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Originally published In Press as doi:10.1074/jbc.M404648200 on July 1, 2004

J. Biol. Chem., Vol. 279, Issue 39, 40715-40722, September 24, 2004
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Cholesterol Rules

DIRECT OBSERVATION OF THE COEXISTENCE OF TWO FLUID PHASES IN NATIVE PULMONARY SURFACTANT MEMBRANES AT PHYSIOLOGICAL TEMPERATURES*

Jorge Bernardino de la Serna{ddagger}, Jesus Perez-Gil{ddagger}, Adam C. Simonsen§, and Luis A. Bagatolli¶||

From the {ddagger}Departmento de Bioquímica, Facultad de Biología, Universidad Complutense, 28040 Madrid, Spain and the MEMPHYS-Center for Biomembrane Physics, the §Department of Physics and the Department of Biochemistry and Molecular Biology, University of Southern Denmark, 5230 Odense, Denmark

Received for publication, April 27, 2004 , and in revised form, June 23, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Pulmonary surfactant, the lipid-protein material that stabilizes the respiratory surface of the lungs, contains approximately equimolar amounts of saturated and unsaturated phospholipid species and significant proportions of cholesterol. Such lipid composition suggests that the membranes taking part in the surfactant structures could be organized heterogeneously in the form of inplane domains, originating from particular distributions of specific proteins and lipids. Here we report novel results concerning the lateral organization of bilayer membranes made of native pulmonary surfactant where the coexistence of two distinct micrometer sized fluid phases (fluid ordered and fluid disordered-like phases) is observed at physiological temperatures by using fluorescence microscopy and atomic force microscopy. Additional experiments using fluorescent-labeled proteins SP-B and SP-C show that at physiological temperatures these hydrophobic proteins are located exclusively in the fluid disordered-like phase. Most interestingly, the microscopic coexistence of fluid phases is maintained up to 37.5 °C, where most fluid ordered phases melt. This observation suggests that the particular composition of this material is naturally designed to be at the "edge" of a lateral structure transition under physiological conditions, likely providing particular structural and dynamic properties for its mechanical function. The observed lateral structure in native pulmonary surfactant membranes is dramatically affected by the extraction of cholesterol, an effect not observed upon extraction of the surfactant proteins. Furthermore, the spreading properties of the native surfactant material at the air-liquid interface were also greatly affected by cholesterol extraction, suggesting a connection between the observed lateral structure and a physiologically relevant function of the material. We suggest that the particular lipid composition of surfactant could be finely tuned to provide, under physiological conditions, a structural scaffold for surfactant proteins to act at appropriate local densities and lipid composition.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Pulmonary surfactant is the main secretory product of the alveolar type II pneumocytes and is required to stabilize the lungs in air-breathing animals (1, 2). This surfactant material is mainly composed of lipids, mainly phospholipids, and small amounts of specifically associated proteins. Among the phospholipids significant amounts of dipalmitoylphosphatidylcholine (DPPC)1 and phosphatidylglycerol are present, both of which are unusual species in most animal membranes. Mono-unsaturated phosphatidylcholines (PC), phosphatidylinositol, and neutral lipids including cholesterol are also present in pulmonary surfactant in varying proportions. The surfactant proteins (SP) SP-A, SP-B, SP-C, and SP-D constitute about 8% of lung surfactant by weight. SP-A and SP-D are large glyco-proteins (>500 kDa) belonging to the superfamily of collectins, and they serve important functions in innate defense mechanisms of the lung (3). The hydrophobic proteins SP-B and SP-C modulate the surface-active properties of surfactant lipids and are strictly required to establish an operational respiratory surface (4). Pulmonary surfactant lipid-protein complex is secreted into the thin aqueous alveolar lining of the lung as multilamellar assemblies, which spontaneously transform into nanotubular membrane-based structures called tubular myelin. These structures are considered reservoirs of highly surface-active components in the pathway that forms surface-active films at the lung air-water interface. Such films can reduce the surface tension to nearly 0 mN/m and in doing so prevent alveolar collapse at low lung volumes (1, 5). Dysfunctions of the surfactant system are relevant in diseases including neonatal and acute respiratory distress syndrome, cystic fibrosis, and pneumonia (6). Several studies (7, 8) have shown that interfacial films made with the hydrophobic fraction (lipids + SP-B + SP-C) of pulmonary surfactant undergo phase separation under lateral compression. In addition, the particular phenomenon of gel/fluid phase separation induced by temperature was also reported in bilayers composed of part of the hydrophobic fraction of pulmonary surfactant at physiological temperatures (9). Even though the particular composition of the lung surfactant suggests that native surfactant membrane-based structures could exhibit lateral segregation phenomena at physiological temperatures, this aspect has been merely speculative up to now. For example, the presence of high amounts of DPPC (~40% weight of the total material) with a melting phase transition above mammalian physiological temperatures (41.5 °C) indicates the possibility of the coexistence of a solid/fluid-like phase (10). On the other hand, the presence of cholesterol along with other unsaturated phospholipid species in the native material suggests the possibility of a fluid ordered/fluid disordered-like phase coexistence (distinguished in part by their cholesterol content) under physiologically relevant conditions (10). Even though lung surfactant has cholesterol concentrations of up to 20–22 mol % (11, 12), there is no clear understanding of how this molecule impacts the lateral structure of the native material. The presence of cholesterol could thus have important consequences for the lateral organization of the native pulmonary surfactant material, illustrating another example of the fundamental role of cholesterol in modulating the lateral structure of membranes, as the raft hypothesis proposes (13, 14). Furthermore, there is presently no clear link between membrane lateral heterogeneity and the physiological function of native pulmonary surfactant material (10).

In recent years, a new experimental strategy, based on the direct visualization of free standing lipid bilayers using giant unilamellar vesicle technology in conjunction with confocal and two photon excitation fluorescence microscopy techniques, has opened the possibility of correlating morphological and dynamical information on lipid membranes at molecular and supramolecular levels. Important novel information such as the morphology of different coexisting lipid phases (such as gel/fluid and fluid ordered/fluid disordered), mechanical properties of membranes displaying phase coexistence, local hydration, and molecular diffusion in lipid domains can be extracted directly from the fluorescence images (9, 15–23). The present study uses the GUV technology in addition to other experimental techniques to provide evidence that a particular lateral structure occurs in native membranes of pulmonary surfactant at physiological temperatures. Additionally we demonstrate that cholesterol plays a critical role in promoting such membrane organization. On the other hand, it is important to note that preparation of GUVs was previously limited to membranes composed of artificial and natural lipid extracts (from lipid solutions in organic solvents). In this study we also present a method to prepare GUVs composed of native membranes without using organic solvents or detergents. To our knowledge, GUV experiments performed with native membrane preparations to further evaluate the concurrent effect of lipids and proteins in lateral segregation phenomena have not yet been reported.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Materials—1,1'-Dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiIC18), 2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine (Bodipy-PC), and isothiocyanate derivatives of Alexa Fluor® 488 and Texas Red® were from Molecular Probes Inc. (Eugene, OR). Methyl-{beta}-cyclodextrin was from Aldrich. DPPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and cholesterol were purchased from Avanti Polar Lipids (Alabaster, AL) and were used without further purification. Native pulmonary surfactant material was obtained from pig lungs as described previously (24). The cholesterol quantitation kits INFINITYTM and IVD were purchased from Sigma and Spinreact (Girona, Spain), respectively.

Preparation of Surfactant and Proteins—Stock suspensions of native pulmonary surfactant material (NPSM) were in 5 mM Tris buffer, pH 7, containing 150 mM NaCl. The total concentration of phospholipid was 0.5 mg/ml, estimated by phosphorus quantitation upon phospholipid mineralization (25). For fluorescence experiments the fluorescent probes were incorporated into NPSM stock suspensions before preparing the giant vesicles. In the case of the lipophilic probes (DiIC18 and Bodipy-PC) a small aliquot (0.5 µl) of an Me2SO solution containing the fluorescent probes (0.125 mM) was added to the NPSM stock suspension (final volume 500 µl) and incubated for 20 min at room temperature. In all cases the percentage of fluorescent probes in the sample was less than 0.1 mol %.

Fluorescently labeled surfactant proteins were prepared by labeling purified SP-B and SP-C solutions in organic solvent, as described previously (26). This procedure allows attachment of a single fluorophore to the N-terminal amine group of the protein. In our studies Alexa 488 and Texas Red were used for SP-C and SP-B, respectively. Labeled proteins were incorporated into NPSM by addition of small aliquots of fluorescent SP-B and SP-C (0.5% protein to lipid by weight) in Me2SO.

Giant Vesicle Preparation—GUVs composed of NPSM organic extract, its lipid fraction, or the ternary mixture DOPC/DPPC/cholesterol were prepared as described previously (16, 27) by using the electroformation method originally developed by Angelova and Dimitrov (28) and Angelova et al. (29). A previously described (16, 27) special temperature-controlled chamber was used for this purpose. Briefly the process can be described in the following three steps. 1) ~3 µl of the stock solution of the lipid or lipid/protein organic solution were spread on the surface of each platinum wire. The chamber was located under a stream of N2 during this procedure and then placed under vacuum overnight to remove the organic solvent. 2) Aqueous solution was added to the chamber (200 mosM sucrose solution prepared with Millipore-filtered water, 17.5 megohms/cm). The sucrose solution was previously heated to the desired temperature (above the lipid mixture phase transition, we used 45 °C), and then sufficient volume was added to cover the platinum wires (~300 µl). 3) The platinum wires were connected immediately to a function generator (Digimess® FG 100, Germany), and a low frequency AC field (sinusoidal wave function with a frequency of 10 Hz and an amplitude of 3 V) was applied for 120 min. After vesicle formation, the AC field was turned off, and the vesicles were collected with a pipette and transferred to a plastic tube.

NPSM GUVs were also prepared using the electroformation method but with some modifications in step 1 of the protocol described above. In this case NPSM GUVs were formed from NPSM suspended in buffer solution (no organic solvents in this case). Briefly, ~3 µl of the stock suspension in buffer was spread on the surface of each platinum wire as small drops. The chamber was then placed under a stream of N2 and subsequently under low vacuum for 30 min to allow the native material to adsorb onto the platinum wire. An important point in this step is to avoid dehydration of the sample to maintain the integrity of the native membranes. Once the material was adsorbed to the Pt wire, we proceed with steps 2 and 3 as described above. As a control experiment, GUVs composed of DOPC/DPPC/cholesterol were also prepared from multilamellar suspensions in aqueous solution as described above for NPSM and then compared with those obtained from the lipid organic extract. No differences in the morphology of the final resulting GUVs were observed between the two described protocols.

Observation of Giant Vesicles—Aliquots of giant vesicles suspended in sucrose were added to an equi-osmolar concentration of glucose solution. Because of the density difference between the two solutions, the vesicles precipitate at the bottom of the chamber which facilitates observation of the GUVs in the inverted confocal microscope. GUV preparations were observed in 8-well plastic chambers (Lab-Tek Brand Products, Naperville IL). The chamber was located in an inverted confocal microscope (Zeiss LSM 510 META) for observation. The excitation wavelengths were 488 (for Alexa 488 and Bodipy-PC) and 543 nm (for DiIC18 and Texas Red). The temperature was controlled from a water bath connected to a homemade device into which the 8-well plastic chamber was inserted. The temperature was measured inside the sample chamber using a digital thermocouple (model 400B, Omega Inc., Stamford, CT) with a precision of 0.1 °C.

Differential Scanning Calorimetry—Experiments were performed in a Science Corp. N-DSCII calorimeter using 15 mM total lipid concentration of native pulmonary surfactant suspension. The scan rate was 0.5 K/min versus buffer. Five temperature scans were collected from each sample between 25 to 70 °C. Neither change in the total concentration nor in the scan rate gave significant changes in the obtained results.

Cholesterol Extraction Experiments—Methyl-{beta}-cyclodextrin/glucose solutions were prepared in Millipore water 17.5 megohms/cm. The osmolarity of these solutions was measured in an osmometer (The Advance Osmometer, model 3D3, Advance Instruments Inc., Norwood, MA) and adjusted to match the osmolarity of the vesicles containing sucrose solution. Vesicles were diluted in the methyl-{beta}-cyclodextrin/glucose-containing solution and observed afterward. Alternatively, aliquots of methyl-{beta}-cyclodextrin solutions were added to the vesicles already dispersed in glucose solution. No differences in the effect of methyl-{beta}-cyclodextrin on the vesicles were observed between the two procedures. Cholesterol was also removed from the lipid fraction of NPSM by hydrophobic chromatography in Sephadex LH-20 (30).

Phospholipid and Cholesterol Determination—Total phospholipid concentration in the different samples was determined by phosphorus analysis as we described above (25). The amount of cholesterol in NPSM and in their different fractions was quantitatively estimated using the following: (i) a colorimetric assay based on the activity of cholesterol oxidase (IVD kit from Spinreact, Girona), and (ii) the determination of peroxide products resulting from cholesterol oxidation (INFINITYTM kit from Sigma). Cholesterol content in the samples is given as cholesterol-to-phospholipid molar ratio. Four different samples were used for total cholesterol quantitation from each of at least three different batches of each of the fractions studied.

Protein Extraction from NPSM—The hydrophobic fraction of NPSM, containing all the lipid species plus the hydrophobic proteins SP-B and SP-C but lacking hydrophilic proteins SP-A and SP-D, was obtained by chloroform/methanol extraction. Separation of lipid from protein components in the organic mixture was achieved by chromatography of the surfactant organic extract through a Sephadex LH-20 column, as described previously (31).

Atomic Force Microscopy of NPSM Bilayers—NPSM aqueous suspensions were spread on top of an ultrapure water subphase in a Langmuir-Blodgett trough (Kibron, µ-trough) until a surface pressure of 1 mN/m was obtained. The film was allowed to equilibrate for 10 min and was subsequently compressed to 42 mN/m. While maintaining this pressure, a film was transferred to a freshly cleaved mica substrate previously immersed into the subphase by lifting the support at a constant speed of 1.5 mm/min. Following the initial deposition, a second layer was transferred onto the first by re-immersion of the coated mica into the subphase at 1.5 mm/s (also at a constant surface pressure of 42 mN/m). Consequently, the supported membranes used in the atomic force microscopy (AFM) experiments are bilayer structures formed by double transfer of the surface film (including any additional associated structure to the monolayer), which is obtained by spreading aqueous suspensions of native surfactant at the air-water interface. Topographical AFM images were obtained under aqueous conditions in a PicoSPM (Molecular Imaging) microscope operated in magnetically activated tapping mode using MAClevers.

Spreading Kinetics—Surface activity of surfactant preparations was assayed by running {pi} -t interfacial adsorption isotherms. In a typical experiment, 3 µl of surfactant suspension (10 mg/ml phospholipid concentration) were spread by direct deposition on top of a subphase 5 mM Tris, pH 7, containing 150 mM NaCl in the Teflon trough (20 cm2) of a thermostated surface balance built by Nima (Coventry, UK), and at 5 cm of a filter paper Wilhelmy plate, before monitoring surface pressure against time. Spreading experiments were done either at 25 or 37 °C. Spreading experiments in the presence of methyl-{beta}-cyclodextrin were carried out by spreading equivalent amounts of native surfactant on top of 5 mM Tris, pH 7, subphases containing 150 mM NaCl, and different concentrations of methyl-{beta}-cyclodextrin. Data shown are representative of three different experiments for two different batches of each assayed surfactant preparation.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Fig. 1 shows images of single GUVs composed of NPSM doped with the fluorescent probes Bodipy-PC and DiIC18. Based on the particular round shape of the domains (18, 20, 21) and the partition properties of the different fluorescent probes (15, 19), we conclude that the lateral organization of the NPSM vesicles corresponds to a fluid ordered/fluid disordered-like phase coexistence. The coexistence of two fluid phases persists from 22 to 37.5 °C. In general, when fluid domains are embedded in a fluid environment, circular domains are formed because both phases are isotropic, and the line energy (tension), which is associated with the rim of two demixing phases, is minimized by optimizing the area-to-perimeter ratio. As observed in Fig. 1C, the domains span the bilayer in agreement with previous observations done in artificial and natural lipid mixtures (15–18). NPSM frequently gave rise to multilamellar giant vesicles (see Fig. 1D), whose morphology resembles the structure of lamellar bodies that are the multilamellar assemblies storing and secreting pulmonary surfactant in the type II pneumocytes of the lung. Coexistence of two fluid phases was observed in every bilayer of these giant multilamellar assemblies. Fig. 2 shows an AFM image of an NPSM-supported membrane, without exogenous fluorescent probes. Such membranes are part of the surface-active film of pulmonary surfactant that spontaneously forms upon adsorption at the interface (32). It is important to emphasize that the samples visualized by AFM are not simple monolayers, as those studied previously in the literature, but bilayers as we perform a double deposition of surfactant films onto mica (see "Experimental Procedures"). AFM scanning of supported membranes demonstrates the presence of circular-shaped domains with sizes comparable with those seen in GUVs, confirming that two fluid phases coexist in the native material. This observation represents an important control experiment because it indicates that the membrane lateral structure in the GUVs is really present in the original material and is not because of the incorporation of fluorescent probes or the way the samples are prepared. AFM also reveals the coexistence of nano-scale structures inside the circular-shaped domains. Topological analysis shows that the distribution of heights observed in the round domains (i.e. higher versus lower heights) is more heterogeneous than the height distribution observed in the more homogeneous intervening background (Fig. 2). In particular, the round domains contain regions of lower heights compared to the height observed in the continuous region. This last finding suggests that 1) the round domains may correspond to a liquid disordered-like phase state and that 2) the region surrounding the round domains may display a liquid ordered-like phase state. The higher heights in the round domains may be due to the presence of surfactant proteins. The complex topography of the liquid-disordered domains is likely connected with particular organization of the many different molecular species (lipids and proteins) in the plane of the membrane. Given the compositional complexity of the material, further experiments to clarify this issue are warranted but are outside of the scope of the present study.



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  		FIG. 1.
Giant vesicles from native pulmonary surfactant membranes. Giant unilamellar (A–C) and multilamellar (D) vesicles formed from native pulmonary surfactant material. A, transmission image; B–D, fluorescent images. The confocal planes in the fluorescent images were obtained at the polar (B) and center (C and D) regions of the giant vesicles. The temperature was 37 °C. The giant vesicles are labeled with the lipophilic fluorescence probes DiIC18 (red) and Bodipy-PC (green). In the NPSM the rounded green areas in the images correspond to fluid disordered regions, and the red background corresponds to fluid ordered phase. The scale bars correspond to 10 µm. Note that the different domains span the lipid bilayer (C and D).

 



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  		FIG. 2.
AFM scanning image from a pulmonary surfactant membrane. A, AFM scanning image of membranes formed by spreading NPSM on top of an air-liquid interface, at a lateral pressure of 42 mN/m. Film transfer was performed using the Langmuir-Blodgett method onto a mica support. The supported membrane was scanned in a liquid cell using tapping mode. B, magnification of the region marked in A. C, topographic profile along the white line included in B. The temperature was 25 °C.

 
The particular domain pattern observed on the GUV surface is likely to be relevant for modulating the lateral distribution of the surfactant hydrophobic proteins SP-B and SP-C in both the native membranes and those formed from surfactant organic extract, as shown in Fig. 3. When fluorescently labeled SP-B and SP-C were incorporated into NPSM GUVs or into GUVs formed from the hydrophobic fraction of NPSM, the fluorescence of these proteins colocalized with the Bodipy-PC-enriched areas (from 22 to 37.5 °C). This last fact indicates that the hydrophobic proteins segregate into fluid disordered regions, in agreement with the observations obtained in the AFM experiments (Fig. 2). The partitioning of SP-B and SP-C in NPSM and in bilayers made of surfactant organic extract resemble the distribution of the proteins observed in monolayer experiments, where both SP-B and SP-C preferentially situate in liquid-expanded areas (33). This distribution is particularly remarkable in the case of SP-C, because this protein should, in principle, have a different local environment in bilayers with respect to monolayers simply because of the different membrane thickness (SP-C in bilayers accommodate in a transmembrane orientation (1)). Our results seem also to discard the potential association of SP-C with cholesterol-enriched liquid ordered regions of the membranes, in contrast with that described for other palmitoylated transmembrane proteins (34). Additionally, we observed that removal of SP-A from NPSM does not affect the partition properties of either SP-B or SP-C proteins (Fig. 3B).



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  		FIG. 3.
Lateral distribution of surfactant proteins SP-B and SP-C in pulmonary surfactant membranes. Fluorescence images of GUVs composed of NPSM (A) or NPSM organic extract (B) upon incorporation of fluorescently labeled proteins SP-B and SP-C (0.5% protein/lipid by weight). The two color images (bottom images) are split into those corresponding to Alexa 488-labeled SP-C (green) and Texas Red-labeled SP-B (red). Scale bars are 10 µm. Images were taken at 37 °C.

 
Fig. 4 illustrates the effect of temperature on the lateral structure of NPSM. The liquid disordered domains showed dynamically fluctuating borderlines when the temperature was raised close to 37.5 °C followed by a dramatic lateral structure change above 37.5 °C. Fig. 4A shows that at 38 °C, most of the liquid ordered phase has melted, yielding less than 10% of the surface covered by a network of filamentous shapes, probably ordered-like phase. Fig. 4B shows a representative differential scanning calorimetry experiment obtained from NPSM solutions. The thermogram is characterized by a broad thermal transition showing the end of the melting process above 37.5 °C, in agreement with the data obtained in GUVs and that previously reported for alveolar surfactant (crude lung wash) dispersion from mongrel dogs (35). Additionally, a very similar effect is observed by using fluorescent-labeled proteins SP-B and SP-C instead of the lipophilic fluorescent probes. From our experimental data we can observe that the proteins remain in the fluid disordered areas at temperatures above the lateral structure change as observed at 37.5 °C (Fig. 4C).



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  		FIG. 4.
Effect of temperature on the structure of native pulmonary surfactant membranes. Representative confocal fluorescence images of giant unilamellar vesicles composed of native pulmonary surfactant material at the indicated temperatures obtained with the lipophilic fluorescent probes DiIC18 and Bodipy-PC (A) and fluorescently labeled SP-B and SP-C (C). The fluorescent image in C is split into two colors corresponding to Alexa 488-labeled SP-C (green) and Texas Red-labeled SP-B (red). Scale bars are 20 µm. Differential scanning calorimetry thermogram of NPSM (B).

 
The similarities between the fluorescent images obtained in the NPSM and those reported previously (18, 20–22) in cholesterol-containing ternary lipid mixtures are remarkable and suggest a potentially important role for cholesterol in the lateral structural organization of NPSM. However, because of the compositional complexity of NPSM, the potential influence of other components, in particular surfactant proteins, on the lateral arrangement of this membranous system had to be evaluated. To address this point we used different experimental strategies involving the extraction of particular components of NPSM. Cholesterol extraction with methyl-{beta}-cyclodextrin and extraction of surfactant proteins (SP-A, SP-B and SP-C) were performed on the NPSM, and the results are summarized in Fig. 5A. Neither the absence of the water-soluble fraction (including the collectin SP-A) nor the extraction of the hydrophobic proteins SP-B and SP-C from the NPSM changed the circular shape of the lipid domains between 22 and 37.5 °C. This result indicates that the presence of the surfactant proteins is not critical to maintain the phase coexistence pattern observed in NPSM. The cholesterol-to-phospholipid molar ratio in all these materials was similar (between 18 and 21 mol %), as summarized in Table I. Fig. 5A also shows that after isothermal cholesterol extraction with methyl-{beta}-cyclodextrin, the phase coexistence pattern characterized by round domains in NPSM changes to elongated irregularly shaped domains, similar to those reported previously in artificial ternary mixtures at very low cholesterol levels (20–22) and in DPPC-containing phospholipid binary mixtures (15, 16). The lateral structure observed in these systems corresponds to the coexistence of gel and fluid phases. To explain further the observed cholesterol-dependent phase changes in NSPM at constant temperature, cholesterol-extraction experiments using methyl-{beta}-cyclodextrin were performed in the well characterized DOPC/DPPC/cholesterol mixture (20–22). This particular lipid mixture was chosen as a model system because the presence of DPPC and cholesterol is similar to that of NPSM. Fig. 5B shows two different phase transitions as a result of increasing methyl-{beta}-cyclodextrin concentration at constant temperature, i.e. fluid ordered -> fluid ordered/fluid disordered and fluid ordered/fluid disordered -> gel/fluid. The last phase transition occurs in a remarkably similar fashion to that observed in NPSM (compare Fig. 5, A and B). The domain compositional information already reported for DOPC/DPPC/cholesterol in the fluid ordered/fluid disordered phase coexistence regime (20–22) suggests that the DPPC-containing fluid ordered phase is enriched in cholesterol compared with the DOPC-enriched fluid disordered phase where the cholesterol content is low. Based on these observations we propose that the fluid ordered phase in NPSM may be enriched in DPPC and cholesterol compared with the fluid disordered phase. Additionally, from our experiments we notice that although the shape of the lipid domains is quite similar for NPSM and the cholesterol-containing ternary mixtures in the same phase coexistence regime (compare Fig. 5A, top left image, with B, center image, and Fig. 5A, bottom left image, with B, bottom image), there are some differences in the partition properties of the fluorescent probes. This observation suggests that the molecular composition of these membrane domains (i.e. fluid ordered, gel, fluid disordered) could play an important role in modulating specific molecular interactions in the plane of the membrane in agreement with previous results observed in lipid mixtures (18).



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  		FIG. 5.
Fluorescent images of GUVs made from different surfactant fractions and lipid mixtures. A, GUV composed of NPSM (top left) and GUV composed of the organic extract of NPSM that contains all the hydrophobic components but no hydrophilic proteins (top right); GUV composed of the lipid fraction of the NPSM organic extract, from which hydrophobic proteins SP-B and SP-C have also been removed (bottom right), and GUV of NPSM treated with 40 mg/ml methyl-{beta}-cyclodextrin (bottom left). Images were taken at 25 °C. The cholesterol-to-phospholipid molar ratio for the samples that display coexistence of two fluid phases (round-shaped domains) are 21% (top left image), 19% (top right image), and 18% (bottom right). B, fluorescence images of GUVs composed of DOPC/DPPC/cholesterol (1:1:40 mol %) before (top) and after exposure to 10 (center) and 40 mg/ml (bottom) of methyl-{beta}-cyclodextrin. Images were taken at 25 °C. All the GUVs in the figure have been labeled with the lipophilic fluorescence probes DiIC18 (red) and Bodipy-PC (green). C, fluorescence images of GUVs composed of a lipid fraction of NPSM that contains no surfactant proteins at different cholesterol molar ratio (with respect to phospholipids). Images from left to right are from GUVs containing 10, 15, 18, 21, and 30 mol % cholesterol. Images were taken at 25 °C. The scale bars correspond to 10 µm.

 


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  		TABLE I
Cholesterol content in NPSM and its fractions

 
We found that the changes observed as the cholesterol concentration is decreased (Fig. 5A) can be reversed by re-introducing cholesterol as shown in Fig. 5C. GUVs formed from surfactant lipid extract partially depleted of cholesterol (cholesterol to phospholipid ratio 10 mol %, instead of 20 mol % as in NPSM) show a similar pattern of gel/fluid phase coexistence as that observed in NPSM GUVs treated with a high concentration of methyl-{beta}-cyclodextrin (compare Fig. 5, A and C). Increasing the cholesterol concentration to 15 mol % induces the appearance of numerous circular-shaped domains. Further increase in cholesterol (up to 30 mol % relative to phospholipids) yields an increase in the area occupied by the fluid-ordered phase (Fig. 5C, red area in the GUVs containing 15–30 mol % cholesterol). Note that size and morphology of the fluid disordered domains become comparable with those observed in NPSM (Fig. 5A) upon addition of 8–10 mol % of cholesterol, which raises the cholesterol content to values in the same range as those in native pulmonary surfactant. From this experiment, we estimate that a cholesterol-to-phospholipid ratio of about 13–15 mol % triggers a phase change from gel/fluid to fluid ordered/fluid disordered phase coexistence. These experiments clearly demonstrate that the cholesterol concentration is a critical parameter in modulating the lateral structure of pulmonary surfactant membranes. Moreover, the cholesterol-dependent phase coexistence pattern reported here may explain why the phase coexistence pattern reported previously in GUVs composed of the bovine lipid extract surfactant BLES®, a clinical preparation, corresponds to gel/fluid phase coexistence (9). BLES® is almost devoid of cholesterol, and in this respect it is similar to NPSM at relatively high methyl-{beta}-cyclodextrin concentration.

One key question is whether the lateral structure of the surfactant material may affect its functional properties. Rapid spreading and adsorption of lung surfactant material to form surface-active films at the air-water interface is an important property that can be used to evaluate the proper function of a given surfactant. Fig. 6 presents spreading-at-interface {pi} -t isotherms of different surfactant suspensions directly deposited on top of open buffered subphases. Both native surfactant as purified from porcine lungs and its reconstituted organic fraction (lipids + SP-B + SP-C) have optimal adsorption activities, very rapidly producing equilibrium surface pressures around 45 mN/m (Fig. 6, A and B). Suspensions lacking proteins show much lower adsorption rates and do not reach surface pressures higher than 30 mN/m after 50 min, indicating the essential role of hydrophobic proteins to catalyze phospholipid interfacial transference. Most interestingly, spreading isotherms of suspensions prepared from the surfactant lipid fraction partially depleted of cholesterol are further impaired, suggesting that cholesterol-containing surfactant structures have somehow better interfacial dynamics. Spreading kinetics were always faster at 37 than at 25 °C, but the difference in the ability of the different samples to reach the highest surface pressures persisted, suggesting that such effect is because of intrinsic structural differences among the samples. Fig. 6C shows spreading kinetics of NPSM suspensions spread on top of subphases containing different concentrations of methyl-{beta}-cyclodextrin. Native surfactant spread rapidly at the first instance, independent of the amount of methyl-{beta}-cyclodextrin present in the subphase. However, the higher the amount of methyl-{beta}-cyclodextrin in the subphase (i.e. the higher amount of cholesterol extracted from the spreading surfactant), the lower the maximum pressure reached at the late spreading stages. Optimal insertion of phospholipids against high pressures seems therefore to require the action of surfactant-specialized structures such as the one described here when both cholesterol and hydrophobic proteins are present.



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  		FIG. 6.
Interfacial spreading {pi} -t isotherms of NPSM and its fractions. {pi} -t isotherms at 25 (A) and 37 °C (B) of NPSM (closed squares), its reconstituted organic extract containing all the lipids plus the hydrophobic proteins (open squares), its reconstituted lipid fraction without proteins (closed circles), and its reconstituted lipid fraction without proteins and partly depleted of cholesterol (10 mol % instead of 20%) (open circles). C, {pi} -t isotherms of NPSM spread at 25 °C on top of subphases containing different concentrations of methyl-{beta}-cyclodextrin (indicated in the figure, in mg/ml). All the experiments were done by spreading 3 µl of an aqueous suspension of the different materials (phospholipid concentration 10 mg/ml) on top of a 20-cm2 thermostated trough containing buffer (5 mM Tris, 150 mM NaCl, pH 7), with or without methyl-{beta}-cyclodextrin, as a subphase. The subphase was equilibrated for 4 min at the desired temperature before initiating sample spreading.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
CONCLUSIONS
 REFERENCES
 
The present study offers a novel view on the importance of cholesterol for the relationships in the structure-function of pulmonary surfactant, and suggests a rationale for the presence of a significant amount of this lipid species in the composition of all the known surfactants. In this paper we demonstrate that the presence of cholesterol promotes a defined lateral structure in surfactant bilayers at physiological temperatures. The particular lateral organization of lipids and proteins in surfactant membranes would be essential to support not only a rapid interfacial adsorption to equilibrium surface pressures (as shown in the Fig. 6), but for the formation of well defined surface-associated structures (such as that shown in the Fig. 2). This last scenario would be competent to support proper dynamic surface behavior when the surfactant material is subjected to rapid dynamic cycling.

Several studies have analyzed previously the effects of cholesterol on the surface activity of surfactant, mostly using model lipid mixtures. However, the conclusions about a potential role of cholesterol in surfactant function have been contradictory. Some studies have concluded that cholesterol has a general beneficial effect on surfactant interfacial adsorption, mostly due to a "fluidizing" effect on DPPC-enriched surfactant lipid systems (36). On the other hand, the surface behavior studies of cholesterol-containing monolayers using model lipid mixtures has come to the conclusion that cholesterol impairs the ability of any lipid mixture to reach and sustain very high surface pressures during compression (37–39). As a consequence of these studies, many of the clinical surfactants used today are depleted of cholesterol, which is considered as a sort of "contaminant" for a good surface activity. Our results suggest that last idea should be re-examined by using native material as model system.

The dependence of the lateral structure of surfactant membranes on cholesterol concentration provides a framework for the physiological meaning of rapid changes in the cholesterol-to-phospholipid ratio reported previously (11) in pulmonary surfactant in response to various physiological stimuli. For example, in homeothermic mammals, the cholesterol content changes with exercise, suggesting that changes in the mechanical properties of surfactant membranes are required to accommodate the changes in ventilation. The cholesterol content in surfactant also varies with the temperature of the environment in both heterothermic species (40) and in animals that enter torpor (41). The proportion of cholesterol correlates with differences in lung structure, both from a phylogenetic and an ontogenetic point of view (42). The amount of cholesterol could be finely tuned in the context of the presence, in given amounts, of other lipid species, such as saturated and unsaturated phospholipids, to provide a structural "scaffold" for surfactant membranes at precise conditions of temperature and breathing dynamics. Potential physiological mechanisms providing tight regulatory control of surfactant composition, especially regarding cholesterol/phospholipid ratio, should be investigated as they could be evolutionarily conserved to fit surfactant material properties to required performance. For example, compositional data reported for surfactant in the literature include remarkable differences in terms of cholesterol-to phospholipid ratio, both among species and among individuals. The possibility that such differences could originate, at least partly, from environmental effects should be explored. These ideas need still to be fully established in future experiments, for example by comparing different surfactant preparations extracted from particular animal species at different environmental conditions.

The thermotropic behavior of NPSM (Fig. 4) suggests that the composition of this material needs to be optimized to be at the "edge" of the liquid ordered/liquid disordered phase coexistence regime at physiological temperatures, which is likely to provide particular structural and dynamical properties for its mechanical function under particular environmental conditions. It is interesting to note that the phase transition event observed for NPSM is different from that observed for lipid mixtures displaying coexistence of two fluid phases (18, 21). In the artificial system the fluid ordered/fluid disordered phase pattern (that is characterized for the presence of circular domains) changes to a single homogeneous phase (the fluid disordered phase), an effect that is not observed in NPSM. The possible maintenance of a minor proportion of ordered-like phase at temperatures a few degrees higher than 38 °C, such as those reached in feverish states, as well as the correlation between the structural features and the functional properties of surfactant material remain to be established. For instance, Inoue et al. (43) showed that lung compliance was consistent with surfactant activity remaining intact above 42 °C. One could speculate that possible regulatory mechanisms might exist to compensate for environmental factors through modification of the relative proportion in surfactant of certain key species, with cholesterol a probable candidate.

Cholesterol has been reported to induce a compression-driven remixing behavior of coexisting phases in monolayers composed of pulmonary surfactant organic extracts (8, 44). Such behavior is probably related to the existence of a cholesterol-dependent critical miscibility pressure in the two-dimensional phase diagrams of simple model systems (45). A possible correlation between the compression-driven remixing and the temperature-induced melting of liquid ordered regions in surfactant bilayers observed in our experiments is difficult to attain because there is not a clear link between lateral pressure conditions in both systems (monolayers and bilayers). However, both phenomena are indicative of surfactant composition being, perhaps, evolutionarily tuned to support a highly dynamic behavior of the surfactant operative structures.

It has been proposed that the regions in pulmonary surfactant interfacial films accumulating unsaturated lipid species and hydrophobic proteins are involved in initiating monolayer-to-bilayer transitions upon compression (during lung deflation) and in forming structures that re-spread efficiently upon expansion (during inspiration) (1, 46). Similarly, we speculate that phase separation observed in surfactant membranes may be relevant to provide particular regions (with particular composition and lateral arrangement) where accumulated SP-B and SP-C may initiate bilayer-to-monolayer molecular transfer, a process that is essential in establishing the surface-active surfactant film in the lung. This last assumption is supported by the connection between the results of the spreading experiments (Fig. 6) and the particular lateral structure of NPSM at different cholesterol-to-phospholipid molar ratios. We propose that in order to show optimal spreading behavior, the surfactant must contain hydrophobic surfactant proteins while simultaneously having a lipid composition permitting the coexistence of fluid phases under physiological conditions. The role of the phase coexistence-containing structures in other surface-active or mechanical properties of surfactant still has to be explored in detail, but the present data indicate that composition of clinical surfactants designed to treat respiratory pathologies such as acute respiratory distress syndrome could still be largely improved.

Finally, our results also provide a clear example of the notion that specialized regions with particular lateral packing properties coexist in natural membranes and control specific molecular interactions in agreement with the raft hypothesis. The raft hypothesis has been widely spread in recent years (13, 14) and suggests that factors modulating lipid segregation and domain dynamics may indirectly regulate specific membrane-associated functions. There are remarkable aspects in this mechanism as we learned from our experiments with pulmonary surfactant membranes. First, the coexistence of defined membrane regions with particular lateral arrangements is triggered by the presence of a few key lipid species (cholesterol, DPPC instead of sphingomyelin, as in the raft hypothesis, and unsaturated phospholipids), independent of the compositional complexity of the material. This last aspect could possibly be connected with events at supramolecular levels such as the mechanical properties of the membrane. Second, the composition of a particular membrane region displaying a particular lateral structure (for example the liquid ordered or liquid disordered regions) would modulate specific molecular interactions in the plane of the membrane (as seen in Fig. 5 with the different distribution of the lipophilic probes between artificial and native membranes displaying the coexistence of two fluid phases), possibly being connected with events occurring at the molecular level. Most interestingly, our results show that the surfactant proteins are not participating substantially in triggering the particular lateral structure observed in this particular membrane. Even though this is a new observation in the framework of the raft hypothesis, additional experiments in other membrane systems are required to generalize this suggestion further.


   CONCLUSIONS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 CONCLUSIONS
REFERENCES
 
We have obtained unambiguous evidence that at physiological temperatures the lateral phase separation observed in native pulmonary surfactant membranes is entirely dependent on the concentration of a few key lipid species, with a particular involvement of cholesterol. Remarkably, this phenomenon is independent of the presence of surfactant proteins. Cholesterol extraction affects the spreading properties of the native material suggesting an interesting correlation between the lateral structure and one of the relevant physiological functions of surfactant. Additionally, the dramatic lateral structure transition observed at 37.5 °C underlines the critical properties associated with surfactant full composition under physiological conditions. Apparently, this material has been evolutionarily optimized to sustain a structure that is at the borderline of a lateral structure transition, likely contributing to essential levels of mechanical plasticity. We believe that our results point to an entirely new perspective on the establishment of structure-function relationships in pulmonary surfactant, which may significantly facilitate the search for new pulmonary surfactant materials effective in the treatment of pathologies such as acute respiratory distress syndrome. In addition, the relevance of our results goes beyond the pulmonary surfactant field, into the general field of biomembranes. According to our results, pulmonary surfactant could be one of the first membranous systems reported where the coexistence of specialized membrane domains exists as a structural basis for its function.


   FOOTNOTES
 
* This work was supported by Dirección General de Educación Superior e Investigaciones Científicas Grant BIO2003-09056 (to J. B. S. and J. P.-G.), Comunidad Autónoma de Madrid Grant 08.2/0054, and by Danish Natural Science Research Council Grant 21-03-0569 (to L. A. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

|| To whom correspondence should be addressed: MEMPHYS-Center for Biomembrane Physics, Dept. of Biochemistry and Molecular Biology, University of Southern Denmark, 5230 Odense M, Denmark. Tel.: 45-65-50-34-76; Fax: 45-66-15-87-60; E-mail: bagatolli{at}memphys.sdu.dk.

1 The abbreviations used are: DPPC, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine; AFM, atomic force microscopy; Bodipy-PC, 2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine; DiIC18, 1,1'-dioctadecyl-3,3,3',3' -tetramethylindocarbocyanine perchlorate; Me2SO, dimethyl sulfoxide; DOPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine; GUV, giant unilamellar vesicle; NPSM, native pulmonary surfactant material; SP, surfactant proteins; PC, phosphatidylcholine. Back


   ACKNOWLEDGMENTS
 
We thank Dr. I. Plasencia for technical assistance with protein and lipid analysis of surfactant fractions and Dr. Lars Duelund for the assistance with differential scanning calorimetry experiments. We also thank Dr. David Jameson (University of Hawaii, Manoa), Dr. Kevin M. W. Keough (Memorial University of Newfoundland), Dr. Ole Mouritsen (University of Southern Denmark), Dr. Felix Goñi (University of Basque Country), Dr. Enrico Gratton (University of Illinois, Urbana-Champaign), and Drs. Sandra Orgeig and Chris Daniels (University of Adelaide) for their useful comments and suggestions on a critical reading of the manuscript. MEMPHYS-Center for Biomembrane Physics is supported by The Danish National Research Foundation.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 

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