Lateral phase separation in interfacial films of pulmonary surfactant.

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

Distinct steps in the adsorption of pulmonary surfactant to an air-liquid interface

To investigate the mechanisms by which vesicles of pulmonary surfactant adsorb to an air-liquid interface, we measured the effect of different phospholipids and of their concentration both in the subphase and at the interface on this process. Adsorbing vesicles contained the hydrophobic surfactant proteins mixed with the following four sets of surfactant phospholipids that varied the content of anionic headgroups and mixed acyl chains independently: the complete set of purified phospholipids (PPL) from calf surfactant; modified PPL (mPPL) from which the anionic phospholipids were removed; a mixture of dipalmitoyl phosphatidylcholine (DPPC) and dipalmitoyl phosphatidylglycerol (DPPG) (9:1, mol:mol); and DPPC alone. The initial reduction in surface tension depended strongly on the anionic phospholipids and the subphase concentration. The acyl groups had no effect. Adsorption beyond the initial stage depended more on the mixed acyl groups, became increasingly independent of subphase concentration, and was determined instead by the interfacial concentration of the surface film. The different constituents produced the same effects in vesicles adsorbing to a clean interface or in a preexisting film to which vesicles of SP:DPPC adsorbed. Adsorption for vesicles of SP:PPL adsorbing to DPPC or of SP:DPPC to PPL above a certain threshold surface concentration followed exactly the same isotherm. Our results fit best with a two-step model for adsorption. The anionic phospholipids first promote the initial juxtaposition of vesicles to the interface. Compounds with mixed acyl constituents at the point of contact between vesicle and interface then facilitate fusion with the surface.     

Discrepancy between phase behavior of lung surfactant phospholipids and the classical model of surfactant function

The studies reported here used fluorescence microscopy and Brewster angle microscopy to test the classical model of how pulmonary surfactant forms films that are metastable at high surface pressures in the lungs. The model predicts that the functional film is liquid-condensed (LC) and greatly enriched in dipalmitoyl phosphatidylcholine (DPPC). Both microscopic methods show that, in monolayers containing the complete set of phospholipids from calf surfactant, an expanded phase persists in coexistence with condensed domains at surface pressures approaching 70 mN/m. Constituents collapsed from the interface above 45 mN/m, but the relative area of the two phases changed little, and the LC phase never occupied more than 30% of the interface. Calculations based on these findings and on isotherms obtained on the continuous interface of a captive bubble estimated that collapse of other constituents increased the mol fraction of DPPC to no higher than 0.37. We conclude that monolayers containing the complete set of phospholipids achieve high surface pressures without forming a homogeneous LC film and with a mixed composition that falls far short of the nearly pure DPPC predicted previously. These findings contradict the classical model.     

Pulmonary Surfactant Model Systems Catch the Specific Interaction of an Amphiphilic Peptide with Anionic Phospholipid

Interfacial behavior was studied in pulmonary surfactant model systems containing an amphiphilic α-helical peptide (Hel 13-5), which consists of 13 hydrophobic and five hydrophilic amino acid residues. Fully saturated phospholipids of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG) were utilized to understand specific interactions between anionic DPPG and cationic Hel 13-5 for pulmonary functions. Surface pressure (π)-molecular area (A) and surface potential (ΔV)-A isotherms of DPPG/Hel 13-5 and DPPC/DPPG (4:1, mol/mol)/Hel 13-5 preparations were measured to obtain basic information on the phase behavior under compression and expansion processes. The interaction leads to a variation in squeeze-out surface pressures against a mole fraction of Hel 13-5, where Hel 13-5 is eliminated from the surface on compression. The phase behavior was visualized by means of Brewster angle microscopy, fluorescence microscopy, and atomic force microscopy. At low surface pressures, the formation of differently ordered domains in size and shape is induced by electrostatic interactions. The domains independently grow upon compression to high surface pressures, especially in the DPPG/Hel 13-5 system. Under the further compression process, protrusion masses are formed in AFM images in the vicinity of squeeze-out pressures. The protrusion masses, which are attributed to the squeezed-out Hel 13-5, grow larger in lateral size with increasing DPPG content in phospholipid compositions. During subsequent expansion up to 35 mN m⁻¹, the protrusions retain their height and lateral diameter for the DPPG/Hel 13-5 system, whereas the protrusions become smaller for the DPPC/Hel 13-5 and DPPC/DPPG/Hel 13-5 systems due to a reentrance of the ejected Hel 13-5 into the surface. In this work we detected for the first time, to our knowledge, a remarkably large hysteresis loop for cyclic ΔV-A isotherms of the binary DPPG/Hel 13-5 preparation. This exciting phenomenon suggests that the specific interaction triggers two completely independent processes for Hel 13-5 during repeated compression and expansion: 1), squeezing-out into the subsolution; and 2), and close packing as a monolayer with DPPG at the interface. These characteristic processes are also strongly supported by atomic force microscopy observations. The data presented here provide complementary information on the mechanism and importance of the specific interaction between the phosphatidylglycerol headgroup and the polarized moiety of native surfactant protein B for biophysical functions of pulmonary surfactants.     

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.     

Quantitative Brewster angle microscopy of the surface film of human broncho-alveolar lavage fluid

The morphology, thickness and surface pressure of the surfactant film of broncho-alveoalar lavage (BAL) fluid from patients with sarcoidosis were investigated during spontaneous adsorption of the BAL's surface active material at the air/aqueous buffer interface at 37 degrees C. The biochemical parameters of the BAL fluid determined were protein (Lowry), total phospholipids (from phosphate after ashing) and the individual phospholipids (HPLC). During the spontaneous adsorption of the pulmonary surfactant the surface pressure increased from initially 26 mN/m to 44 mN/m in the equilibrium state. Simultaneously to the increase of the surface pressure, a continuous increase of the reflectivity signal was observed by quantitative Brewster angle microscopy (BAM). The film thickness is calculated from the reflectivity values using an optical model. The effect of the uncertainty of the refractive index, which has to be estimated, is discussed. The BAM images show the inhomogeneous nature of the surfactant film with three distinct phases of different reflectivity, even at relatively low surface pressures. For the brightest phase, the thickness amounts to approximately 12 nm in the equilibrium state of adsorption. This suggests a multilamellar structure. Additionally, we found visual evidence for an adsorption mechanism involving the spreading of vesicles at the interface, in agreement with published results. Differences in the morphology and thickness of the pulmonary surfactant film reported in the literature are obviously due to the varying experimental conditions and materials. We think that the experimental conditions chosen in our study provide a more realistic view of the structure in the lungs in vivo.     

Reversible Formation of Nanodomains in Monolayers of DPPC Studied by Kinetic Modeling

Dipalmitoylphosphatidylcholine (DPPC) is the most abundant component in pulmonary surfactants and is believed to be responsible for maintaining low surface tension in alveoli during breathing. In this work, a kinetic model is introduced that describes the phase separation in DPPC films that produces the liquid-condensed (LC) and liquid-expanded (LE) fractions, which differ according to the area density of DPPC. The phase separation in an initially homogeneous film has been investigated numerically. Furthermore, explicit simulations of periodic compression-expansion cycles are reported. In this process, a moderate change of the surface area resulted in a dramatic change in the total amount of LC fraction, as well as in the surface morphology. Depending on the extent of the film's compression, the simulated surface morphologies comprised individual nanosized LC domains embedded in the LE fraction, interconnected networks of such domains, or continuous LC films with nanopores. Equilibration of the total area of the LC nanodomains occurred over a few milliseconds, indicating that the rate of the LE-LC phase transformation is sufficient for maintaining low surface tension during breathing, and that nanoscale LC domains are likely to play a major role in this process. Unlike the total content of the LC fraction, which stabilized quickly, the average size of LC nanodomains showed a tendency to increase slowly, at a rate determined by the diffusivity of DPPC. The computed average domain size seems to be compatible with published experiments for DPPC films. The numeric results also elucidate the distinction between thermodynamically determined and kinetically limited structural properties during phase separation in the major structure-forming component of pulmonary surfactants.     

Interactions of pulmonary surfactant protein A with phospholipid monolayers change with pH.

The interaction of pulmonary surfactant protein A (SP-A) labeled with Texas Red (TR-SP-A) with monolayers containing zwitterionic and acidic phospholipids has been studied at pH 7.4 and 4.5 using epifluorescence microscopy. At pH 7.4, TR-SP-A expanded the pi-A isotherms of film of dipalmitoylphosphatidylcholine (DPPC). It interacted at high concentration at the edges of condensed-expanded phase domains, and distributed evenly at lower concentration into the fluid phase with increasing pressure. At pH 4.5, TR-SP-A expanded DPPC monolayers to a slightly lower extent than at pH 7.4. It interacted primarily at the phase boundaries but it did not distribute into the fluid phase with increasing pressure. Films of DPPC/dipalmitoylphosphatidylglycerol (DPPG) 7:3 mol/mol were somewhat expanded by TR-SP-A at pH 7.4. The protein was distributed in aggregates only at the condensed-expanded phase boundaries at all surface pressures. At pH 4.5 TR-SP-A caused no expansion of the pi-A isotherm of DPPC/DPPG, but its fluorescence was relatively homogeneously distributed throughout the expanded phase at all pressures studied. These observations can be explained by a combination of factors including the preference for SP-A aggregates to enter monolayers at packing dislocations and their disaggregation in the presence of lipid under increasing pressure, together with the influence of pH on the aggregation state of SP-A and the interaction of SP-A with zwitterionic and acidic lipid.     

Metal nanoparticle pollutants interfere with pulmonary surfactant function in vitro

Reported associations between air pollution and pulmonary and cardiovascular diseases prompted studies on the effects of gold nanoparticles (Au NP) on pulmonary surfactant function. Low levels (3.7 mol % Au/lipid, 0.98% wt/wt) markedly inhibited adsorption of a semisynthetic pulmonary surfactant (dipalmitoyl-phosphatidylcholine (DPPC)/palmitoyl-oleoyl-phosphatidylglycerol/surfactant protein B (SP-B); 70:30:1 wt %). Au NP also impeded the surfactant's ability to reduce surface tension (γ) to low levels during film compression and to respread during film expansion. Transmission electron microscopy showed that Au NP generated by a seed-growth method were spherical with diameters of ∼15 nm. Including palmitoyl-oleoyl-phosphatidylglycerol appeared to coat the NP with at least one lipid bilayer but did not affect NP shape or size. Similar overall observations occurred with dimyristoyl phosphatidylglycerol. Dipalmitoyl-phosphatidylglycerol was less effective in NP capping, although similar sized NP were formed. Including SP-B (1% wt/wt) appears to induce the formation of elongated strands of interacting threads with the fluid phosphatidylglycerols (PG). Including DPPC resulted in formation of aggregated, less spherical NP with a larger size distribution. With DPPC, strand formation due to SP-B was not observed. Agarose gel electrophoresis studies demonstrated that the aggregation induced by SP-B blocked migration of PG-coated NP. Migration was also influenced by the fluidity of the PGs. It is concluded that Au NP can interact with and sequester pulmonary surfactant phospholipids and, if inhaled from the atmosphere, could impede pulmonary surfactant function in the lung.     

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.     

Atomic force microscopy studies of functional and dysfunctional pulmonary surfactant films. I. Micro- and nanostructures of functional pulmonary surfactant films and the effect of SP-A

Monolayers of a functional pulmonary surfactant (PS) can reach very low surface tensions well below their equilibrium value. The mechanism by which PS monolayers reach such low surface tensions and maintain film stability remains unknown. As shown previously by fluorescence microscopy, phospholipid phase transition and separation seem to be important for the normal biophysical properties of PS. This work studied phospholipid phase transitions and separations in monolayers of bovine lipid extract surfactant using atomic force microscopy. Atomic force microscopy showed phospholipid phase separation on film compression and a monolayer-to-multilayer transition at surface pressure 40-50 mN/m. The tilted-condensed phase consisted of domains not only on the micrometer scale, as detected previously by fluorescence microscopy, but also on the nanometer scale, which is below the resolution limits of conventional optical methods. The nanodomains were embedded uniformly within the liquid-expanded phase. On compression, the microdomains broke up into nanodomains, thereby appearing to contribute to tilted-condensed and liquid-expanded phase remixing. Addition of surfactant protein A altered primarily the nanodomains and promoted the formation of multilayers. We conclude that the nanodomains play a predominant role in affecting the biophysical properties of PS monolayers and the monolayer-to-multilayer transition.     

Distribution of the surfactant-associated protein C within a lung surfactant model film investigated by near-field optical microscopy

Lung surfactant films at the air/water interface exhibit the particularity that surfactant molecules are expelled from the surface monolayer into a surface associated multilamellar phase during compression. They are able to re-enter the surface film during the following expansion. The underlying mechanism for this behavior is not fully understood yet. However, an important role is ascribed to the surfactant-associated protein C (SP-C). Here, we studied a model lung surfactant, consisting of dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylglycerol (DPPG), and SP-C, by means of scanning near-field optical microscopy (SNOM). Attaching a fluorescent dye to the protein allowed the localization of its lateral distribution at various surface pressures with high resolution. At an early stage of compression, the film appears demixed into a pure lipid phase and a protein-enriched phase. Within the latter phase, protein aggregations are revealed. They show a uniform density, having three times the fluorescence intensity of their surroundings. Across the phase boundary between the lipid phase and the protein-rich phase, there is a protein density gradient rather than an abrupt border. When the film is highly compressed, we observe the formation of multilamellar structures that are fluorescent. They are often surrounded by a slightly fluorescent monolayer. The fluorescence of the multilayer stacks (i. e., the protein content per unit area) is proportional to the height of the stacks.     

Fluorescently labeled pulmonary surfactant protein C in spread phospholipid monolayers.

Pulmonary surfactant, a lipid-protein complex, secreted into the fluid lining of lungs prevents alveolar collapse at low lung volumes. Pulmonary surfactant protein C (SP-C), an acylated, hydrophobic, alpha-helical peptide, enhances the surface activity of pulmonary surfactant lipids. Fluorescein-labeled SP-C (F-SP-C) (3, 6, 12 wt%) in dipalmitoylphosphatidylcholine (DPPC), and DPPC:dipalmitoylphosphatidylglycerol (DPPG) [DPPC:DPPG 7:3 mol/mol] in spread monolayers was studied by epifluorescence microscopy. Mass spectometry of F-SP-C indicated that the protein is partially deacylated and labeled with 1 mol fluorescein/1 mol protein. The protein partitioned into the fluid, or liquid expanded, phase. Increasing amounts of F-SP-C in DPPC or DPPC:DPPG monolayers decreased the size and total amounts of the condensed phase at all surface pressures. Calcium (1.6 mM) increased the amount of the condensed phase in monolayers of DPPC:DPPG but not of DPPC alone, and such monolayers were also perturbed by F-SP-C. The study indicates that SP-C perturbs the packing of neutral and anionic phospholipid monolayers even when the latter systems are condensed by calcium, indicating that interactions between SP-C and the lipids are predominantly hydrophobic in nature.     

Phase behaviour and morphology of binary mixtures of DPPC with stearonitrile, stearic acid, and octadecanol at the air-water interface

The behaviour of dipalmitoylphosphatidylcholine (DPPC), mixed with stearonitrile (SN), was investigated at the air-water interface by surface pressure-area (pi-A) measurements and by direct visualisation of monolayers by Brewster angle microscopy (BAM). The pi-A-X diagram of system DPPC/SN was compared with the corresponding diagrams of systems DPPC/stearic acid (SA) and DPPC/octadecanol (OD) at 20 degrees C. Monolayers of the three systems reach the closest packing of alkyl chains in the 0.4-0.6 range of XDPPC. Thermodynamic analysis indicates miscibility in the three binary systems with negative deviations from the ideal behaviour. Morphological features of system DPPC/SN change significantly with XDPPC and temperature in the range 10-30 degrees C. At 10 and 20 degrees C mixed monolayers form condensed states from low pi all over the composition range. At 30 degrees C, the liquid-expanded (LE)--liquid-condensed (LC) phase transition occurs at increasing pi with XDPPC. The shape and size of condensed domains change with XDPPC and pi. Contrarily to the behaviour of pure components, mixed monolayers of DPPC/SN exhibit orientational order in the 0.2-0.6 mol fraction range of DPPC. BAM observation confirmed the partial miscibility indicated by GE data in a limited range of compositions at 30 degrees C.     

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.     

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     

Effect of Pulmonary Surfactant Protein SP-B on the Micro- and Nanostructure of Phospholipid Films

Monolayers of dipalmitoylphosphatidylcholine (DPPC) and DPPC/dipalmitoylphosphatidylglycerol (DPPG) (7:3, w/w) in the absence or in the presence of 2, 5, 10, or 20 weight percent of porcine surfactant protein SP-B were spread at the air-liquid interface of a surface balance, compressed up to surface pressures in the liquid-expanded/liquid-condensed (LE-LC) plateau of the isotherm, transferred onto mica supports, and analyzed by scanning force microscopy. In the absence of protein, the films showed micrometer-sized condensed domains with morphology and size that were analogous to those observed in situ at the air-liquid interface by epifluorescence microscopy. Scanning force microscopy permits examination of the coexisting phases at a higher resolution than previously achieved with fluorescent microscopy. Both LE and LC regions of DPPC films were heterogeneous in nature. LC microdomains contained numerous expanded-like islands whereas regions apparently liquid-expanded were covered by a condensed-like framework of interconnected nanodomains. Presence of increasing amounts of pulmonary surfactant protein SP-B affected the distribution of the LE and LC regions of DPPC and DPPC/DPPG films both at the microscopic and the nanoscopic level. The condensed microdomains became more numerous but their size decreased, resulting in an overall reduction of the amount of total LC phase in both DPPC and DPPC/DPPG films. At the nanoscopic level, SP-B also caused a marked reduction of the size of the condensed-like nanodomains in the LE phase and an increase in the length of the LE/LC interface. SP-B promotes a fine nanoscopic framework of lipid and lipid-protein nanodomains that is associated with a substantial mechanical resistance to film deformation and rupture as observed during film transference and manipulation. The effect of SP-B on the nanoscopic structure of the lipid films was greater in DPPC/DPPG than in pure DPPC films, indicating additional contributions of electrostatic lipid-protein interactions. The alterations of the nanoscopic structures of phospholipid films by SP-B provide the structural framework for the protein simultaneously sustaining structural stability as well as dynamical flexibility in surfactant films at the extreme conditions imposed by the respiratory mechanics. SP-B also formed segregated two-dimensional clusters that were associated with the boundaries between LC microdomains and the LE regions of DPPC and DPPC/DPPG films. The presence of these clusters at protein-to-lipid proportions above 2% by weight suggests that the concentration of SP-B in the surfactant lipid-protein complexes may be close to the solubility limit of the protein in the lipid films.     

Analysis of lung surfactant model systems with time-of-flight secondary ion mass spectrometry

An often-used model lung surfactant containing dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylglycerol (DPPG), and the surfactant protein C (SP-C) was analyzed as Langmuir-Blodgett film by spatially resolved time-of-flight secondary ion mass spectrometry (TOF-SIMS) to directly visualize the formation and composition of domains. Binary lipid and lipid/SP-C systems were probed for comparison. TOF-SIMS spectra revealed positive secondary ions (SI) characteristic for DPPC and SP-C, but not for DPPG. SI mapping results in images with domain structures in DPPC/DPPG and DPPG/SP-C, but not in DPPC/SP-C films. We are able to distinguish between the fluid and condensed areas probably due to a matrix effect. These findings correspond with other imaging techniques, fluorescence light microscopy (FLM), scanning force microscopy (SFM), and silver decoration. The ternary mixture DPPC/DPPG/SP-C transferred from the collapse region exhibited SP-C-rich domains surrounding pure lipid areas. The results obtained are in full accordance with our earlier SFM picture of layered protrusions that serve as a compressed reservoir for surfactant material during expansion. Our study demonstrates once more that SP-C plays a unique role in the respiration process.     

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.     

Multilayer formation upon compression of surfactant monolayers depends on protein concentration as well as lipid composition. An atomic force microscopy study

The determinants for the formation of multilayers upon compression of surfactant monolayers were investigated by compressing films, beyond the squeeze-out plateau, to a surface tension of 22 millinewtons/m. Atomic force microscopy was used to visualize the topography of lipid films containing varying amounts of native surfactant protein B (SP-B). These films were compared with films containing synthetic peptides based on the N terminus of human SP-B: monomeric mSP-B-(1-25) or dimeric dSP-B-(1-25). The formation of typical hexagonal network structures as well as the height of protrusions were shown to depend on the concentration of SP-B. Protrusions of bilayer height were formed from physiologically relevant concentrations of 0.2-0.4 mol % (4.5-8.5 wt %) SP-B upwards. Much higher concentrations of SP-B-(1-25) peptides were needed to obtain network structures, and protrusion heights were not equal to those found for films with native SP-B. A striking observation was that while protrusions formed in films of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)/1,2-dipalmitoyl-sn-glycero-3-(phospho-rac-(1-glycerol)) (DPPG) (80/20) had single bilayer thickness, those formed in DPPC/1-palmitoyl-2-oleoyl-sn-glycero-3-(phospho-rac-(1-glycerol)) (80/20) had various heights of multilayers, whereas those seen in DPPC/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/DPPG (60/20/20) were mainly of bilayer height. For the first time direct observations by atomic force microscopy show (i) that a certain minimal concentration of SP-B is required for the formation of layered protrusions upon film compression, (ii) that protrusion height depends on whether the phospholipids contain an unsaturated fatty acyl chain, and (iii) that protrusion height also depends on whether the unsaturated acyl chain is present in phosphatidylcholine or in phosphatidylglycerol.