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.     

Differential effects of surfactant protein A on regional organization of phospholipid monolayers containing surfactant protein B or C

Epifluorescence microscopy combined with a surface balance was used to study monolayers of dipalmitoylphosphatidylcholine (DPPC)/egg phosphatidylglycerol (PG) (8:2, mol/mol) plus 17 wt % SP-B or SP-C spread on subphases containing SP-A in the presence or absence of 5 mM Ca(2+). Independently of the presence of Ca(2+) in the subphase, SP-A at a bulk concentration of 0.68 microg/ml adsorbed into the spread monolayers and caused an increase in the molecular areas in the films. Films of DPPC/PG formed on SP-A solutions showed a pressure-dependent coexistence of liquid-condensed (LC) and liquid-expanded (LE) phases. Apart from these surface phases, a probe-excluding phase, likely enriched in SP-A, was seen in the films between 7 mN/m < or = pi < or = 20 mN/m. In monolayers of SP-B/(DPPC/PG) spread on SP-A, regardless of the presence of calcium ions, large clusters of a probe-excluding phase, different from probe-excluding lipid LC phase, appeared and segregated from the LE phase at near-zero surface pressures and coexisted with the conventional LE and LC phases up to approximately 35 mN/m. Varying the levels of either SP-A or SP-B in films of SP-B/SP-A/(DPPC/PG) revealed that the formation of the probe-excluding clusters distinctive for the quaternary films was influenced by the two proteins. Concanavalin A in the subphase could not replace SP-A in its ability to modulate the textures of films of SP-B/(DPPC/PG). In films of SP-C/SP-A/(DPPC/PG), in the absence of calcium, regions consisting of a probe-excluding phase, likely enriched in SP-A, were detected at surface pressures between 2 mN/m and 20 mN/m in addition to the lipid LE and LC phases. Ca(2+) in the subphase appeared to disperse this phase into tiny probe-excluding particles, likely comprising Ca(2+)-aggregated SP-A. Despite their strikingly different morphologies, the films of DPPC/PG that contained combinations of SP-B/SP-A or SP-C/SP-A displayed similar distributions of LC and LE phases with LC regions occupying a maximum of 20% of the total monolayer area. Combining SP-A and SP-B reorganized the morphology of monolayers composed of DPPC and PG in a Ca(2+)-independent manner that led to the formation of a separate potentially protein-rich phase in the films.     

Adsorption of pulmonary surfactant protein SP-A to monolayers of phospholipids containing hydrophobic surfactant protein SP-B or SP-C: potential differential role for tertiary interaction of lipids, hydrophobic proteins, and SP-A

Surface balance techniques were used to study the interactions of surfactant protein SP-A with monolayers of surfactant components preformed at the air-water interface. SP-A adsorption into the monolayers was followed by monitoring the increase in the surface pressure Deltapi after injection of SP-A beneath the films. Monolayers of dipalmitoylphosphatidylcholine (DPPC):egg phosphatidylglycerol (PG) (8:2, mol/mol) spread at initial surface pressure pi(i) = 5 mN/m did not promote the adsorption of SP-A at a subphase concentration of 0.68 microg/mL as compared to its adsorption to the monolayer-free surface. Surfactant proteins, SP-B or SP-C, when present in the films of DPPC:PG spread at pi(i) = 5 mN/m, enhanced the incorporation of SP-A in the monolayers to a similar extent; the Deltapi values being dependent on the levels of SP-B or SP-C, 3-17 wt %, in the lipid films. Calcium in the subphase did not affect the intrinsic surface activity of SP-A but reduced the Deltapi values produced by the adsorption of the protein to all the preformed films independently of their compositions and charges. The divalent ions likely modified the interaction of SP-A with the monolayers through their effects on the conformation, self-association, and charge state of SP-A. Values of Deltapi produced by adsorption of SP-A to the films of DPPC:PG with or without SP-B or SP-C were a function of the initial surface pressure of the films, pi(i). In the range of pressures 5 相似文献   

Surfactant protein A forms extensive lattice-like structures on 1,2-dipalmitoylphosphatidylcholine/rough-lipopolysaccharide-mixed monolayers

Due to the inhalation of airborne particles containing bacterial lipopolysaccharide (LPS), these molecules might incorporate into the 1,2-dipalmitoylphosphatidylcholine (DPPC)-rich monolayer and interact with surfactant protein A (SP-A), the major surfactant protein component involved in host defense. In this study, epifluorescence microscopy combined with a surface balance was used to examine the interaction of SP-A with mixed monolayers of DPPC/rough LPS (Re-LPS). Binary monolayers of Re-LPS plus DPPC showed negative deviations from ideal behavior of the mean areas in the films consistent with partial miscibility and attractive interaction between the lipids. This interaction resulted in rearrangement and reduction of the size of DPPC-rich solid domains in DPPC/Re-LPS monolayers. The adsorption of SP-A to these monolayers caused expansion in the lipid molecular areas. SP-A interacted strongly with Re-LPS and promoted the formation of DPPC-rich solid domains. Fluorescently labeled Texas red-SP-A accumulated at the fluid-solid boundary regions and formed networks of interconnected filaments in the fluid phase of DPPC/Re-LPS monolayers in a Ca(2+)-independent manner. These lattice-like structures were also observed when TR-SP-A interacted with lipid A monolayers. These novel results deepen our understanding of the specific interaction of SP-A with the lipid A moiety of bacterial LPS.     

Epifluorescence microscopic studies of monolayers containing mixtures of dioleoyl- and dipalmitoylphosphatidylcholines.

Monolayers of dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine (DOPC), and some mixtures of these lipids were investigated using an epifluorescence microscopic surface balance. Monolayers were visualized at 23 +/- 1 degree C through the fluorescence of 1 mol% of two different fluorescent probes, 1-palmitoyl-2-(12-[(7-nitro-2-1,3-benzoxadizole-4- yl)amino]dodecanoyl)phosphatidylcholine (NBD-PC), which partitions into the liquid expanded (LE) or disordered lipid phase and 3,3'-dioctadecyloxacarbocyanine perchlorate (DiO-C18), which preferentially associates with the liquid condensed (LC) phase or lipid with ordered chains. LC domains were observed in pure DPPC monolayers at relatively low surface pressures (pi), and these domains grew with increasing surface pressure. Only liquid expanded phase was observed in pure DOPC monolayers up to the point of monolayer collapse. In monolayers containing 29:70:1, 49:50:1, and 69:30:1 (mol/mol/mol) of DPPC:DOPC:probe the domains of LC phase were smaller than those seen in DPPC monolayers at equivalent surface pressures. Quantitative analysis of the visual fields shown by the mixed monolayers showed a distribution of sizes of condensed domains at any given pi. At pi = 30 mN m-1, liquid-expanded, or fluid, regions occupied more than 70% of the total monolayer area in all three mixtures studied, whereas DPPC monolayers were more than 75% condensed or solid at that pressure. For monolayers of DPPC:DOPC:NBD-PC 49:50:1 and 69:30:1 the average domain size and the percentage of the total area covered with LC, or rigid, areas increased to a maximum at pi around 35 mN m-1 followed by a decrease at higher pi.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pulmonary surfactant protein A interacts with gel-like regions in monolayers of pulmonary surfactant lipid extract

Epifluorescence microscopy was used to investigate the interaction of pulmonary surfactant protein A (SP-A) with spread monolayers of porcine surfactant lipid extract (PSLE) containing 1 mol % fluorescent probe (NBD-PC) spread on a saline subphase (145 mM NaCl, 5 mM Tris-HCl, pH 6.9) containing 0, 0.13, or 0.16 microg/ml SP-A and 0, 1.64, or 5 mM CaCl(2). In the absence of SP-A, no differences were noted in PSLE monolayers in the absence or presence of Ca(2+). Circular probe-excluded (dark) domains were observed against a fluorescent background at low surface pressures (pi approximately 5 mN/m) and the domains grew in size with increasing pi. Above 25 mN/m, the domain size decreased with increasing pi. The amount of observable dark phase was maximal at 18% of the total film area at pi approximately 25 mN/m, then decreased to approximately 3% at pi approximately 40 mN/m. The addition of 0.16 microg/ml SP-A with 0 or 1.64 mM Ca(2+) in the subphase caused an aggregation of dark domains into a loose network, and the total amount of dark phase was increased to approximately 25% between pi of 10-28 mN/m. Monolayer features in the presence of 5 mM Ca(2+) and SP-A were not substantially different from those spread in the absence of SP-A, likely due to a self-association and aggregation of SP-A in the presence of higher concentrations of Ca(2+). PSLE films were spread on a subphase containing 0.16 microg/ml SP-A with covalently bound Texas Red (TR-SP-A). In the absence of Ca(2+), TR-SP-A associated with the reorganized dark phase (as seen with the lipid probe). The presence of 5 mM Ca(2+) resulted in an appearance of TR-SP-A in the fluid phase and of aggregates at the fluid/gel phase boundaries of the monolayers. This study suggests that SP-A associates with PSLE monolayers, particularly with condensed or solid phase lipid, and results in some reorganization of rigid phase lipid in surfactant monolayers.     

Interactions of pulmonary surfactant protein SP-A with monolayers of dipalmitoylphosphatidylcholine and cholesterol: roles of SP-A domains

Pulmonary surfactant protein A (SP-A) is an oligomeric glycoprotein that binds dipalmitoylphosphatidylcholine (DPPC). Interactions of rat SP-A and recombinant SP-As with pure and binary monolayers of DPPC and cholesterol were studied using a rhomboid surface balance at 37 degrees C. A marked inflection at equilibrium surface tension (23 mN/m) in surface tension-area isotherm of a pure DPPC film was abolished by rat SP-A. The inflection was decreased and shifted to 18 mN/m with wild-type recombinant SP-A (SP-Ahyp). Both rat SP-A and SP-Ahyp decreased surface area reduction required for pure DPPC films to reach near zero surface tension from 30 to 25%. SP-Ahyp, E195Q,R197D, mutated in carbohydrate recognition domain (CRD) known to be essential for SP-A-vesicle interactions, conveyed a detrimental effect on DPPC surface activity. SP-ADeltaG8-P80, with deletion of collagen-like domain, had little effect. Both SP-Ahyp, C6S (Ser substitution for Cys6) and SP-Ahyp,DeltaN1-A7 (N-terminal segment deletion) which appear mainly as monomers on non-reducing SDS-PAGE analysis, increased required surface area reduction for minimal surface tension. All SP-As reduced collapse surface tension of a pure cholesterol film from 27 to 23 mN/m in the presence of Ca2+. When mixed films were formed by successive spreading of DPPC/SP-A/cholesterol, rat SP-A, SP-Ahyp, or SP-ADeltaG8-P80 blocked the interaction of cholesterol with DPPC; SP-Ahyp,E195Q,R197D could not impede the interaction; SP-Ahyp,C6S or SP-Ahyp,DeltaN1-A7 only partially blocked the interaction, and cholesterol appeared to stabilize SP-Ahyp,C6S-DPPC association. These results demonstrate the importance of CRD and N-terminal dependent oligomerization in SP-A-phospholipid associations. The findings further indicate that SP-A-cholesterol interactions differ from SP-A-DPPC interactions and may be nonspecific.     

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.     

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.     

Anaesthetic — phospholipid interaction

Binding of the positively charged drug chlorpromazine to phospholipid monolayers was investigated. A preferential uptake was observed near the phase transtion of the corresponding lipid. Cholesterol considerably diminishes the chlorpromazine uptake, again particularly near a lipid phase transition. The binding properties depend on the chlorpromazine concentration in the subphase. A critical concentration is 5·10^-5M, where higher uptake occurs in the liquid condensed than in the liquid expanded state of the monolayer at pressures of about 10 mN/m. Dipalmitoylphosphatidylcholine monolayers spread on a subphase containing chlorpromazine are comparable to monolayers at higher temperature but in the absence of chlorpromazine. These data are in agreement with previous fluorescence and electron paramagnetic resonance experiments on lipid bilayer membranes (Luxnat and Galla 1986).Abbreviations CPZ chlorpromazine - DPPC dipalmitoylphosphatidylcholine - DMPC dimyristoylphosphatidylcholine - LE liquid expanded - LC liquid condensed     

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.     

Formation of three-dimensional protein-lipid aggregates in monolayer films induced by surfactant protein B

This study focuses on the structural organization of surfactant protein B (SP-B) containing lipid monolayers. The artificial system is composed of the saturated phospholipids dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG) in a molar ratio of 4:1 with 0.2 mol% SP-B. The different "squeeze-out" structures of SP-B were visualized by scanning probe microscopy and compared with structures formed by SP-C. Particularly, the morphology and material properties of mixed monolayers containing 0.2 mol% SP-B in a wide pressure range of 10 to 54 mN/m were investigated revealing that filamentous domain boundaries occur at intermediate surface pressure (15-30 mN/m), while disc-like protrusions prevail at elevated pressure (50-54 mN/m). In contrast, SP-C containing lipid monolayers exhibit large flat protrusions composed of stacked bilayers in the plateau region (app. 52 mN/m) of the pressure-area isotherm. By using different scanning probe techniques (lateral force microscopy, force modulation, phase imaging) it was shown that SP-B is dissolved in the liquid expanded rather than in the liquid condensed phase of the monolayer. Although artificial, the investigation of this system contributes to further understanding of the function of lung surfactant in the alveolus.     

Thermal stability and DPPC/Ca2+ interactions of pulmonary surfactant SP-A from bulk-phase and monolayer IR spectroscopy.

Surfactant protein A (SP-A), the most abundant pulmonary surfactant protein, is implicated in multiple biological functions including surfactant homeostasis, biophysical activity, and host defense. SP-A forms ternary complexes with lipids and Ca2+ which are important for protein function. The current study uses infrared (IR) transmission spectroscopy to investigate the bulk-phase interaction between SP-A, 1,2-dipalmitoylphosphatidylcholine (DPPC), and Ca2+ ions along with IR reflection-absorption spectroscopy (IRRAS) to examine protein secondary structure and lipid orientational order in monolayer films in situ at the air/water interface. The amide I contour of SP-A reveals two features at 1653 and 1636 cm(-1) arising from the collagen-like domain and a broad feature at 1645 cm(-1) suggested to arise from the carbohydrate recognition domain (CRD). SP-A secondary structure is unchanged in lipid monolayers. Thermal denaturation of SP-A in the presence of either DPPC or Ca2+ ion reveals a sequence of events involving the initial melting of the collagen-like region, followed by formation of intermolecular extended forms. Interestingly, these spectral changes were inhibited in the ternary system, showing that the combined presence of both DPPC and Ca2+ confers a remarkable thermal stability upon SP-A. The ternary interaction was revealed by the enhanced intensity of the asymmetric carboxylate stretching vibration. The IRRAS measurements indicated that incorporation of SP-A into preformed DPPC monolayers at a surface pressure of 10 mN/m induced a decrease in the average acyl chain tilt angle from 35 degrees to 28 degrees. In contrast, little change in chain tilt was observed at surface pressures of 25 or 40 mN/m. These results are consistent with and extend the fluorescence microscopy studies of Keough and co-workers [Ruano, M. L. F., et al. (1998) Biophys. J. 74, 1101-1109] in which SP-A was suggested to accumulate at the liquid-expanded/liquid-condensed boundary. Overall these experiments reveal the remarkable stability of SP-A in diverse, biologically relevant environments.     

Pulmonary surfactant proteins SP-B and SP-C in spread monolayers at the air-water interface: III. Proteins SP-B plus SP-C with phospholipids in spread monolayers.

Spread binary monolayers of surfactant-associated proteins SP-B and SP-C were formed at the air-water interface. Surface pressure measurements showed no interactions between the hydrophobic proteins. The effects of a mixture of SP-B plus SP-C (2:1, w/w) on the properties of monolayers of dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylglycerol (DPPG), and DPPC:DPPG (7:3, mol:mol) were studied. During compression of ternary and quaternary films, containing less than 0.4 mol% or 5 weight% total protein, the proteins were not squeezed out and appeared to remain associated with the film until collapse at surface pressures of about 65-70 mN.m-1. At initial concentrations of total protein of about 0.9 mol% or 10 weight%, exclusion of protein-lipid complexes was observed at 40-50 mN.m-1. Larger amounts of phospholipid were removed by proteins from (SP-B:SP-C)/DPPG films than from (SP-B:SP-C)/DPPC ones. Separate squeeze-out of SP-B (or SP-B plus DPPC) at about 40 mN.m-1, followed by exclusion of SP-C (or SP-C plus DPPC) at about 50 mN.m-1, was observed in (SP-B:SP-C)/DPPC films. This led to a conclusion that there was independent behavior of SP-B and SP-C in (SP-B:SP-C)/DPPC monolayers. The quaternary (SP-B:SP-C)/(DPPC:DPPG) films showed qualitatively similar process of squeeze-out of the proteins. In the ternary mixtures of SP-B plus SP-C with DPPG separate exclusion of SP-B was not detected; rather, the data was consistent with exclusion of a (SP-B:SP-C)/DPPG complex at about 50 mN.m-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Submicron structure in L-alpha-dipalmitoylphosphatidylcholine monolayers and bilayers probed with confocal, atomic force, and near-field microscopy.

Langmuir-Blodgett (LB) monolayers and bilayers of L-alpha-dipalmitoylphosphatidylcholine (DPPC), fluorescently doped with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (diIC18), are studied by confocal microscopy, atomic force microscopy (AFM), and near-field scanning optical microscopy (NSOM). Beyond the resolution limit of confocal microscopy, both AFM and NSOM measurements of mica-supported lipid monolayers reveal small domains on the submicron scale. In the NSOM studies, simultaneous high-resolution fluorescence and topography measurements of these structures confirm that they arise from coexisting liquid condensed (LC) and liquid expanded (LE) lipid phases, and not defects in the monolayer. AFM studies of bilayers formed by a combination of LB dipping and Langmuir-Schaefer monolayer transfer exhibit complex surface topographies that reflect a convolution of the phase structure present in each of the individual monolayers. NSOM fluorescence measurements, however, are able to resolve the underlying lipid domains from each side of the bilayer and show that they are qualitatively similar to those observed in the monolayers. The observation of the small lipid domains in these bilayers is beyond the spatial resolving power of confocal microscopy and is complicated in the topography measurements taken with AFM, illustrating the utility of NSOM for these types of studies. The data suggest that the small LC and LE lipid domains are formed after lipid transfer to the substrate through a dewetting mechanism. The possible extension of these measurements to probing for lipid phase domains in natural biomembranes is discussed.     

Persistence of phase coexistence in disaturated phosphatidylcholine monolayers at high surface pressures

Prior reports that the coexistence of the liquid-expanded (LE) and liquid-condensed (LC) phases in phospholipid monolayers terminates in a critical point have been compromised by experimental difficulties with Langmuir troughs at high surface pressures and temperatures. The studies reported here used the continuous interface of a captive bubble to minimize these problems during measurements of the phase behavior for monolayers containing the phosphatidylcholines with the four different possible combinations of palmitoyl and/or myristoyl acyl residues. Isothermal compression produced surface pressure-area curves for dipalmitoyl phosphatidylcholine (DPPC) that were indistinguishable from previously published data obtained with Langmuir troughs. During isobaric heating, a steep increase in molecular area corresponding to the main LC-LE phase transition persisted for all four compounds to 45 mN/m, at which collapse of the LE phase first occurred. No other discontinuities to suggest other phase transitions were apparent. Isobars for DPPC at higher pressures were complicated by collapse of the monolayer, but continued to show evidence up to 65 mN/m for at least the onset of the LC-LE transition. The persistence of the main phase transition to high surface pressures suggests that a critical point for these monolayers of disaturated phospholipids is either nonexistent or inaccessible at an air-water interface.     

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

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

Fluidization of a dipalmitoyl phosphatidylcholine monolayer by fluorocarbon gases: potential use in lung surfactant therapy

Fluorocarbon gases (gFCs) were found to inhibit the liquid-expanded (LE)/liquid-condensed (LC) phase transition of dipalmitoyl phosphatidylcholine (DPPC) Langmuir monolayers. The formation of domains of an LC phase, which typically occurs in the LE/LC coexistence region upon compression of DPPC, is prevented when the atmosphere above the DPPC monolayer is saturated with a gFC. When contacted with gFC, the DPPC monolayer remains in the LE phase for surface pressures lower than 38 mN m(-1), as assessed by compression isotherms and fluorescence microscopy (FM). Moreover, gFCs can induce the dissolution of preexisting LC phase domains and facilitate the respreading of the DPPC molecules on the water surface, as shown by FM and grazing incidence x-ray diffraction. gFCs have thus a highly effective fluidizing effect on the DPPC monolayer. This gFC-induced fluidizing effect was compared with the fluidizing effect brought about by a mixture of unsaturated lipids and proteins, namely the two commercially available lung surfactant substitutes, Curosurf and Survanta, which are derived from porcine and bovine lung extracts, respectively. The candidate FCs were chosen among those already investigated for biomedical applications, and in particular for intravascular oxygen transport, i.e., perfluorooctyl bromide, perfluorooctylethane, bis(perfluorobutyl)ethene, perfluorodecalin, and perfluorooctane. The fluidizing effect is most effective with the linear FCs. This study suggests that FCs, whose biocompatibility is well documented, may be useful in lung surfactant substitute compositions.     

Microstructure and dynamic surface properties of surfactant protein SP-B/dipalmitoylphosphatidylcholine interfacial films spread from lipid-protein bilayers

 Suspensions of dipalmitoylphosphatidylcholine (DPPC) bilayers containing 5, 10 or 20% (w/w) surfactant protein SP-B have been reconstituted and spread at air-liquid interfaces. Compression isotherms of DPPC/SP-B monolayers spread from these preparations were qualitatively comparable to the isotherms of the corresponding DPPC/SP-B monolayers spread from solvents. SP-B was squeezed-out at higher pressures from vesicle-spread films than from solvent-spread monolayers. SP-B caused a marked decrease on the rate of relaxation of DPPC collapse phases to equilibrium pressures in all the lipid/protein films assayed. This stabilizing effect was higher in vesicle-spread than in solvent-spread monolayers. Inclusion in the films of traces of the fluorescent probe NBD-PC (1 mol%) and use of a fluorescent derivative of SP-B labeled with a rhodamine derivative, Texas Red, allowed for direct observation of protein and lipid domains at the interface by epifluorescence microscopy. Upon compression, SP-B altered the packing of phospholipids in the bilayer-spread films, observed as a SP-B-induced reduction of the area of liquid-condensed domains, in a way similar to its effect in solvent-spread monolayers. SP-B was not associated with condensed regions of the films. Fluorescence images from vesicle-spread films showed discrete fluorescent aggregates that could be consistent with the existence of lipid-protein vesicles in close association with the monolayer. Both the retention of SP-B at higher surface pressures and the greater stability of collapse phases of DPPC/SP-B films prepared by spreading from liposomes in comparison to those spread from solvents can be interpreted as a consequence of formation of complex bilayer-monolayer interacting systems. Received: 1 December 1999 / Revised version: 2 March 2000 / Accepted: 2 March 2000     

Thermal response of Langmuir-Blodgett films of dipalmitoylphosphatidylcholine studied by atomic force microscopy and force spectroscopy

The topographic evolution of supported dipalmitoylphosphatidylcholine (DPPC) monolayers with temperature has been followed by atomic force microscopy in liquid environment, revealing the presence of only one phase transition event at approximately 46 degrees C. This finding is a direct experimental proof that the two phase transitions observed in the corresponding bilayers correspond to the individual phase transition of the two leaflets composing the bilayer. The transition temperature and its dependency on the measuring medium (liquid saline solution or air) is discussed in terms of changes in van der Waals, hydration, and hydrophobic/hydrophilic interactions, and it is directly compared with the transition temperatures observed in the related bilayers under the same experimental conditions. Force spectroscopy allows us to probe the nanomechanical properties of such monolayers as a function of temperature. These measurements show that the force needed to puncture the monolayers is highly dependent on the temperature and on the phospholipid phase, ranging from 120+/-4 pN at room temperature (liquid condensed phase) to 49+/-2 pN at 65 degrees C (liquid expanded phase), which represents a two orders-of-magnitude decrease respective to the forces needed to puncture DPPC bilayers. The topographic study of the monolayers in air around the transition temperature revealed the presence of boundary domains in the monolayer surface forming 120 degrees angles between them, thus suggesting that the cooling process from the liquid-expanded to the liquid-condensed phase follows a nucleation and growth mechanism.