The periodicity and architecture of lipid retained and extracted lung surfactant and its origin from multilamellar bodies.

Lipid and carbohydrate retaining procedures were utilized to retain these components of surfactant in thin section. The origin of tubular myelin surfactant from multilamellar bodies was postulated on morphological evidence to be : an expansion of lamellae in pairs caused by the absorption of a homogeneous, probably carbohydratebased, material of undetermined origin; immediately after the pairs expanded to 438 Å, the lamella (lipid bilayer) that was most peripheral in the multilamellar body underwent a molecular rearrangement to form the two cross lipid bilayers; the four lipid bilayers formed a square tubule 413 Å of side and 0.16–1.8 μ long. The homogeneous material included within each tubule (the surfactant matrix) did not completely fill the intratubular space, but a centrally located, spherical, 57 Å diameter, electron transparent, area was observed. The 413 Å square tubules were separated on all sides by a 25 Å fatty acid tail area. The phospholipid head areas were 46 Å and the surfactant matrix was 132 Å between the electron transparent, center area and the phospholipid heads layer.Extraction with several organic solvents was also studied: slight ethanol extraction—alteration of surfactant matrix and loss of transparent, central area. Moderate ethanol extraction—alteration of surfactant matrix to form artifactual tubules and filaments, disruption of lipid bilayers. Severe extraction—ethanol produced loss of integrity of both matrix and lipid bilayers; vinylcyclohexane dioxide or methacrylate produced complete loss of all lipid bilayers, but surfactant matrix retained tubular configuration ; DMP-30 produced complete loss of lipid bilayers, and although most of the matrix material was retained, all areas appeared fractured and randomly oriented.     

The three-dimensional aspect of mammalian lung multilamellar bodies

The three-dimensional aspect of rat and monkey lung multilamellar bodies was demonstrated in lipid retained thin sections. The glutaraldehyde and urea lipid retention embedment and an Epon 812 resin polar dehydrant procedure were utilized to retain lamellar lipids for precise morphological study. The unextracted multilamellar bodies were found to conform to a general, though complex, threedimensional structure. A model that demonstrated that structure was derived. Freezeetch and extracted material were shown to support the model. Mature multilamellar bodies were from 1.2–1.6 μ in diameter and were 1.0–1.6 μ high. Each body contained a matrix core that included from 2–25 vesicular bodies and was in contact with the limiting membrane at the matrix plate. Most bodies had from 25–70 lamellae attached for 360 ° to the projection plate. Microtubules were seen in communication with the matrix core. When sectioned in longitudinal section, lamellae projected from the base plate and coursed parallel to the limiting membrane of the top half of the body. Any cross-section produced circular lamellae without apparent attachment. Oblique sections sometimes produced both ‘stacked’ and ‘circular’ lamellae. Four postulates of multilamellar body formation were discussed in light of these findings.     

The ultrastructure of multilamellar bodies and surfactant in the human lung

Summary Normal tissues from human lungs were dehydrated through Epon 812 resin to retain many of the lipids and carbohydrates in thin section. The three-dimensional structure of the multilamellar body was determined. The paired layer of phospholipid heads (PH) is 36Å thick; the layer of fatty-acid tails (FA) is 31Å, the same as reported previously for non-human primates and rodents. The human multilamellar body is apparently unique: the lamellae of the major focus divide into two or three lamellae; the matrix material of the core is without vesicular bodies and a projection core is present. When compared with those of the rat, human tissues contain a greater number of lamellar foci and fewer lamellae per focus. The presence of a peripheral layer of lamellae, an ever-present external limiting membrane, and the fusion of multilamellar bodies are also characteristic. Tubular myelin surfactant has the same appearance as in other mammals.Multilamellar bodies were observed in direct communication with Golgi vesicles. Their origin from multivesicular bodies and their maturation through secretion and exocytosis were demonstrated.Untransformed multilamellar bodies in the alveolar space demonstrated three periodicities (P): (1) compact regular lamellae, PH = 36Å, FA = 31Å, P = 66Å; (2) compact broad lamellae, PH = 72Å, FA = 22Å, P = 94Å; (3) loose lamellae, PH = 36Å, FA = 36Å, FA = 31Å with a variable interlamellar space.Appreciation is expressed to Nuket Olson and Phil Offenhauser for their technical assistance. Supported by a grant from the American Lung Association     

The high resolution ultrastructure of the periodicity and architecture of lipid-retained and extracted lung multilamellar body laminations

The three-dimensional aspect of rat and monkey lung multilamellar bodies was demonstrated in lipid retained thin sections. The glutaraldehyde and urea lipid retention embedment and an Epon 812 resin polar dehydrant procedure were utilized to retain lamellar lipids for precise morphological study. The unextracted multilamellar bodies were found to conform to a general, though complex, threedimensional structure. A model that demonstrated that structure was derived. Freezeetch and extracted material were shown to support the model. Mature multilamellar bodies were from 1.2–1.6 μ in diameter and were 1.0–1.6 μ high. Each body contained a matrix core that included from 2–25 vesicular bodies and was in contact with the limiting membrane at the matrix plate. Most bodies had from 25–70 lamellae attached for 360 ° to the projection plate. Microtubules were seen in communication with the matrix core. When sectioned in longitudinal section, lamellae projected from the base plate and coursed parallel to the limiting membrane of the top half of the body. Any cross-section produced circular lamellae without apparent attachment. Oblique sections sometimes produced both ‘stacked’ and ‘circular’ lamellae. Four postulates of multilamellar body formation were discussed in light of these findings.     

Immunocytochemical localization of lysozyme and surfactant protein A in rat type II cells and extracellular surfactant forms.

Using immunogold labeling of fixed, cryosubstituted tissue sections, we compared the distribution of lysozyme, an oxidant-sensitive lamellar body protein, with that of surfactant protein A (SP-A) in rat Type II cells, extracellular surfactant forms, and alveolar macrophages. Morphometric analysis of gold particle distribution revealed that lysozyme and SP-A were present throughout the secretory and endosomal pathways of Type II cells, with prominent localization of lysozyme in the peripheral compartment of lamellar bodies. All extracellular surfactant forms were labeled for both proteins with preferential labeling of tubular myelin and unilamellar vesicles. Labeling of tubular myelin for SP-A was striking when compared with that of lamellar bodies and other extracellular surfactant forms. Lamellar body-like forms and multilamellar structures were uniformly labeled for lysozyme, suggesting that this protein is rapidly redistributed within these forms after secretion of lysozyme-laden lamellar bodies. By contrast, increased labeling for SP-A was observed over peripheral membranes of lamellar body-like forms and multilamellar structures, apparently reflecting progressive SP-A enrichment of these membranes during tubular myelin formation. The results indicate that lysozyme is an integral component of the lamellar body peripheral compartment and secreted surfactant membranes, and support the concept that lysozyme may participate in the structural organization of lung surfactant.     

Ultrastructural alterations of pulmonary surfactant in rat lungs after inhibition of protein synthesis by puromycin.

The effects of systematically administered puromycin on the fine structure of the lung were studied. The effects varied depending on the duration of exposure and the time interval between the last injection and sacrifice. After short term exposure most surfactant had separated from the epithelial surface and profound alterations in the tubular myelin structure were seen. After moderate duration of exposure a previously undescribed multilamellar lining layer was observed which was often detached from the alveolar epithelium. Six hours after the last injection the regular tubular myelin pattern reappeared. Puromycin treatment results in inhibition of various proteins synthesized by type II epithelial cells. Inhibition of synthesis of some proteins, most probably that of glycoprotein A, causes a primary effect on the structure of surfactant. The loss of at least some of the cytoskeletal proteins in Type II epithelial cells apparently results in interference with exocytosis of lamellar body contents.     

Monomolecular surface film and tubular myelin figures of the pulmonary surfactant in hamster lung

Summary Perfusion fixation via pulmonary trunk was applied to the alveolar lining layer in situ at different lung volumes using a fixative containing tannic acid-ferrocyanide osmium. The monomolecular surface film and hypophasic tubular myelin figures were enhanced. In the range of transpulmonary pressure (1–10 cmH₂O), the surface film appeared in the form of a single, electron-dense leaflet, 2.7±0.6 nm (M±SD) in thickness while trilaminar membrane structure was retained in all parts of the tubular myelin figures of the hypophase. The surface film was attached underneath at right angles with trilaminar membranes which formed the outermost parts of the tubular myelin. Such structural continuity was taken to support a view that the phospholipid unit membrane of the tubular myelin figure would be transformed at the hydrophobic phase into a pair of monomolecular leaflets, eventually forming the surface film.     

Uptake of lung surfactant subfractions into lamellar bodies of adult rabbit lungs

The goals of this investigation were to determine whether subfractions of alveolar surfactant that have different physical and biochemical properties are preferentially taken up from the alveolar air space into lamellar bodies and to correlate the magnitude of the uptake with the properties of the fractions. Radiolabeled subfractions were obtained by differential centrifugation of lavage fluid from rabbits that had been intravenously injected with radioactive palmitate. The subfractions were P (pellet) 3 (1,000 g, 20 min), P4 (60,000 g, 60 min), P5 (100,000 g, 16 h). Subfractions were instilled into the lungs of anesthetized spontaneously breathing adult rabbits, and lavage and lamellar body fractions were isolated at later times. P3 and P4 were taken up to a larger extent than was P5 or liposomes prepared from a P4 lipid extract. The fractions that were preferentially taken up (P3 and P4) contained surfactant apoprotein (APO) 36, tubular myelin, multilamellar vesicles, and were rapidly adsorbed to an air-water interface. P3 also contained APO 10. These results demonstrate that different forms of surfactant are recycled at different rates and suggest that there is specificity in the recycling process.     

Immunocytochemical localization of the major surfactant apoproteins in type II cells, Clara cells, and alveolar macrophages of rat lung

The adsorptive properties of phospholipids of pulmonary surfactant are markedly influenced by the presence of three related proteins (26-38 KD, reduced) found in purified surfactant. Whether these proteins are pre-assembled with lipids before secretion is uncertain but would be expected for a lipoprotein secretion. We performed indirect immunocytochemistry on frozen thin sections of rat lung to identify cells and intracellular organelles that contain these proteins. The three proteins, purified from lavaged surfactant, were used to generate antisera in rabbits. Immunoblotting of rat surfactant showed that the IgG reacted with the three proteins and a 55-60 KD band which may be a polymer of the lower MW species. Specific gold labeling occurred over alveolar type II cells, bronchiolar Clara cells, alveolar macrophages, and tubular myelin. In type II cells labeling occurred in synthetic organelles and lamellar bodies, which contain surfactant lipids. Lamellar body labeling was increased fivefold by pre-treating tissue sections with a detergent. Multivesicular bodies and some small apical vesicles in type II cells were also labeled. Secondary lysosomes of alveolar macrophages were immunoreactive. Labeling in Clara cells exceeded that of type II cells, with prominent labeling in secretory granules, Golgi apparatus, and endoplasmic reticulum. These observations clarify the organelles and pathways utilized in the elaboration of surfactant. After synthesis, the proteins move, probably via multivesicular bodies, to lamellar bodies. Both lipids and proteins are present in tubular myelin. Immunologically identical or closely similar proteins are synthesized by Clara cells and secreted from granules which appear not to contain lipid. The role of these proteins in bronchiolar function is unknown.     

A peculiar mode of formation of the surface lining layer in the lungs of Salamandra salamandra

The pneumocytes of the larva of Salamandra salamandra contain numerous lamellar bodies and their precursors: electron-dense bodies at various stages of development. Both lamellar bodies and electron-dense bodies occur inside the fluid-filled lung. The former are spherical or bell-shaped and possess concentrically arranged smooth membranes, 8 nm thick; the latter have paracrystalline cores composed of alternately oriented clear and dark striations (3.6–3.9 nm and 2.6–3.6 nm, respectively). On all sides such cores separate membranes, which assume a concentric orientation. No tubular myelin was observed in any phase of the transformation of lamellar bodies and electron-dense bodies into the surface lining layer. Fixation of the lungs of adult individuals with tannic acid-containing fixative visualized the surface lining layer, but not tubular myelin.     

Accessibility of Galactosyl Ceramides to Probe Reagents in Central Nervous System Myelin

The suitability of isolated central nerve myelin preparations for probe labelling studies was assessed and the accessibility of galactosyl ceramides in myelin to galactose oxidase and sodium periodate was determined. Isolated myelin preparations present a uniform external membrane surface to added probes because lamellae in the myelin sheath separate at their external apposition surfaces exclusively during isolation. The cytoplasmic apposition remains intact in isolated myelin. Cationised ferritin can gain access along external apposition regions of inner lamellae in multilamellar fragments of isolated myelin, indicating that proteins and lipids on the external membrane surface will be accessible to probes. Over 50% of the total galactosyl ceramides of myelin are accessible to galactose oxidase attack; hydroxy fatty acid- and nonhydroxy fatty acid-containing cerebrosides are equally attacked. Sodium periodate attacks over 90% of the galactosyl ceramides in isolated myelin at 20°C and electron micrographs of the periodate-treated myelin reveal changes at the external apposition only. Galactosyl ceramides in vesicles of myelin lipid vesicles are not so readily attacked by periodate. The disposition of galactosyl ceramides in the myelin lamellae is discussed.     

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.     

Stabilization of large multilamellar liposomes by human serum in vitro

The leakage of 5,6-carboxyfluorescein from large multilamellar liposomes prepared from dipalmitoylphosphatidylcholine (without or with cholesterol) was investigated in vitro in the presence of human serum. Below the phospholipid phase transition temperature, the rate of dye release is retarted 3–8-fold in the presence of up to 25% human serum in the incubation medium, as compared to the release in isotonic phosphate-buffered saline. This effect is significantly augmented by incorporation of 50 mol% cholesterol into the lipid bilayer. At and above the phase transition temperature, the initial rapid dye leakage in the presence of serum is followed by a slow long-term release. Incubation of the liposomes with serum is assumed to result in the association of serum proteins with the outermost lipid bilayer which in turn will lead to their stabilization, while the inner lamellae are not immediately accessible to the serum proteins. The permeability of the outer protein-rich lipid bilayer appears to be restricted, as concluded from the decreased dye release in the presence of serum. Massive leakage from multilamellar liposomes appears to be primarily due to bilayer defects occurring in the lipid transition region rather than being caused by protein-lipid interactions. The results of our in vitro experiments are discussed in terms of the potential usefulness of multilamellar liposomes as drug carriers in vivo for local and topical applications.     

Lamellar bodies of cultured human fetal lung: content of surfactant protein A (SP-A), surface film formation and structural transformation in vitro

Lamellar bodies were isolated from dexamethasone and T3-treated explant cultures of human fetal lung, using sucrose density-gradient centrifugation. We examined their content of surfactant apoprotein A (SP-A), and their ability to form surface films and to undergo structural transformation in vitro. SP-A measured by ELISA composed less than 2% of total protein within lamellar bodies; this represented, as a minimum estimate, a 2-12-fold enrichment over homogenate. One- and two-dimensional gel electrophoresis also suggested that SP-A was a minor protein component of lamellar bodies. Adsorption of lamellar bodies to an air/water interface was moderately rapid, but accelerated dramatically upon addition of exogenous SP-A in ratios of 1:2-16 (SP-A:phospholipid, w/w). Similar adsorption patterns were seen for lamellar bodies from fresh adult rat and rabbit lung. Lamellar bodies incubated under conditions that promote formation of tubular myelin underwent structural rearrangement only in the presence of exogenous SP-A, with extensive formation of multilamellate whorls of lipid bilayers (but no classical tubular myelin lattices). We conclude that lamellar bodies are enriched in SP-A, but have insufficient content of SP-A for structural transformation to tubular myelin and rapid surface film formation in vitro.     

Extramembraneous particles and structural variations of tubular myelin figures in rat lung surfactant

Tubular myelin figures of pulmonary surfactant were examined by electron microscopy after fixation in glutaraldehyde and postfixation in an osmium tetroxide-ferrocyanide mixture. Bilayered membranes were seen as parallel arrays or as lattices with spacings varying from about 36 to 50 nm. This method also produced good visualization of drumstick-like particles, 5 nm in diameter and about 15 nm in length. The particles were regularly spaced at intervals of 16 nm in rows along the rectangular angles of myelin membranes. Depending on the size of the tubules the particles contacted each other in the center of the tubules at low diameters (tubular diameter less than 40 nm) and formed a continuous filamentous central core, or they were separated from one another (tubular diameter greater than 40 nm). In the latter case the central core had a hollow appearance. Based on further findings employing tannic acid, lipid extraction with 2,2-dimethoxypropane, and a ruthenium red-osmium tetroxide technique for the demonstration of polyanionic proteins it is suggested that these particles are protein in nature and that they are involved in the formation and maintenance of the structure of tubular myelin. A new concept of the ultrastructure of tubular myelin figures is proposed.     

Lipid-DNA complex formation: reorganization and rupture of lipid vesicles in the presence of DNA as observed by cryoelectron microscopy.

Cryoelectron microscopy has been used to study the reorganization of unilamellar cationic lipid vesicles upon the addition of DNA. Unilamellar DNA-coated vesicles, as well as multilamellar DNA lipid complexes, could be observed. Also, DNA induced fusion of unilamellar vesicles was found. DNA appears to adsorb to the oppositely charged lipid bilayer in a monolayer of parallel helices and can act as a molecular "glue" enforcing close apposition of neighboring vesicle membranes. In samples with relatively high DNA content, there is evidence for DNA-induced aggregation and flattening of unilamellar vesicles. In these samples, multilamellar complexes are rare and contain only a small number of lamellae. At lower DNA contents, large multilamellar CL-DNA complexes, often with >10 bilayers, are formed. The multilamellar complexes in both types of sample frequently exhibit partially open bilayer segments on their outside surfaces. DNA seems to accumulate or coil near the edges of such unusually terminated membranes. Multilamellar lipid-DNA complexes appear to form by a mechanism that involves the rupture of an approaching vesicle and subsequent adsorption of its membrane to a "template" vesicle or a lipid-DNA complex.     

The effect of N-(1-methyldodecyl)-N,N-dimethylaminoxide on the conformation of hydrocarbon chains in phospholipid bilayers isolated from Escherichia coli

Using the method of ESR spectroscopy of stearic acid spin probes labeled by the doxyl group on the 12th or 16th carbon, it has been found that bactericidal surfactant N-(1-methyldodecyl)-N,N-dimethylamine oxide increases the effective energy difference between trans- and gauche conformers Eg and decreases the probability of gauche conformers formation pg in lipid hydrocarbon chains in multilamellar liposomes prepared from Escherichia coli-isolated phospholipids, at low surfactant concentrations. Above the surfactant: phospholipid molar ratio of 1:14 to 1:17, the value of Eg decreases and that of pg increases. The results are interpreted using the cluster model of lipid bilayer. At low concentrations the surfactant molecules are inserted into the dynamical defects between the clusters, thereby increasing the packing density of chains in the bilayer. At high concentrations the surfactant molecules penetrate into the clusters perturbing the dense packing of chains in clusters.     

Phospholipid composition of lamellar bodies formed by fetal rabbit lung type II cells in organ culture

Lung tissue obtained from fetal rabbits of 23 days gestational age was maintained in organ culture to study the in vitro formation of lamellar body phospholipids. During the culture period, the epithelium of the prealveolar ducts of the explants differentiated to form type II pneumonocytes. After 8 days in culture, the explants were harvested, homogenized, and two lamellar body fractions were isolated by sucrose density gradient centrifugation. The lamellar body fraction which best retained the distinct multilamellar structure was recovered at the interface between a solution of buffer without sucrose and buffer containing 0.41 m sucrose. The phospholipid compositions of both lamellar body fractions were similar to those reported for lamellar bodies and surfactant isolated from fetal rabbit lung, with the exception of a slightly higher phosphatidylethanolamine content. The disaturated phosphatidylcholine content of the lamellar body fractions, expressed as a percentage of total lipid phosphorus, was not influenced by the presence of palmitate in the medium.     

Effects of the lung surfactant protein?B construct Mini-B on lipid bilayer order and topography

The hydrophobic lung surfactant protein, SP-B, is essential for survival. Cycling of lung volume during respiration requires a surface-active lipid-protein layer at the alveolar air-water interface. SP-B may contribute to surfactant layer maintenance and renewal by facilitating contact and transfer between the surface layer and bilayer reservoirs of surfactant material. However, only small effects of SP-B on phospholipid orientational order in model systems have been reported. In this study, N-terminal (SP-B(8-25)) and C-terminal (SP-B(63-78)) helices of SP-B, either linked as Mini-B or unlinked but present in equal amounts, were incorporated into either model phospholipid mixtures or into bovine lipid extract surfactant in the form of vesicle dispersions or mechanically oriented bilayer samples. Deuterium and phosphorus nuclear magnetic resonance (NMR) were used to characterize effects of these peptides on phospholipid chain orientational order, headgroup orientation, and the response of lipid-peptide mixtures to mechanical orientation by mica plates. Only small effects on chain orientational order or headgroup orientation, in either vesicle or mechanically oriented samples, were seen. In mechanically constrained samples, however, Mini-B and its component helices did have specific effects on the propensity of lipid-peptide mixtures to form unoriented bilayer populations which do not exchange with the oriented fraction on the timescale of the NMR experiment. Modification of local bilayer orientation, even in the presence of mechanical constraint, may be relevant to the transfer of material from bilayer reservoirs to a flat surface-active layer, a process that likely requires contact facilitated by the formation of highly curved protrusions.     

Surfactant protein A is localized at the corners of the pulmonary tubular myelin lattice

Immunogold labeling on sections of a freeze-substituted tubular myelin-enriched fraction isolated from a bronchoalveolar lavage of rat lung showed that surfactant protein A (SP-A) occurs predominantly at the corners of the tubular myelin lattice. Seventy-nine percent of the gold particles were located within 20 nm from a corner. Extracellular SP-A was detected only in the tubular myelin lattice and not in vesicles or secreted lamellar bodies. Ultra-thin cryosections of rat lung fixed in vivo showed that intracellular SP-A was distributed homogeneously over the stacked membranes of lamellar bodies in alveolar Type II cells. The presence of SP-A at the corners of the tubular myelin lattice suggests an important role of this protein in the formation and/or maintenance of this highly ordered lattice.