Secondary structure in lung surfactant SP-B peptides: IR and CD studies of bulk and monolayer phases

Pulmonary surfactant protein SP-B is known to facilitate adsorption and spreading of surfactant components across the air/water interface. This property appears essential for in vivo function in the alveolar subphase and at the air/alveolar surface. Three peptides with amino acid sequences based on SP-B containing predicted alpha-helical regions (SP-B(1--20), SP-B(9--36A), SP-B(40--60A)) have been synthesized to probe structure-function relationships and protein-lipid interaction in bulk phase and monolayer environments. IR and CD studies are reported along with traditional surface pressure-molecular area (pi-A) isotherms and IR reflection-absorption spectroscopy (IRRAS) investigations conducted at the air/water interface. In bulk phase, helix-promoting environments (methanol and aqueous dispersions of lipid vesicles), SP-B(1--20) and SP-B(9--36A) contained significant amounts of alpha-helical structure, whereas varying degrees of alpha-helix, random coil, and beta-sheet were observed in aqueous solutions and monolayers. The most striking behavior was observed for SP-B(9--36A), which displayed reversible surface pressure-induced beta-sheet formation. Bulk phase lipid melting curves and monolayer experiments with peptide-lipid mixtures showed subtle differences in the degree of bulk phase interaction and substantial differences in peptide surface activity. The uniqueness of IRRAS is emphasized as the importance of evaluating secondary structure in both bulk phase and monolayer environments for lung surfactant peptide mimics is demonstrated.     

Probing perturbation of bovine lung surfactant extracts by albumin using DSC and 2H-NMR

Lung surfactant (LS), a lipid-protein mixture, forms films at the lung air-water interface and prevents alveolar collapse at end expiration. In lung disease and injury, the surface activity of LS is inhibited by leakage of serum proteins such as albumin into the alveolar hypophase. Multilamellar vesicular dispersions of a clinically used replacement, bovine lipid extract surfactant (BLES), to which (2% by weight) chain-perdeuterated dipalmitoylphosphatidycholine (DPPG mixtures-d(62)) had been added, were studied using deuterium-NMR spectroscopy ((2)H-NMR) and differential scanning calorimetry (DSC). DSC scans of BLES showed a broad gel to liquid-crystalline phase transition between 10-35 degrees C, with a temperature of maximum heat flow (T(max)) around 27 degrees C. Incorporation of the DPPC-d(62) into BLES-reconstituted vesicles did not alter the T(max) or the transition range as observed by DSC or the hydrocarbon stretching modes of the lipids observed using infrared spectroscopy. Transition enthalpy change and (2)H-NMR order parameter profiles were not significantly altered by addition of calcium and cholesterol to BLES. (2)H-NMR spectra of the DPPC-d(62) probes in these samples were characteristic of a single average lipid environment at all temperatures. This suggested either continuous ordering of the bilayer through the transition during cooling or averaging of the DPPC-d(62) environment by rapid diffusion between small domains on a short timescale relative to that characteristic of the (2)H-NMR experiment. Addition of 10% by weight of soluble bovine serum albumin (1:0.1, BLES/albumin, dry wt/wt) broadened the transition slightly and resulted in the superposition of (2)H-NMR spectral features characteristic of coexisting fluid and ordered phases. This suggests the persistence of phase-separated domains throughout the transition regime (5-35 degrees C) of BLES with albumin. The study suggests albumin can cause segregation of protein bound-lipid domains in surfactant at NMR timescales (10(-5) s). Persistent phase separation at physiological temperature may provide for a basis for loss of surface activity of surfactant in dysfunction and disease.     

Molecular organization of the tear fluid lipid layer

The tear fluid protects the corneal epithelium from drying out as well as from invasion by pathogens. It also provides cell nutrients. Similarly to lung surfactant, it is composed of an aqueous phase covered by a lipid layer. Here we describe the molecular organization of the anterior lipid layer of the tear film. Artificial tear fluid lipid layers (ATFLLs) composed of egg yolk phosphatidylcholine (60 mol %), free fatty acids (20 mol %), cholesteryl oleate (10 mol %), and triglycerides (10 mol %) were deposited on the air-water interface and their physico-chemical behavior was compared to egg-yolk phosphatidylcholine monolayers by using Langmuir-film balance techniques, x-ray diffraction, and imaging techniques as well as in silico molecular level simulations. At low surface pressures, ATFLLs were organized at the air-water interface as heterogeneous monomolecular films. Upon compression the ATFLLs collapsed toward the air phase and formed hemispherelike lipid aggregates. This transition was reversible upon relaxation. These results were confirmed by molecular-level simulations of ATFLL, which further provided molecular-scale insight into the molecular distributions inside and dynamics of the tear film. Similar type of behavior is observed in lung surfactant but the folding takes place toward the aqueous phase. The results provide novel information of the function of lipids in the tear fluid.     

Formation of Folds and Vesicles by Dipalmitoylphosphatidylcholine Monolayers Spread in Excess

Lipid monolayers exist in several biological systems, including the stratum corneum of the skin, the fluid tear film of the eye, the Eustachian tube of the ear, and airway and alveolar pulmonary surfactants. In this paper, the monolayer-to-bilayer transition was studied using dipalmitoylphosphatidylcholine (DPPC) as the model. Depositing DPPC organic solvent solutions in excess at an air:buffer interface led to the formation of elongated structures which could be imaged on carbon grids by transmission electron microscopy. The structures appeared to be DPPC folds protruding into the sol. The structures were frequently ordered with respect to one another, suggesting that they arose during lateral compression due to excess DPPC and are characteristic of a type of monolayer collapse phase. In some cases, series of short folds in an extended line and series of vesicles in line or parallel to the folds were observed. This suggests the elongated folds are unstable and can resolve by forming vesicles. Fold formation occurred at defined lipid concentrations above which more vesicles were observed. Surfactant protein-A did not influence fold or vesicle formation but bound to the edges of these structures preferentially. It is concluded that DPPC monolayers can form bilayers spontaneously in the absence of surfactant apoproteins, other proteins or agents. Received: 18 May 2000/Revised: 20 November 2000     

Molecular weight dependence of the depletion attraction and its effects on the competitive adsorption of lung surfactant

Albumin competes with lung surfactant for the air-water interface, resulting in decreased surfactant adsorption and increased surface tension. Polyethylene glycol (PEG) and other hydrophilic polymers restore the normal rate of surfactant adsorption to the interface, which re-establishes low surface tensions on compression. PEG does so by generating an entropic depletion attraction between the surfactant aggregates and interface, reducing the energy barrier to adsorption imposed by the albumin. For a fixed composition of 10 g/L (1% wt.), surfactant adsorption increases with the 0.1 power of PEG molecular weight from 6 kDa-35 kDa as predicted by simple excluded volume models of the depletion attraction. The range of the depletion attraction for PEG with a molecular weight below 6 kDa is less than the dimensions of albumin and there is no effect on surfactant adsorption. PEG greater than 35 kDa reaches the overlap concentration at 1% wt. resulting in both decreased depletion attraction and decreased surfactant adsorption. Fluorescence images reveal that the depletion attraction causes the surfactant to break through the albumin film at the air-water interface to spread as a monolayer. During this transition, there is a coexistence of immiscible albumin and surfactant domains. Surface pressures well above the normal equilibrium surface pressure of albumin are necessary to force the albumin from the interface during film compression.     

Fluorescence light microscopy of pulmonary surfactant at the air-water interface of an air bubble of adjustable size

The structural dynamics of pulmonary surfactant was studied by epifluorescence light microscopy at the air-water interface of a bubble as a model close to nature for an alveolus. Small unilamellar vesicles of dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylglycerol, a small amount of a fluorescent dipalmitoylphosphatidylcholine-analog, and surfactant-associated protein C were injected into the buffer solution. They aggregated to large clusters in the presence of Ca(2+) and adsorbed from these units to the interface. This gave rise to an interfacial film that eventually became fully condensed with dark, polygonal domains in a fluorescent matrix. When now the bubble size was increased or decreased, respectively, the film expanded or contracted. Upon expansion of the bubble, the dark areas became larger to the debit of the bright matrix and reversed upon contraction. We were able to observe single domains during the whole process. The film remained condensed, even when the interface was increased to twice its original size. From comparison with scanning force microscopy directly at the air-water interface, the fluorescent areas proved to be lipid bilayers associated with the (dark) monolayer. In the lung, such multilayer phase acts as a reservoir that guarantees a full molecular coverage of the alveolar interface during the breathing cycle and provides mechanical stability to the film.     

Optical measurement of surface tension in a miniaturized air-liquid interface and its application in lung physiology

We have previously shown that lamellar body-like particles, the form in which pulmonary surfactant is secreted, spontaneously disintegrate when they contact an air-liquid interface, eventually creating an interfacial film. Here, we combined these studies with a new technique enabling the simultaneous and non-invasive measurement of surface tension (gamma). This method is a refinement of the pendant-drop principle. A sapphire cone with a 300-microm aperture keeps the experimental fluid by virtue of surface coherence in a fixed and nearly planar position above the objective of an inverted microscope. The radius of curvature of the fluid meniscus is related to gamma and determines the pattern of light back-reflection upon epi-illumination. This method, which we name "inverted interface", has several novel aspects, in particular its microscopic dimensions. When using lamellar body-like particles freshly released by alveolar type II cells, we found that their conversion at the interface resulted in gamma-reduction close to 30 mN/m. After a fast initial decay, gamma-decrease proceeded slowly and in proportion to single particle conversions. These conversions ceased with time whereas gamma decreased further, probably due to reorganization of the already deposited material. The present investigation indicates that surface film formation by adsorption of large surfactant aggregates is an important mechanism by which gamma is reduced in the lung.     

Inhibition of pulmonary surfactant adsorption by serum and the mechanisms of reversal by hydrophilic polymers: theory

A theory based on the Smolukowski analysis of colloid stability shows that the presence of charged, surface-active serum proteins at the alveolar air-liquid interface can severely reduce or eliminate the adsorption of lung surfactant from the subphase to the interface, consistent with the observations reported in the companion article (pages 1769-1779). Adding nonadsorbing, hydrophilic polymers to the subphase provides a depletion attraction between the surfactant aggregates and the interface, which can overcome the steric and electrostatic resistance to adsorption induced by serum. The depletion force increases with polymer concentration as well as with polymer molecular weight. Increasing the surfactant concentration has a much smaller effect than adding polymer, as is observed. Natural hydrophilic polymers, like the SP-A present in native surfactant, or hyaluronan, normally present in the alveolar fluids, can enhance adsorption in the presence of serum to eliminate inactivation.     

Infrared spectroscopic investigations of pulmonary surfactant. Surface film transitions at the air-water interface and bulk phase thermotropism.

The molecular structure of the phospholipid component of intact pulmonary surfactant isolated from bovine lung lavage has been examined by Fourier transform infrared spectroscopy. Two different physical states of the surfactant were examined by means of different infrared spectroscopic sampling techniques. Transmission infrared experiments were used to study the surfactant in the bulk phase. In these experiments, the thermotropic behavior of the bulk surfactant was monitored by temperature-induced variations in the phospholipid acyl chain CH2 stretching frequencies. A broad phase transition (confirmed by differential scanning calorimetry) was noted with an onset temperature near 15 degrees C and a completion temperature near 42 degrees C. In addition to the bulk transmission experiments, external reflection infrared spectroscopy was used to examine surfactant films in situ at the air-water interface. As surface pressure was increased from 0 to 43 dyn/cm, a gradual and continuous decrease in the CH2 stretching frequency was noted for the surfactant. Thus, under surface pressures which correspond to large lung volumes in vivo, the surfactant acyl chains exist mostly in the ordered (trans) configuration. The frequency shift in the CH2 stretching mode is consistent with a continuous ordering of the acyl chains upon compression over the pressure range 0-43 dyn/cm, and implies that a weakly cooperative phase transition occurs in the hydrocarbon region of the surface film. The surface film transition is especially noted in the pressure-area curve of the surfactant and approximates in two dimensions the broad thermotropic phase transition of the bulk phase surfactant.(ABSTRACT TRUNCATED AT 250 WORDS)     

Environmental tobacco smoke effects on lung surfactant film organization

Adsorption of the clinical lung surfactants (LS) Curosurf or Survanta from aqueous suspension to the air-water interface progresses from multi-bilayer aggregates through multilayer films to a coexistence between multilayer and monolayer domains. Exposure to environmental tobacco smoke (ETS) alters this progression as shown by Langmuir isotherms, fluorescence microscopy and atomic force microscopy (AFM). After 12 h of LS exposure to ETS, AFM images of Langmuir-Blodgett deposited films show that ETS reduces the amount of material near the interface and alters how surfactant is removed from the interface during compression. For Curosurf, ETS prevents refining of the film composition during cycling; this leads to higher minimum surface tensions. ETS also changes the morphology of the Curosurf film by reducing the size of condensed phase domains from 8-12 μm to ∼ 2 μm, suggesting a decrease in the line tension between the domains. The minimum surface tension and morphology of the Survanta film are less impacted by ETS exposure, although the amount of material associated with the film is reduced in a similar way to Curosurf. Fluorescence and mass spectra of Survanta dispersions containing native bovine SP-B treated with ETS indicate the oxidative degradation of protein aromatic amino acid residue side chains. Native bovine SP-C isolated from ETS exposed Survanta had changes in molecular mass consistent with deacylation of the lipoprotein. Fourier Transform Infrared Spectroscopy (FTIR) characterization of the hydrophobic proteins from ETS treated Survanta dispersions show significant changes in the conformation of SP-B and SP-C that correlate with the altered surface activity and morphology of the lipid-protein film.     

Size Influences the Effect of Hydrophobic Nanoparticles on Lung Surfactant Model Systems

The alveolar lung surfactant (LS) is a complex lipid protein mixture that forms an interfacial monolayer reducing the surface tension to near zero values and thus preventing the lungs from collapse. Due to the expanding field of nanotechnology and the corresponding unavoidable exposure of human beings from the air, it is crucial to study the potential effects of nanoparticles (NPs) on the structural organization of the lung surfactant system. In the present study, we investigated both, the domain structure in pure DPPC monolayers as well as in lung surfactant model systems. In the pure lipid system we found that two different sized hydrophobic polymeric nanoparticles with diameter of ∼12 nm and ∼136 nm have contrasting effect on the functional and structural behavior. The small nanoparticles inserted into fluid domains at the LE-LC phase transition are not visibly disturbing the phase transition but disrupting the domain morphology of the LE phase. The large nanoparticles led to an expanded isotherm and to a significant decrease in the line tension and thus to a drastic disruption of the domain structures at a much lower number of nanoparticles with respect to the lipid. The surface activity of the model LS films again showed drastic variations due to presence of different sized NPs illustrated by the film balance isotherms and the atomic force microscopy. AFM revealed laterally profuse multilayer protrusion formation on compression but only in the presence of 136 nm sized nanoparticles. Moreover we investigated the vesicle insertion process into a preformed monolayer. A severe inhibition was observed only in the presence of ∼136 nm NPs compared to minor effects in the presence of ∼12 nm NPs. Our study clearly shows that the size of the nanoparticles made of the same material determines the interaction with biological membranes.     

Towards the Molecular Mechanism of Pulmonary Surfactant Protein SP-B: At the Crossroad of Membrane Permeability and Interfacial Lipid Transfer

Pulmonary surfactant is a lipid-protein complex that coats the alveolar air-liquid interface, enabling the proper functioning of lung mechanics. The hydrophobic surfactant protein SP-B, in particular, plays an indispensable role in promoting the rapid adsorption of phospholipids into the interface. For this, formation of SP-B ring-shaped assemblies seems to be important, as oligomerization could be required for the ability of the protein to generate membrane contacts and to mediate lipid transfer among surfactant structures. SP-B, together with the other hydrophobic surfactant protein SP-C, also promotes permeability of surfactant membranes to polar molecules although the molecular mechanisms underlying this property, as well as its relevance for the surface activity of the protein, remain undefined. In this work, the contribution of SP-B and SP-C to surfactant membrane permeability has been further investigated, by evaluation of the ability of differently-sized fluorescent polar probes to permeate through giant vesicles with different lipid/protein composition. Our results are consistent with the generation by SP-B of pores with defined size in surfactant membranes. Furthermore, incubation of surfactant with an anti-SP-B antibody not only blocked membrane permeability but also affected lipid transfer into the air-water interface, as observed in a captive bubble surfactometer device. Our findings include the identification of SP-C and anionic phospholipids as modulators required for maintaining native-like permeability features in pulmonary surfactant membranes. Proper permeability through membrane assemblies could be crucial to complement the overall role of surfactant in maintaining alveolar equilibrium, beyond its biophysical function in stabilizing the respiratory air-liquid interface.     

The bilayer melting transition in lung surfactant bilayers: the role of cholesterol

Aqueous dispersions of a porcine lung surfactant (PLS) extract with and without cholesterol supplementation were analyzed by X-ray scattering. Lamellar liquid-crystalline and gel-type bilayer phases are formed, as in pure phosphatidylcholine (PC)-cholesterol systems. This PLS extract, developed for clinical applications, has a cholesterol content of less than 1% (w/w). Above the limit of swelling, the bilayer structure shows a melting (main) transition during heating at about 34 degrees C. When 13 mol% cholesterol was added to PLS, so that the cholesterol content of natural lung surfactant was reached, the X-ray scattering pattern showed pronounced changes. The main transition temperature was reduced to the range 20-25 degrees C, whereas according to earlier studies of disaturated PC-cholesterol bilayers in water the main transition remains almost constant when the amount of solubilized cholesterol is increased. Furthermore, the changes in scattering pattern at passing this transition in PLS-cholesterol samples were much smaller than at the same transition in PLS samples. These effects of cholesterol solubilization can be related to phase segregation within the bilayers, known from pure PC-cholesterol systems. One phase, solubilizing about 8 mol% cholesterol, exhibits a melting transition, whereas the other bilayer phase, with a liquid-crystalline disordered conformation, has a cholesterol content in the range 20-30 mol% and this phase shows no thermal transition. The relative amount of bilayer lipids that is transformed at the main transition in the PLS-cholesterol sample is therefore only half compared to that in PLS samples. The reduction in transition temperature in the segregated bilayer of lung surfactant lipids is probably an effect of enrichment of disaturated PC species in the phase, which is poor in cholesterol. This work indicates that cholesterol in lung surfactant regulates the crystallization behavior.     

Atomic force microscopy studies of functional and dysfunctional pulmonary surfactant films, II: albumin-inhibited pulmonary surfactant films and the effect of SP-A

Pulmonary surfactant (PS) dysfunction because of the leakage of serum proteins into the alveolar space could be an operative pathogenesis in acute respiratory distress syndrome. Albumin-inhibited PS is a commonly used in vitro model for studying surfactant abnormality in acute respiratory distress syndrome. However, the mechanism by which PS is inhibited by albumin remains controversial. This study investigated the film organization of albumin-inhibited bovine lipid extract surfactant (BLES) with and without surfactant protein A (SP-A), using atomic force microscopy. The BLES and albumin (1:4 w/w) were cospread at an air-water interface from aqueous media. Cospreading minimized the adsorption barrier for phospholipid vesicles imposed by preadsorbed albumin molecules, i.e., inhibition because of competitive adsorption. Atomic force microscopy revealed distinct variations in film organization, persisting up to 40 mN/m, compared with pure BLES monolayers. Fluorescence confocal microscopy confirmed that albumin remained within the liquid-expanded phase of the monolayer at surface pressures higher than the equilibrium surface pressure of albumin. The remaining albumin mixed with the BLES monolayer so as to increase film compressibility. Such an inhibitory effect could not be relieved by repeated compression-expansion cycles or by adding surfactant protein A. These experimental data indicate a new mechanism of surfactant inhibition by serum proteins, complementing the traditional competitive adsorption mechanism.     

Interaction of the C-Terminal Peptide of Pulmonary Surfactant Protein B (SP-B) with a Bicellar Lipid Mixture Containing Anionic Lipid

The hydrophobic lung surfactant SP-B is essential for respiration. SP-B promotes spreading and adsorption of surfactant at the alveolar air-water interface and may facilitate connections between the surface layer and underlying lamellar reservoirs of surfactant material. SP-B_63–78 is a cationic and amphipathic helical peptide containing the C-terminal helix of SP-B. ²H NMR has been used to examine the effect of SP-B_63–78 on the phase behavior and dynamics of bicellar lipid dispersions containing the longer chain phospholipids DMPC-d ₅₄ and DMPG and the shorter chain lipid DHPC mixed with a 3∶1∶1 molar ratio. Below the gel-to-liquid crystal phase transition temperature of the longer chain components, bicellar mixtures form small, rapidly reorienting disk-like particles with shorter chain lipid components predominantly found around the highly curved particle edges. With increasing temperature, the particles coalesce into larger magnetically-oriented structures and then into more extended lamellar phases. The susceptibility of bicellar particles to coalescence and large scale reorganization makes them an interesting platform in which to study peptide-induced interactions between lipid assemblies. SP-B_63–78 is found to lower the temperature at which the orientable phase transforms to the more extended lamellar phase. The peptide also changes the spectrum of motions contributing to quadrupole echo decay in the lamellar phase. The way in which the peptide alters interactions between bilayered micelle structures may provide some insight into some aspects of the role of full-length SP-B in maintaining a functional surfactant layer in lungs.     

The influence of water on the phase transition of sheep lung surfactant. A possible mechanism for surfactant phase transitions in vivo

The effect of water on the thermal properties of sheep lung surfactant lipids was determined by differential scanning calorimetry. Dry surfactant exhibited a phase transition with an upper limit of about 54 degrees C, whereas that of the fully hydrated surfactant was about 30 degrees C. The effect of water was confined to a range of hydration values from 0 to 25%. The results indicate that pulmonary surfactant lipids are capable of undergoing both thermotropic and lyotropic mesomorphism in vitro. The degree of hydration of the surfactant could influence its in vivo biophysical role in alveolar dynamics. Indeed, small changes in the surfactant to water ratio induced by regional differences in the surfactant concentration at the alveolar surface during alveolar expansion and contraction could be sufficient to trigger isothermal phase transitions in the surfactant lipids. This would allow changes to occur in the equilibrium between solidus and fluidus surfactant during the respiratory cycle.     

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.     

Perfluorooctyl bromide dispersions in aqueous media for biomedical applications.

In studying perfluorooctyl bromide (PFOB) dispersions in aqueous media, we have used two types of surfactant: egg yolk phospholipids (EYP) and polyglycerol esters (PGE). Our interest in these dispersions arises from their potential biomedical applications as imaging solutions and oxygen-carrying solutions (i.e., blood substitutes). For EYP systems, we have identified the dispersion structure as consisting of (a) PFOB droplets (250-nm diameter) stabilized by a phospholipid monolayer adsorbed irreversibly at the o/w interface and (b) small empty phospholipid vesicles. With both surfactants commercial preparations yielded stable systems, while purified samples, being non-dispersible, could not be made to act as emulsifiers. In both cases, minor components in the commercial surfactant were found to be necessary for the formation of a stable dispersion, enabling the transport of the pure surfactant to the PFOB/water interface.     

The melting of pulmonary surfactant monolayers.

Monomolecular films of phospholipids in the liquid-expanded (LE) phase after supercompression to high surface pressures (pi), well above the equilibrium surface pressure (pi(e)) at which fluid films collapse from the interface to form a three-dimensional bulk phase, and in the tilted-condensed (TC) phase both replicate the resistance to collapse that is characteristic of alveolar films in the lungs. To provide the basis for determining which film is present in the alveolus, we measured the melting characteristics of monolayers containing TC dipalmitoyl phosphatidylcholine (DPPC), as well as supercompressed 1-palmitoyl-2-oleoyl phosphatidylcholine and calf lung surfactant extract (CLSE). Films generated by appropriate manipulations on a captive bubble were heated from < or =27 degrees C to > or =60 degrees C at different constant pi above pi(e). DPPC showed the abrupt expansion expected for the TC-LE phase transition, followed by the contraction produced by collapse. Supercompressed CLSE showed no evidence of the TC-LE expansion, arguing that supercompression did not simply convert the mixed lipid film to TC DPPC. For both DPPC and CLSE, the melting point, taken as the temperature at which collapse began, increased at higher pi, in contrast to 1-palmitoyl-2-oleoyl phosphatidylcholine, for which higher pi produced collapse at lower temperatures. For pi between 50 and 65 mN/m, DPPC melted at 48-55 degrees C, well above the main transition for bilayers at 41 degrees C. At each pi, CLSE melted at temperatures >10 degrees C lower. The distinct melting points for TC DPPC and supercompressed CLSE provide the basis by which the nature of the alveolar film might be determined from the temperature-dependence of pulmonary mechanics.     

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.