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.     

An elevated level of cholesterol impairs self-assembly of pulmonary surfactant into a functional film

In adult respiratory distress syndrome, the primary function of pulmonary surfactant to strongly reduce the surface tension of the air-alveolar interface is impaired, resulting in diminished lung compliance, a decreased lung volume, and severe hypoxemia. Dysfunction coincides with an increased level of cholesterol in surfactant which on its own or together with other factors causes surfactant failure. In the current study, we investigated by atomic force microscopy and Kelvin-probe force microscopy how the increased level of cholesterol disrupts the assembly of an efficient film. Functional surfactant films underwent a monolayer-bilayer conversion upon contraction and resulted in a film with lipid bilayer stacks, scattered over a lipid monolayer. Large stacks were at positive electrical potential, small stacks at negative potential with respect to the surrounding monolayer areas. Dysfunctional films formed only few stacks. The surface potential of the occasional stacks was also not different from the surrounding monolayer. Based on film topology and potential distribution, we propose a mechanism for formation of stacked bilayer patches whereby the helical surfactant-associated protein SP-C becomes inserted into the bilayers with defined polarity. We discuss the functional role of the stacks as mechanically reinforcing elements and how an elevated level of cholesterol inhibits the formation of the stacks. This offers a simple biophysical explanation for surfactant inhibition in adult respiratory distress syndrome and possible targets for treatment.     

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.     

Effect of hydrophobic surfactant protein SP-C on binary phospholipid monolayers. Molecular machinery at the air/water interface

Fluorescent and modified dark-field microscopies were used to investigate the phase behavior of physiologically relevant lipid/protein monomolecular films containing surfactant protein C(SP-C). Synthetic human SP-C(1-34) was labeled at its N-terminus using the fluorescent probe 6-(((4(4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene-3-yl)phenoxy)acetyl)amino)hexanoic acid (BODIPY/TR-X). Using dual fluorescent labeling (lipid and protein) in the monolayers, we have correlated (at physiologically small concentrations of the protein) the lipid phase separation and protein distribution in situ. A comparison of the lipid and protein dye fluorescent micrographs indicates that SP-C(1-34) is preferentially associated with the disordered lipid phase. Three concepts arise from our results. (1) The presence of SP-C alone does not result in the complete dissolution of condensed phase domains in a fashion that we have previously reported for the entire hydrophobic surfactant protein (SP-B/C) fraction (Biophys. J. 77 (1999) 903). Rather, the use of relatively high amounts ( approximately 10 wt.%) of the labeled SP-C protein is needed to reproduce the fluorescence monolayer morphology previously observed for small concentrations ( approximately 1 wt.%) of the natural SP-B/C mixture. (2) Scattered light, dark-field microscopy performed using grazing angle laser illumination reveals the presence of surface-associated, three-dimensional (3D) structures of micrometer-sized dimensions when the labeled BODIPY/TR-X:SP-C(1-34) protein is included in the monolayer, as previously observed with the naturally isolated SP-B/C mixture. The 3D structures are associated exclusively with the presence of the SP-C protein in disordered monolayer phases. (3) To explain these results, we have derived a molecular model accounting for the structure and physico-chemical properties of the SP-C protein in terms of its energetics. The molecular events involved in the SP-C-mediated production of the 3D surface particles are explained using the analogy of a simple molecular machine, namely a loaded spring. This interpretation is supported by an energetic analysis that suggests the major factor contributing to the formation of the 3D particles is the energy liberated by re-expansion of the surrounding phospholipid film into the area vacated by the SP-C protein as it re-orients away from the surface.     

Effect of hydrophobic surfactant peptides SP-B and SP-C on binary phospholipid monolayers. I. Fluorescence and dark-field microscopy.

The influence of the hydrophobic proteins SP-B and SP-C, isolated from pulmonary surfactant, on the morphology of binary monomolecular lipid films containing phosphocholine and phosphoglycerol (DPPC and DPPG) at the air-water interface has been studied using epifluorescence and dark-field microscopy. In contrast to previously published studies, the monolayer experiments used the entire hydrophobic surfactant protein fraction (containing both the SP-B and SP-C peptides) at physiologically relevant concentrations (approximately 1 wt %). Even at such low levels, the SP-B/C peptides induce the formation of a new phase in the surface monolayer that is of lower intrinsic order than the liquid condensed (LC) phase that forms in the pure lipid mixture. This presumably leads to a higher structural flexibility of the surface monolayer at high lateral pressure. Variation of the subphase pH indicates that electrostatic interaction dominates the association of the SP-B/C peptides with the lipid monolayer. As evidenced from dark-field microscopy, monolayer material is excluded from the DPPC/DPPG surface film on compression and forms three-dimensional, surface-associated structures of micron dimensions. Such exclusion bodies formed only with SP-B/C peptides. This observation provides the first direct optical evidence for the squeeze-out of pulmonary surfactant material in situ at the air-water interface upon increasing monolayer surface pressures.     

Secondary structure and lipid interactions of the N-terminal segment of pulmonary surfactant SP-C in Langmuir films: IR reflection-absorption spectroscopy and surface pressure studies

Pulmonary surfactant, a thin lipid/protein film lining mammalian lungs, functions in vivo to reduce the work of breathing and to prevent alveolar collapse. Analogues of two hydrophobic surfactant proteins, SP-B and SP-C, have been incorporated into therapeutic agents for respiratory distress syndrome, a pathological condition resulting from deficiency in surfactant. To facilitate rational design of therapeutic agents, a molecular level understanding of lipid interaction with surfactant proteins or their analogues in aqueous monolayer films is necessary. The current work uses infrared reflection-absorption spectroscopy (IRRAS) to determine peptide conformation and the effects of S-palmitoylation on the lipid interactions of a synthetic 13 residue N-terminal peptide [SP-C13(palm)(2)] of SP-C, in mixtures with 1,2-dipalmitoylphosphatidylcholine (DPPC) or 1,2-dipalmitoylphosphatidylglycerol (DPPG). Two Amide I' features, at approximately 1655 and approximately 1639 cm(-1) in the peptide IRRAS spectra, are assigned to alpha-helical peptide bonds in hydrophobic and aqueous environments, respectively. In binary DPPC/SP-C13(palm)(2) films, the proportion of hydrated/hydrophobic helix increases reversibly with surface pressure (pi), suggestive of the peptide being squeezed out from hydrophobic regions of the monolayer. No such effect was observed for DPPG/peptide monolayers, indicative of stronger, probably electrostatic, interactions. Depalmitoylation produced a weakened interaction with either phospholipid as deduced from IRRAS spectra and from pi-area isotherms. S-Palmitoylation may modulate peptide hydration and conformation in the N-terminal region of SP-C and may thus permit the peptide to remain in the film at the high surface pressures present during lung compression. The unique capability of IRRAS to detect the surface pressure dependence of protein or peptide structure/interactions in a physiologically relevant model for surfactant is clearly demonstrated.     

Kinetics of phospholipid insertion into monolayers containing the lung surfactant proteins SP-B or SP-C

The lung surfactant proteins SP-B and SP-C are pivotal for fast and reversible lipid insertion at the air/liquid interface, a prerequisite for functional lung activity. We used a model system consisting of a preformed monolayer at the air/liquid interface supplemented with surfactant protein SP-B or SP-C and unilamellar vesicles injected into the subphase of a film balance. The content of SP-B or SP-C was similar to that found in lung lavage. In order to elucidate distinct steps of lipid insertion, we measured the time-dependent pressure increase as a function of the initial surface pressure, the temperature and the phosphatidylglycerol content by means of surface tension measurements and scanning force microscopy (SFM). The results of the film balance study are indicative of a two-step mechanism in which initial adsorption of vesicles to the protein-containing monolayer is followed by rupture and integration of lipid material. Furthermore, we found that vesicle adsorption on a preformed monolayer supplemented with SP-B or SP-C is strongly enhanced by negatively charged lipids as provided by DPPG and the presence of Ca2+ ions in the subphase. Hence, long-range electrostatic interactions are thought to play an important role in attracting vesicles to the surface, being the initial step in replenishment of lipid material. While insertion into the monolayer is independent of the type of protein SP-B or SP-C, initial adsorption is faster in the presence of SP-B than SP-C. We propose that the preferential interaction between SP-B and negatively charged DPPG leads to accumulation of negative charges in particular regions, causing strong adhesion between DPPG-containing vesicles and the monolayer mediated by Ca2+ ions, which eventually causes flattening and rupture of attached liposomes as observed by in situ SFM.     

Surfactant protein interactions with neutral and acidic phospholipid films

The captive bubble tensiometer was employed to study interactions of phospholipid (PL) mixtures of dipalmitoylphosphatidylcholine (DPPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) or 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPG) at 50 microg/ml with physiological levels of the surfactant protein (SP) A SP-B, and SP-C alone and in combination at 37 degrees C. All surfactant proteins enhanced lipid adsorption to equilibrium surface tension (gamma), with SP-C being most effective. Kinetics were consistent with the presence of two adsorption phases. Under the conditions employed, SP-A did not affect the rate of film formation in the presence of SP-B or SP-C. Little difference in gamma(min) was observed between the acidic POPG and the neutral POPC systems with SP-B or SP-C with and without SP-A. However, gamma(max) was lower with the acidic POPG system during dynamic, but not during quasi-static, cycling. Considerably lower compression ratios were required to generate low gamma(min) values with SP-B than SP-C. DPPC-POPG-SP-B was superior to the neutral POPC-SP-B system. Although SP-A had little effect on film formation with SP-B, surface activity during compression was enhanced with both PL systems. In the presence of SP-C, lower compression ratios were required with the acidic system, and with this mixture, SP-A addition adversely affected surface activity. The results suggest specific interactions between SP-B and phosphatidylglycerol, and between SP-B and SP-A. These observations are consistent with the presence of a surface-associated surfactant reservoir which is involved in generating low gamma during film compression and lipid respreading during film expansion.     

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.     

Production and characterisation of recombinant forms of human pulmonary surfactant protein C (SP-C): Structure and surface activity

Surfactant protein C (SP-C) is an essential component for the surface tension-lowering activity of the pulmonary surfactant system. It contains a valine-rich alpha helix that spans the lipid bilayer, and is one of the most hydrophobic proteins known so far. SP-C is also an essential component of various surfactant preparations of animal origin currently used to treat neonatal respiratory distress syndrome (NRDS) in preterm infants. The limited supply of this material and the risk of transmission of infectious agents and immunological reactions have prompted the development of synthetic SP-C-derived peptides or recombinant humanized SP-C for inclusion in new preparations for therapeutic use. We describe herein the recombinant production in bacterial cultures of SP-C variants containing phenylalanines instead of the palmitoylated cysteines of the native protein, as fusions to the hydrophilic nuclease A (SN) from Staphylococcus aureus. The resulting chimerae were partially purified by affinity chromatography and subsequently subjected to protease digestion. The SP-C forms were recovered from the digestion mixtures by organic extraction and further purified by size exclusion chromatography. The two recombinant SP-C variants so obtained retained more than 50% alpha-helical content and showed surface activity comparable to the native protein, as measured by surface spreading of lipid/protein suspensions and from compression pi-A isotherms of lipid/protein films. Compared to the protein purified from porcine lungs, the recombinant SP-C forms improved movement of phospholipid molecules into the interface (during adsorption), or out from the interfacial film (during compression), suggesting new possibilities to develop improved therapeutic preparations.     

Pulmonary surfactant protein SP-C causes packing rearrangements of dipalmitoylphosphatidylcholine in spread monolayers.

The hydrophobic pulmonary surfactant protein SP-C has been isolated from porcine lung surfactant, and it has been incorporated into monolayers of dipalmitoylphosphatidylcholine (DPPC). The monolayers, which contained 1 mol% of a fluorescently-labeled phosphatidylcholine, were observed under various states of compression in an epifluorescence surface balance. SP-C altered the packing arrangements of DPPC in the monolayer, causing the production of many more, smaller condensed lipid domains in its presence than in its absence.     

Monolayer–multilayer transitions in a lung surfactant model: IR reflection–absorption spectroscopy and atomic force microscopy

A hydrophobic pulmonary surfactant protein, SP-C, has been implicated in surface-associated activities thought to facilitate the work of breathing. Model surfactant films composed of lipids and SP-C display a reversible transition from a monolayer to surface-associated multilayers upon compression and expansion at the air/water (A/W) interface. The molecular-level mechanics of this process are not yet fully understood. The current work uses atomic force microscopy on Langmuir–Blodgett films to verify the formation of multilayers in a dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylglycerol, cholesterol, and SP-C model system. Isotherms of SP-C-containing films are consistent with exclusion and essentially complete respreading during compression and expansion, respectively. Multilayer formation was not detected in the absence of SP-C. Most notable are the results from IR reflection–absorption spectroscopy (IRRAS) conducted at the A/W interface, where the position and intensity of the Amide I band of SP-C reveal that the predominantly helical structure changes its orientation in monolayers versus multilayers. IRRAS measurements indicate that the helix tilt angle changed from approximately 80° in monolayers to a transmembrane orientation in multilayers. The results constitute the first quantitative measure of helix orientation in mixed monolayer/multilamellar domains at the A/W interface and provide insight into the molecular mechanism for SP-C-facilitated respreading of surfactant.     

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.     

Production and characterisation of recombinant forms of human pulmonary surfactant protein C (SP-C): Structure and surface activity

Surfactant protein C (SP-C) is an essential component for the surface tension-lowering activity of the pulmonary surfactant system. It contains a valine-rich α helix that spans the lipid bilayer, and is one of the most hydrophobic proteins known so far. SP-C is also an essential component of various surfactant preparations of animal origin currently used to treat neonatal respiratory distress syndrome (NRDS) in preterm infants. The limited supply of this material and the risk of transmission of infectious agents and immunological reactions have prompted the development of synthetic SP-C-derived peptides or recombinant humanized SP-C for inclusion in new preparations for therapeutic use.We describe herein the recombinant production in bacterial cultures of SP-C variants containing phenylalanines instead of the palmitoylated cysteines of the native protein, as fusions to the hydrophilic nuclease A (SN) from Staphylococcus aureus. The resulting chimerae were partially purified by affinity chromatography and subsequently subjected to protease digestion. The SP-C forms were recovered from the digestion mixtures by organic extraction and further purified by size exclusion chromatography. The two recombinant SP-C variants so obtained retained more than 50% α-helical content and showed surface activity comparable to the native protein, as measured by surface spreading of lipid/protein suspensions and from compression π-A isotherms of lipid/protein films. Compared to the protein purified from porcine lungs, the recombinant SP-C forms improved movement of phospholipid molecules into the interface (during adsorption), or out from the interfacial film (during compression), suggesting new possibilities to develop improved therapeutic preparations.     

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.     

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.     

The phase behavior of lipid monolayers containing pulmonary surfactant protein C studied by fluorescence light microscopy

Three compounds of the pulmonary surfactant – dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylglycerol (DPPG), and the surfactant associated protein C (SP-C) – were spread at the air-water interface of a Langmuir trough as a model system to mimic the properties of natural surfactant. Fluorescence microscopical images of the film formed at the interface were obtained during compression using a fluorescence dye bound covalently either to phosphatidylcholine or to SP-C. The images were quantified using statistical methods in respect to relative areas and relative fluorescence intensities of the domains found. In the early stage of compression, film pressure rose slightly and was accompanied by a phase separation which could be recognized in the images by the formation of bright and dark domains. On further compression, after a steep increase of film pressure, a plateau region of constant film pressure started abruptly. During compression in the plateau region, fluorescence intensity of the bright domain formed in the early stage of compression increased. The increasing fluorescence intensity, the non-Gaussian intensity distribution of the bright domain, and the small mean molecular area of the film in the plateau region gave rise to the assumption that multilayer structures were formed in the late stage of compression. The formation of the multilayer structures was fully reversible in repeated compression-expansion cycles including the plateau region of the phase diagram. The ability of lipid/SP-C mixtures to form reversible multilayer structures during compression may be relevant to stability in lungs during expiration and inhalation. Received: 13 February 1997 / Accepted: 22 May 1997     

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.     

External reflection absorption infrared spectroscopy study of lung surfactant proteins SP-B and SP-C in phospholipid monolayers at the air/water interface.

The interactions of the hydrophobic pulmonary surfactant proteins SP-B and SP-C with 1,2-dipalmitoylphosphatidylcholine in mixed, spread monolayer films have been studied in situ at the air/water interface with the technique of external reflection absorption infrared spectroscopy (IRRAS). SP-C has a mostly alpha-helical secondary structure both in the pure state and in the presence of lipids, whereas SP-B secondary structure is a mixture of alpha-helical and disordered forms. When films of SP-B/1,2-dipalmitoylphosphatidylcholine are compressed to surface pressures (pi) greater than approximately 40-43 mN/m, the protein is partially (15-35%) excluded from the surface, as measured by intensity ratios of the peptide bond amide l/lipid C==O stretching vibrations. The extent of exclusion increases as the protein/lipid ratio in the film increases. In contrast, SP-C either remains at the surface at high pressures or leaves accompanied by lipids. The amide l peak of SP-C becomes asymmetric as a result of the formation of intermolecular sheet structures (1615-1630 cm-1) suggestive of peptide aggregation. The power of the IRRAS experiment for determination of film composition and molecular structure, i.e., as a direct test of the squeeze-out hypothesis of pulmonary surfactant function, is evident from this work.     

The molecular mechanism of monolayer-bilayer transformations of lung surfactant from molecular dynamics simulations

The aqueous lining of the lung surface exposed to the air is covered by lung surfactant, a film consisting of lipid and protein components. The main function of lung surfactant is to reduce the surface tension of the air-water interface to the low values necessary for breathing. This function requires the exchange of material between the lipid monolayer at the interface and lipid reservoirs under dynamic compression and expansion of the interface during the breathing cycle. We simulated the reversible exchange of material between the monolayer and lipid reservoirs under compression and expansion of the interface. We used a mixture of dipalmitoyl-phosphatidylcholine, palmitoyl-oleoyl-phosphatidylglycerol, cholesterol, and surfactant-associated protein C as a functional analog of mammalian lung surfactant. In our simulations, the monolayer collapses into the water subphase on compression and forms bilayer folds. On monolayer reexpansion, the material is transferred from the folds back to the interface. The simulations indicate that the connectivity of the bilayer aggregates to the monolayer is necessary for the reversibility of the monolayer-bilayer transformation. The simulations also show that bilayer aggregates are unstable in the air subphase and stable in the water subphase.