Lipid oxidation and signalling in programmed cell death

The pulmonary surfactant lines as a complex monolayer of lipids and proteins the alveolar epithelial surface. The monolayer dynamically adapts the surface tension of this interface to the varying surface areas during inhalation and exhalation. Its presence in the alveoli is thus a prerequisite for a proper lung function. The lipid moiety represents about 90% of the surfactant and contains mainly dipalmitoylphosphatidylcholine (DPPC) and phosphatidylglycerol (PG). The surfactant proteins involved in the surface tension adaption are called SP-A, SP-B and SP-C. The aim of the present investigation is to analyse the properties of monolayer films made from pure SP-C and from mixtures of DPPC, DPPG and SP-C in order to mimic the surfactant monolayer with minimal compositional requirement. Pressure-area diagrams were taken. Ellipsometric measurements at the air-water interface of a Langmuir film balance allowed measurement of the changes in monolayer thickness upon compression. Isotherms of pure SP-C monolayers exhibit a plateau between 22 and 25 mN/m. A further plateau is reached at higher compression. Structures of the monolayer formed during compression are reversible during expansion. Together with ellipsometric data which show a stepwise increase in film thickness (coverage) during compression, we conclude that pure SP-C films rearrange reversibly into multilayers of homogenous thickness.

Lipid monolayers collapse locally and irreversibly if films are compressed to approximately 0–4 nm²/molecule. In contrast, mixed DPPG/SP-C monolayers with less than 5 mol% protein collapse in a controlled and reversible way. The pressure-area diagrams exhibit a plateau at 20 mN/m, indicating partial demixing of SP-C and DPPG. The thickness isotherm obtained by ellipsometry indicates a transformation into multilayer structures. In DPPC/DPPG/SP-C mixtures again a reversible collapse was observed but without a drastic increase in surface layer thickness which may be due to the formation of protrusion under the surface. Thus lipid monolayers containing small amounts of SP-C may mimic the lung surfactant.     

Bacterial lipopolysaccharide promotes destabilization of lung surfactant-like films

The airspaces are lined with a dipalmitoylphosphatidylcholine (DPPC)-rich film called pulmonary surfactant, which is named for its ability to maintain normal respiratory mechanics by reducing surface tension at the air-liquid interface. Inhaled airborne particles containing bacterial lipopolysaccharide (LPS) may incorporate into the surfactant monolayer. In this study, we evaluated the effect of smooth LPS (S-LPS), containing the entire core oligosaccharide region and the O-antigen, on the biophysical properties of lung surfactant-like films composed of either DPPC or DPPC/palmitoyloleoylphosphatidylglycerol (POPG)/palmitic acid (PA) (28:9:5.6, w/w/w). Our results show that low amounts of S-LPS fluidized DPPC monolayers, as demonstrated by fluorescence microscopy and changes in the compressibility modulus. This promoted early collapse and prevented the attainment of high surface pressures. These destabilizing effects could not be relieved by repeated compression-expansion cycles. Similar effects were observed with surfactant-like films composed of DPPC/POPG/PA. On the other hand, the interaction of SP-A, a surfactant membrane-associated alveolar protein that also binds to LPS, with surfactant-like films containing S-LPS increased monolayer destabilization due to the extraction of lipid molecules from the monolayer, leading to the dissolution of monolayer material in the aqueous subphase. This suggests that SP-A may act as an LPS scavenger.     

Surface respreading after collapse of monolayers containing major lipids of pulmonary surfactant

Isotherms have been obtained near 37 degrees C for a series of repetitive compressions and expansions of monolayers that contain major components of lung surfactant. The minimum surface tension or maximum surface pressure which could be achieved under conditions of dynamic compression, and the rate of return of lipid from excluded phase to the monolayers were measured. Monolayers of pure 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), or of DPPC plus 10 or 30 mol% of the calcium salt of 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-glycerol (POPG) (POPG-Ca) achieved very high surface pressures or low surface tensions (near 0 mN m-1), but they showed no return of material from the collapse phases under the test conditions. Monolayers of POPG-Ca alone collapsed at relatively low surface pressures (high surface tensions), but showed good return of material from the collapse phase into the monolayer. Monolayers containing more complex mixtures of lipids (DPPC, phosphatidylglycerol (PG), unsaturated phosphatidylcholine (PC), cholesterol (chol] in ratios similar to those found in surfactant achieved minimum surface tensions intermediate between those of monolayers with less complex compositions. These more complex mixtures showed a better rate of return of lipids from the collapse phases to the monolayer than did simple DPPC-POPG mixtures. 31P-NMR and differential scanning calorimetric investigations of the mixture DPPC/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine(POPC)/POP G/DPPG/chol (10:4:2:1:3) showed that in the bulk phase at 37 degrees C, it was in bilayers in the liquid-crystalline state.     

Surface activity in vitro: role of surfactant proteins

Pattle, who provided some of the initial direct evidence for the presence of pulmonary surfactant in the lung, was also the first to show surfactant was susceptible to proteases such as trypsin. Pattle concluded surfactant was a lipoprotein. Our group has investigated the roles of the surfactant proteins (SP-) SP-A, SP-B, and SP-C using a captive bubble tensiometer. These studies show that SP-C>SP-B>SP-A in enhancing surfactant lipid adsorption (film formation) to the equilibrium surface tension of approximately 22-25 mN/m from the 70 mN/m of saline at 37 degrees C. In addition to enhancing adsorption, surfactant proteins can stabilize surfactant films so that lateral compression induced through surface area reduction results in the lowering of surface tension (gamma) from approximately 25 mN/m (equilibrium) to values near 0 mN/m. These low tensions, which are required to stabilize alveoli during expiration, are thought to arise through exclusion of fluid phospholipids from the surface monolayer, resulting in an enrichment in the gel phase component dipalmitoylphosphatidylcholine (DPPC). The results are consistent with DPPC enrichment occurring through two mechanisms, selective DPPC adsorption and preferential squeeze-out of fluid components such as unsaturated phosphatidylcholine (PC) and phosphatidylglycerol (PG) from the monolayer. Evidence for selective DPPC adsorption arises from experiments showing that the surface area reductions required to achieve gamma near 0 mN/m with DPPC/PG samples containing SP-B or SP-A plus SP-B films were less than those predicted for a pure squeeze-out mechanism. Surface activity improves during quasi-static or dynamic compression-expansion cycles, indicating the squeeze-out mechanism also occurs. Although SP-C was not as effective as SP-B in promoting selective DPPC adsorption, this protein is more effective in promoting the reinsertion of lipids forced out of the surface monolayer following overcompression at low gamma values. Addition of SP-A to samples containing SP-B but not SP-C limits the increase in gamma(max) during expansion. It is concluded that the surfactant apoproteins possess distinct overlapping functions. SP-B is effective in selective DPPC insertion during monolayer formation and in PG squeeze-out during monolayer compression. SP-A can promote adsorption during film formation, particularly in the presence of SP-B. SP-C appears to have a superior role to SP-B in formation of the surfactant reservoir and in reinsertion of collapse phase lipids.     

Pulmonary surfactant proteins SP-B and SP-C in spread monolayers at the air-water interface: II. Monolayers of pulmonary surfactant protein SP-C and phospholipids.

The interaction of the hydrophobic pulmonary surfactant protein SP-C with dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylglycerol (DPPG) and DPPC:DPPG (7:3, mol:mol) in spread monolayers at the air-water interface has been studied. At low concentrations of SP-C (about 0.5 mol% or 3 weight%protein) the protein-lipid films collapsed at surface pressures of about 70 mN.m-1, comparable to those of the lipids alone. At initial protein concentrations higher than 0.8 mol%, or 4 weight%, the isotherms displayed kinks at surface pressures of about 50 mN.m-1 in addition to the collapse plateaux at the higher pressures. The presence of less than 6 mol%, or 27 weight%, of SP-C in the protein-lipid monolayers gave a positive deviation from ideal behavior of the mean areas in the films. Analyses of the mean areas in the protein-lipid films as functions of the monolayer composition and surface pressure showed that SP-C, associated with some phospholipid (about 8-10 lipid molecules per molecule of SP-C), was squeezed out from the monolayers at surface pressures of about 55 mN.m-1. The results suggest a potential role for SP-C to modify the composition of the monolayer at the air-water interface in the alveoli.     

Pulmonary surfactant proteins SP-B and SP-C in spread monolayers at the air-water interface: I. Monolayers of pulmonary surfactant protein SP-B and phospholipids.

The effects of pulmonary surfactant protein SP-B on the properties of monolayers of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG), and a mixture of DPPC:DPPG (7:3, mol:mol) were studied using spread films at the air-water interface. The addition of SP-B to the phospholipid monolayers gave positive deviations from additivity of the mean areas in the films. At low protein concentrations (less than 45% amino acid residues which corresponds to 0.5 mol% or 10 weight% SP-B) monolayers of SP-B/DPPC, SP-B/DPPG and SP-B/(DPPC:DPPG) collapsed at surface pressures of about 70 mN.m-1, comparable to those of the lipids alone. At higher concentrations of SP-B in the protein-lipid monolayers, kink points appeared in the isotherms at about 40-45 mN.m-1, implying possible exclusion of material from the films, hence, changes in the original monolayer compositions. Calculated analyses of the monolayer compositions as a function of surface pressure indicated that nearly pure SP-B, associated with small amounts of phospholipid (2-3 lipid molecules per SP-B dimer), was lost from SP-B/DPPC, SP-B/DPPG, and SP-B/(DPPC:DPPG) films at surface pressures higher than 40-45 mN.m-1. The results are consistent with a low effectiveness of SP-B in removing saturated phospholipids, DPPC or DPPG, from the spread SP-B/phospholipid films.     

Phospholipid packing and hydration in pulmonary surfactant membranes and films as sensed by LAURDAN

The efficiency of pulmonary surfactant to stabilize the respiratory surface depends critically on the ability of surfactant to form highly packed films at the air-liquid interface. In the present study we have compared the packing and hydration properties of lipids in native pulmonary surfactant and in several surfactant models by analyzing the pressure and temperature dependence of the fluorescence emission of the LAURDAN (1-[6-(dimethylamino)-2-naphthyl]dodecan-1-one) probe incorporated into surfactant interfacial films or free-standing membranes. In interfacial films, compression-driven changes in the fluorescence of LAURDAN, evaluated from the generalized polarization function (GPF), correlated with changes in packing monitored by surface pressure. Compression isotherms and GPF profiles of films formed by native surfactant or its organic extract were compared at 25 or 37 °C to those of films made of dipalmitoylphosphatidylcholine (DPPC), palmitoyloleoylphosphatidylcholine (POPC), DPPC/phosphatidylglycerol (PG) (7:3, w/w), or the mixture DPPC/POPC/palmitoyloleoylphosphatidylglycerol (POPG)/cholesterol (Chol) (50:25:15.10), which simulates the lipid composition of surfactant. In general terms, compression of surfactant films at 25 °C leads to LAURDAN GPF values close to those obtained from pure DPPC monolayers, suggesting that compressed surfactant films reach a dehydrated state of the lipid surface, which is similar to that achieved in DPPC monolayers. However, at 37 °C, the highest GPF values were achieved in films made of full surfactant organic extract or the mixture DPPC/POPC/POPG/Chol, suggesting a potentially important role of cholesterol to ensure maximal packing/dehydration under physiological constraints. Native surfactant films reached high pressures at 37 °C while maintaining relatively low GPF, suggesting that the complex three-dimensional structures formed by whole surfactant might withstand the highest pressures without necessarily achieving full dehydration of the lipid environments sensed by LAURDAN. Finally, comparison of the thermotropic profiles of LAURDAN GPF in surfactant model bilayers and monolayers of analogous composition shows that the fluorophore probes an environment that is in average intrinsically more hydrated at the interface than inserted into free-standing bilayers, particularly at 37 °C. This effect suggests that the dependence of membrane and surfactant events on the balance of polar/non-polar interactions could differ in bilayer and monolayer models, and might be affected differently by the access of water molecules to confined or free-standing lipid structures.     

Interaction of lung surfactant proteins with anionic phospholipids.

Langmuir isotherms, fluorescence microscopy, and atomic force microscopy were used to study lung surfactant specific proteins SP-B and SP-C in monolayers of dipalmitoylphosphatidylglycerol (DPPG) and palmitoyloleoylphosphatidylglycerol (POPG), which are representative of the anionic lipids in native and replacement lung surfactants. Both SP-B and SP-C eliminate squeeze-out of POPG from mixed DPPG/POPG monolayers by inducing a two- to three-dimensional transformation of the fluid-phase fraction of the monolayer. SP-B induces a reversible folding transition at monolayer collapse, allowing all components of surfactant to remain at the interface during respreading. The folds remain attached to the monolayer, are identical in composition and morphology to the unfolded monolayer, and are reincorporated reversibly into the monolayer upon expansion. In the absence of SP-B or SP-C, the unsaturated lipids are irreversibly lost at high surface pressures. These morphological transitions are identical to those in other lipid mixtures and hence appear to be independent of the detailed lipid composition of the monolayer. Instead they depend on the more general phenomena of coexistence between a liquid-expanded and liquid-condensed phase. These three-dimensional monolayer transitions reconcile how lung surfactant can achieve both low surface tensions upon compression and rapid respreading upon expansion and may have important implications toward the optimal design of replacement surfactants. The overlap of function between SP-B and SP-C helps explain why replacement surfactants lacking in one or the other proteins often have beneficial effects.     

Effects of cholesterol on surface activity and surface topography of spread surfactant films

Pulmonary surfactant forms a monolayer of lipids and proteins at the alveolar air/liquid interface. Although cholesterol is a natural component of surfactant, its function in surface dynamics is unclear. To further elucidate the role of cholesterol in surfactant, we used a captive bubble surfactometer (CBS) to measure surface activity of spread films containing dipalmitoylphosphatidylcholine/1-palmitoyl-2-oleoylphosphatidylcholine/1-palmitoyl-2-oleoylphosphatidylglycerol (DPPC/POPC/POPG, 50/30/20 molar percentages), surfactant protein B (SP-B, 0.75 mol %), and/or surfactant protein C (SP-C, 3 mol %) with up to 20 mol % cholesterol. A cholesterol concentration of 10 mol % was optimal for reaching and maintaining low surface tensions in SP-B-containing films but led to an increase in maximum surface tension in films containing SP-C. No effect of cholesterol on surface activity was found in films containing both SP-B and SP-C. Atomic force microscopy (AFM) was used, for the first time, to visualize the effect of cholesterol on topography of SP-B- and/or SP-C-containing films compressed to a surface tension of 22 mN/m. The protrusions found in the presence of cholesterol were homogeneously dispersed over the film, whereas in the absence of cholesterol the protrusions tended to be more clustered into network structures. A more homogeneous dispersion of surfactant lipid components may facilitate lipid insertion into the surfactant monolayer. Our data provide additional evidence that natural surfactant, containing SP-B and SP-C, is superior to surfactants lacking one of the components, and furthermore, this raises the possibility that the cholesterol found in surfactant of warm-blooded mammals does not have a function in surface activity.     

Lipid compositional analysis of pulmonary surfactant monolayers and monolayer-associated reservoirs

Pulmonary surfactant is a lipid:protein complex containing dipalmitoyl-phosphatidylcholine (DPPC) as the major component. Recent studies indicate adsorbed surfactant films consist of a surface monolayer and a monolayer-associated reservoir. It has been hypothesized that the monolayer and its functionally contiguous reservoir may be enriched in DPPC relative to bulk phase surfactant. We investigated the compositional relationship between the monolayer and its reservoir using paper-supported wet bridges to transfer films from adsorbing dishes to clean surfaces on spreading dishes. Spreading films appear to form monolayers in the spreading dishes. We employed bovine lipid extract surfactant [BLES(chol)] containing [3H]DPPC and either [14C]palmitoyl, oleoyl-phosphatidylcholine (POPC), [14C]dipalmitoyl-phosphatidylglycerol (DPPG), [14C]palmitoyl, oleoyl-phosphatidylglycerol (POPG), or [14C]cholesterol. Radiolabeled phosphatidylglycerols were prepared using phospholipase D. The studies demonstrated that the [3H]DPPC-[14C] POPC ratios were the same in the prepared BLES dispersions as in Langmuir-Blodgett films, indicating a lack of DPPC selectivity during film formation. Furthermore, identical 3H-14C isotopic ratios were observed with DPPC and either 14C-labeled POPC, DPPG, POPG, or cholesterol in the original dispersions, the bulk phases in adsorption dish D1, and monolayers recovered from spreading dish D2. These relationships remained unperturbed with 2-fold increases in bulk concentrations in D1 and 10-fold variations in D1-D2 surface area. These results indicate adsorbed surfactant monolayers and their associated reservoirs possess similar lipid compositions and argue against selective adsorption of DPPC.     

Effect of the hydrophobic surfactant proteins on the surface activity of spread films in the captive bubble surfactometer

The main function of pulmonary surfactant, a mixture of lipids and proteins, is to reduce the surface tension at the air/liquid interface of the lung. The hydrophobic surfactant proteins SP-B and SP-C are required for this process. When testing their activity in spread films in a captive bubble surfactometer, both SP-B and SP-C showed concentration dependence for lipid insertion as well as for lipid film refinement. Higher activity in DPPC refinement of the monolayer was observed for SP-B compared with SP-C. Further differences between both proteins were found, when subphase phospholipid vesicles, able to create a monolayer-attached lipid reservoir, were omitted. SP-C containing monolayers showed gradually increasing minimum surface tensions upon cycling, indicating that a lipid reservoir is required to prevent loss of material from the monolayer. Despite reversible cycling dynamics, SP-B containing monolayers failed to reach near-zero minimum surface tensions, indicating that the reservoir is required for stable films.     

Liquid-crystalline collapse of pulmonary surfactant monolayers

During exhalation, the surfactant film of lipids and proteins that coats the alveoli in the lung is compressed to high surface pressures, and can remain metastable for prolonged periods at pressures approaching 70 mN/m. Monolayers of calf lung surfactant extract (CLSE), however, collapse in vitro, during an initial compression at approximately 45 mN/m. To gain information on the source of this discrepancy, we investigated how monolayers of CLSE collapse from the interface. Observations with fluorescence, Brewster angle, and light scattering microscopies show that monolayers containing CLSE, CLSE-cholesterol (20%), or binary mixtures of dipalmitoyl phosphatidylcholine(DPPC)-dihydrocholesterol all form bilayer disks that reside above the monolayer. Upon compression and expansion, lipids flow continuously from the monolayer into the disks, and vice versa. In several respects, the mode of collapse resembles the behavior of other amphiphiles that form smectic liquid-crystal phases. These findings suggest that components of surfactent films must collapse collectively rather than being squeezed out individually.     

Influence of lipid saturation grade and headgroup charge: a refined lung surfactant adsorption model

Rapid adsorption of surfactant material to the air/liquid interface of the lung is essential for maintaining normal lung function. The detailed mechanism of this process, however, remains unclear. In this study, we elucidate the influence of lipid saturation grade and headgroup charge of surface layer lipids on surfactant protein (SP)-induced vesicle insertion into monolayers spread at the air/water interface of a film balance. We used dipalmitoylphosphatidlycholine (DPPC),1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) as monolayer lipids doped with either hydrophobic surfactant-specific protein SP-B or SP-C (0.2 and 0.4 mol %, respectively). Vesicles consisting of DPPC/DPPG (4:1, mol ratio) were injected into a stirred subphase to quantify adsorption kinetics. Based on kinetic film balance and fluorescence measurements, a refined model describing distinct steps of vesicle adsorption to surfactant monolayers is presented. First, in a protein-independent step, lipids from vesicles bridged to the interfacial film by Ca²⁺ ions are inserted into defects of a disordered monolayer at low surface pressures. Second, in a SP-facilitated step, active material insertion involving an SP-B- or SP-C-induced flip-flop of lipids occurs at higher surface pressures. Negatively charged lipids obviously influence the threshold pressures at which this second protein-mediated adsorption mechanism takes place.     

Effects of lung surfactant proteins, SP-B and SP-C, and palmitic acid on monolayer stability

Langmuir isotherms and fluorescence and atomic force microscopy images of synthetic model lung surfactants were used to determine the influence of palmitic acid and synthetic peptides based on the surfactant-specific proteins SP-B and SP-C on the morphology and function of surfactant monolayers. Lung surfactant-specific protein SP-C and peptides based on SP-C eliminate the loss to the subphase of unsaturated lipids necessary for good adsorption and respreading by inducing a transition between monolayers and multilayers within the fluid phase domains of the monolayer. The morphology and thickness of the multilayer phase depends on the lipid composition of the monolayer and the concentration of SP-C or SP-C peptide. Lung surfactant protein SP-B and peptides based on SP-B induce a reversible folding transition at monolayer collapse that allows all components of surfactant to be retained at the interface during respreading. Supplementing Survanta, a clinically used replacement lung surfactant, with a peptide based on the first 25 amino acids of SP-B also induces a similar folding transition at monolayer collapse. Palmitic acid makes the monolayer rigid at low surface tension and fluid at high surface tension and modifies SP-C function. Identifying the function of lung surfactant proteins and lipids is essential to the rational design of replacement surfactants for treatment of respiratory distress syndrome.     

Combinations of fluorescently labeled pulmonary surfactant proteins SP-B and SP-C in phospholipid films

Hydrophobic pulmonary surfactant (PS) proteins B (SP-B) and C (SP-C) modulate the surface properties of PS lipids. Epifluorescence microscopy was performed on solvent-spread monolayers of fluorescently labeled porcine SP-B (R-SP-B, labeled with Texas Red) and SP-C (F-SP-C, labeled with fluorescein) in dipalmitoylphosphatidylcholine (DPPC) (at protein concentrations of 10 and 20 wt%, and 10 wt% of both) under conditions of cyclic compression and expansion. Matrix-assisted laser desorption/ionization (MALDI) spectroscopy of R-SP-B and F-SP-C indicated that the proteins were intact and labeled with the appropriate fluorescent probe. The monolayers were compressed and expanded for four cycles at an initial rate of 0.64 A2 x mol(-1) x s(-1) (333 mm2 x s x [-1]) up to a surface pressure pi approximately 65 mN/m, and pi-area per residue (pi-A) isotherms at 22 +/- 1 degrees C were obtained. The monolayers were microscopically observed for the fluorescence emission of the individual proteins present in the film lipid matrix, and their visual features were video recorded for image analysis. The pi-A isotherms of the DPPC/protein monolayers showed characteristic "squeeze out" effects at pi approximately 43 mN/m for R-SP-B and 55 mN/m for F-SP-C, as had previously been observed for monolayers of the native proteins in DPPC. Both proteins associated with the expanded (fluid) phase of DPPC monolayers remained in or associated with the monolayers at high pi (approximately 65 mN/m) and redispersed in the monolayer upon its reexpansion. At comparable pi and area/molecule of the lipid, the proteins reduced the amounts of condensed (gel-like) phase of DPPC monolayers, with F-SP-C having a greater effect on a weight basis than did R-SP-B. In any one of the lipid/protein monolayers the amounts of the DPPC in condensed phase were the same at equivalent pi during compression and expansion and from cycle to cycle. This indicated that only minor loss of components from these systems occurred between compression-expansion cycles. This study indicates that hydrophobic PS proteins associate with the fluid phase of DPPC in films, some proteins remain at high surface pressures in the films, and such lipid-protein films can still attain high pi during compression.     

Lateral phase separation in interfacial films of pulmonary surfactant.

To determine if lateral phase separation occurs in films of pulmonary surfactant, we used epifluorescence microscopy and Brewster angle microscopy (BAM) to study spread films of calf lung surfactant extract (CLSE). Both microscopic methods demonstrated that compression produced domains of liquid-condensed lipids surrounded by a liquid-expanded film. The temperature dependence of the pressure at which domains first emerged for CLSE paralleled the behavior of its most prevalent component, dipalmitoyl phosphatidylcholine (DPPC), although the domains appeared at pressures 8-10 mN/m higher than for DPPC over the range of 20-37 degrees C. The total area occupied by the domains at room temperature increased to a maximum value at 35 mN/m during compression. The area of domains reached 25 +/- 5% of the interface, which corresponds to the predicted area of DPPC in the monolayer. At pressures above 35 mN/m, however, both epifluorescence and BAM showed that the area of the domains decreased dramatically. These studies therefore demonstrate a pressure-dependent gap in the miscibility of surfactant constituents. The monolayers separate into two phases during compression but remain largely miscible at higher and lower surface pressures.     

Role of pulmonary surfactant components in surface film formation and dynamics

Pulmonary surfactant is a mixture of lipids and proteins which is secreted by the epithelial type II cells into the alveolar space. Its main function is to reduce the surface tension at the air/liquid interface in the lung. This is achieved by forming a surface film that consists of a monolayer which is highly enriched in dipalmitoylphosphatidylcholine and bilayer lipid/protein structures closely attached to it. The molecular mechanisms of film formation and of film adaptation to surface changes during breathing in order to remain a low surface tension at the interface, are unknown. The results of several model systems give indications for the role of the surfactant proteins and lipids in these processes. In this review, we describe and compare the model systems that are used for this purpose and the progress that has been made. Despite some conflicting results using different techniques, we conclude that surfactant protein B (SP-B) plays the major role in adsorption of new material into the interface during inspiration. SP-C's main functions are to exclude non-DPPC lipids from the interface during expiration and to attach the bilayer structures to the lipid monolayer. Surfactant protein A (SP-A) appears to promote most of SP-B's functions. We describe a model proposing that SP-A and SP-B create DPPC enriched domains which can readily be adsorbed to create a DPPC-rich monolayer at the interface. Further enrichment in DPPC is achieved by selective desorption of non-DPPC lipids during repetitive breathing cycles.     

Folding of lipid monolayers containing lung surfactant proteins SP-B1-25 and SP-C studied via coarse-grained molecular dynamics simulations

To explore the role of lung surfactant proteins SP-B and SP-C in storing and redelivering lipid from lipid monolayers during the compression and re-expansion occurring in lungs during breathing, we simulate the folding of lipid monolayers with and without these proteins. We utilize the MARTINI coarse-grained force field to simulate monolayers containing pure dipalmitoylphosphatidylcholine (DPPC) and DPPC mixed with palmitoyloleoylphosphatidylglycerol (POPG), palmitic acid (PA), and/or peptides. The peptides considered include the 25-residue N-terminal fragment of SP-B (SP-B_1-25), SP-C, and several SP-B_1-25 mutants in which charged and hydrophilic residues are replaced by hydrophobic ones, or vice-versa. We observe two folding mechanisms: folding by the amplification of undulations and folding by nucleation about a defect. The first mechanism is observed in monolayers containing either POPG or peptides, while the second mechanism is observed only with peptides present, and involves the lipid-mediated aggregation of the peptides into a defect, from which the fold can nucleate. Fold nucleation from a defect displays a dependence on the hydrophobic character of the peptides; if the number of hydrophobic residues is decreased significantly, monolayer folding does not occur. The addition of POPG or peptides to the DPPC monolayer has a fluidizing effect, which assists monolayer folding. In contrast, the addition of PA has a charge-dependent condensing affect on DPPC monolayers containing SP-C. The peptides appear to play a significant role in the folding process, and provide a larger driving force for folding than POPG. In addition to promoting fold formation, the peptides also display fusogenic behavior, which can lead to surface refining.     

Effects of a cationic and hydrophobic peptide, KL4, on model lung surfactant lipid monolayers.

We report on the surface behavior of a hydrophobic, cationic peptide, [lysine-(leucine)4]4-lysine (KL4), spread at the air/water interface at 25 degrees C and pH 7.2, and its effect at very low molar ratios on the surface properties of the zwitterionic phospholipid 1,2-dipalmitoylphosphatidylcholine (DPPC), and the anionic forms of 1-palmitoyl-2-oleoylphosphatidylglycerol (POPG) and palmitic acid (PA), in various combinations. Surface properties were evaluated by measuring equilibrium spreading pressures (pi(e)) and surface pressure-area isotherms (pi-A) with the Wilhelmy plate technique. Surface phase separation was observed with fluorescence microscopy. KL4 itself forms a single-phase monolayer, stable up to a surface pressure pi of 30 mN/m, and forms an immiscible monolayer mixture with DPPC. No strong interaction was detected between POPG and KL4 in the low pi region, whereas a stable monolayer of the PA/KL4 binary mixture forms, which is attributed to ionic interactions between oppositely charged PA and KL4. KL4 has significant effects on the DPPC/POPG mixture, in that it promotes surface phase separation while also increasing pi(e) and pi(max), and these effects are greatly enhanced in the presence of PA. In the model we have proposed, KL4 facilitates the separation of DPPC-rich and POPG/PA-rich phases to achieve surface refinement. It is these two phases that can fulfill the important lung surfactant functions of high surface pressure stability and efficient spreading.     

Multilayer structures in lipid monolayer films containing surfactant protein C: effects of cholesterol and POPE

The influence of cholesterol and POPE on lung surfactant model systems consisting of DPPC/DPPG (80:20) and DPPC/DPPG/surfactant protein C (80:20:0.4) has been investigated. Cholesterol leads to a condensation of the monolayers, whereas the isotherms of model lung surfactant films containing POPE exhibit a slight expansion combined with an increased compressibility at medium surface pressure (10-30 mN/m). An increasing amount of liquid-expanded domains can be visualized by means of fluorescence light microscopy in lung surfactant monolayers after addition of either cholesterol or POPE. At surface pressures of 50 mN/m, protrusions are formed which differ in size and shape as a function of the content of cholesterol or POPE, but only if SP-C is present. Low amounts of cholesterol (10 mol %) lead to an increasing number of protrusions, which also grow in size. This is interpreted as a stabilizing effect of cholesterol on bilayers formed underneath the monolayer. Extreme amounts of cholesterol (30 mol %), however, cause an increased monolayer rigidity, thus preventing reversible multilayer formation. In contrast, POPE, as a nonbilayer lipid thought to stabilize the edges of protrusions, leads to more narrow protrusions. The lateral extension of the protrusions is thereby more influenced than their height.