated animals. The drug does not affect the incorporation of labeled substrates into disaturated PC in either cell type. However, in alveolar macrophages there is a decrease in the rate of intracellular degradation of recently synthesized disaturated PC in chlorphentermine-treated animals. The drug also inhibits the phospholipase-induced catabolism of rat surfactant disaturated PC which occurs during incubation of alveolar lavage fluid in vitro at 37 degrees C. When the lavage fluid is divided into subfractions by differential centrifugation, a larger percentage of the phospholipid is distributed in the less sedimentable subfractions in chlorphentermine-treated animals relative to controls, suggesting the accumulation of older surfactant materials. These results suggest that chlorphentermine-induced phospholipidosis of pulmonary surfactant materials is due to decreased rates of phospholipid degradation.     

Phosphatidylglycerol in lung surfactant. II. Subcellular distribution and mechanism of biosynthesis in vitro.

Lamellar inclusion bodies, apparent precursors for alveolar surfactant lining, have remarkably similar phospholipid composition to surfactant from alveolar lavage, but distinctly different from other fractions studied: mitochondria, microsomal fraction containing endoplasmic reticulum membranes, plasma membranes and nuclei. Surfactant contained (as % of total phospholipid phosphate): 75.5-77.0% lecithin, 11.0-11.2% phosphatidylglycerol, 4.2-4.6% phosphatidylethanolamine, 3.0-3.2% phosphatidylinositol, 1.5-1.7% bis-(monoacylglycerol) phosphate, 1.2-1.9% phosphatidylserine, and 0.7-1.5% sphingomyelin. Fatty acids of phosphatidylglycerol from lamellar bodies were similar to those from microsomes but different from those in mitochondria. Lung homogenate in continuous sucrose density gradient displayed two major activity peaks of phosphatidylglycerol synthesis: the heavier from mitochondria; the lighter from endoplasmic reticulum. Studies on mechanism of phosphatidylglycerol synthesis in vitro revealed (in these two fractions) CDP-diglyceride and sn-glycerol phosphate precursors to phosphatidylglycerol phosphate, that hydrolysed to phosphatidylglycerol. In microsomes disaturated CDP-diglycerides were 1.6-1.9 times more active substrates than in mitochondria, whereas CDP-diglycerides from egg lecithin were almost equally active. In contrast to lung mitochondria no cardiolipin synthesis was detected in microsomes. The highest specific activities for phosphatidate cytidyltransferase, CDP-diglyceride-inositol phosphatidyltransferase, choline phosphotransferase, and phosphatidylethanolamine methyltransferase were all found in microsomes. The present in vitro studies and additional evidence (M. Hallman and L. Gluck, (1975) Fed. Proc. 34, 274) support the hypothesis that de novo synthesis of surfactant lecithin phosphatidylinositol and phosphatidylglycerol takes place in the endoplasmic reticulum of alveolar cells.     

Cellular uptake and processing of surfactant lipids and apoprotein SP-A by rat lung

The intracellular pathways and the kinetics of metabolism of surfactant apoprotein and lipid, which may be recycled from the alveolar space, are largely unknown. We used a lipid-apoprotein complex made from liposomes of pure lipids in a ratio found in mammalian pulmonary surfactant plus surfactant apoprotein (SP-A, Mr = 26,000-36,000) to test some possible relationships in the recycling of these major surfactant components between intrapulmonary compartments. After intratracheal instillation of 80 microliters of an apoprotein-liposome mixture with separate radiolabels in the lipid and the apoprotein, rats were killed at times from 8 min to 4 h later. The lungs were lavaged with saline, and subcellular fractions were isolated on discontinuous sucrose density gradients. Both the [14C]lipid radiolabel and the 125I-apoprotein radiolabel demonstrated a time-dependent increase in radioactivity recovered in a lamellar body-enriched fraction. Uptake of the radiolabels into other subcellular fractions did not exhibit a clear-cut time dependence; more of the protein than the lipid radiolabel was found in the Golgi-rich and microsomal fractions. We conclude that both the lipid and apoprotein portions of lung surfactant are taken up by lung cells and are incorporated into secretory granules of the cells.     

Secretion of surfactant by primary cultures of alveolar type II cells isolated from rats

Pulmonary surfactant conventionally is prepared from material obtained by endobronchial lavage. Although it has been assumed that the components of surfactant are secreted by alveolar type II cells, direct proof of this assumption has not been available. Furthermore, it is possible that the final material obtained by lavage has been modified after secretion or altered during the isolation procedure. It has been shown previously that type II cells, after 1 day in primary culture, secrete saturated phosphatidylcholine, one of the lipid components of surfactant. Because saturated phosphatidylcholine is not unique to surfactant and because type II cells in culture lose differentiated characteristics over the first several days in culture, it has not previously been established how closely the secretory products of cultures of type II cells resemble surfactant as obtained by endobronchial lavage. We therefore studied the morphologic, physical and chemical characteristics of the material that type II cells secrete under basal conditions and after stimulation with terbutaline or 12-O-tetradecanoyl-13-phorbol acetate. The secreted material resembled surfactant obtained by lavage; it was similar morphologically to the lamellar material and tubular myelin seen in the fluid-filled alveoli of fetal rats, it lowered surface tension to 5 mN per meter, and it contained the 72000 dalton apolipoprotein of surfactant (as measured by the 'rocket' immunoelectrophoresis technique). When cells were incubated for 22 h with [1-(14)C]acetate, the distribution of radioactivity in the secreted material was very similar to the phospholipid composition of rat surfactant. We conclude that the material secreted by alveolar type II cells after 1 day in primary culture is similar to surfactant obtained by endobronchial lavage.     

ISOLATION AND CHARACTERIZATION OF LAMELLAR BODIES AND TUBULAR MYELIN FROM RAT LUNG HOMOGENATES

Three surface-active fractions which differ in their morphology have been isolated from rat lung homogenates by ultracentrifugation in a discontinuous sucrose density gradient. In order of increasing density, the fractions consisted, as shown by electron microscopy, primarily of common myelin figures, lamellar bodies, and tubular myelin figures. The lipid of all three fractions contained approximately 94% polar lipids and 2% cholesterol. In the case of the common myelin figures and the lamellar bodies, the polar lipids consisted of 73% phosphatidylcholines, 9% phosphatidylserines and inositols, and 8% phosphatidylethanolamines. In the case of the tubular myelin figures, the respective percentages were 58, 19, and 5. Over 90% of the fatty acids of the lecithins of all three fractions were saturated. Electrophoresis of the proteins of the fractions in sodium dodecyl sulfate or Triton X-100 revealed that the lamellar bodies and the tubular myelin figures differed in the mobilities of their proteins. The common myelin figures, however, contained proteins from both of the other fractions. These data indicate that, whereas the lipids of the extracellular, alveolar surfactant(s) originate in the lamellar bodies, the proteins arise from another source. It is further postulated that the tubular myelin figures represent a liquid crystalline state of the alveolar surface-active lipoproteins.