Effects of lung surfactant proteins, SP-B and SP-C, and palmitic acid on monolayer stability

Langmuir isotherms and fluorescence and atomic force microscopy images of synthetic model lung surfactants were used to determine the influence of palmitic acid and synthetic peptides based on the surfactant-specific proteins SP-B and SP-C on the morphology and function of surfactant monolayers. Lung surfactant-specific protein SP-C and peptides based on SP-C eliminate the loss to the subphase of unsaturated lipids necessary for good adsorption and respreading by inducing a transition between monolayers and multilayers within the fluid phase domains of the monolayer. The morphology and thickness of the multilayer phase depends on the lipid composition of the monolayer and the concentration of SP-C or SP-C peptide. Lung surfactant protein SP-B and peptides based on SP-B induce a reversible folding transition at monolayer collapse that allows all components of surfactant to be retained at the interface during respreading. Supplementing Survanta, a clinically used replacement lung surfactant, with a peptide based on the first 25 amino acids of SP-B also induces a similar folding transition at monolayer collapse. Palmitic acid makes the monolayer rigid at low surface tension and fluid at high surface tension and modifies SP-C function. Identifying the function of lung surfactant proteins and lipids is essential to the rational design of replacement surfactants for treatment of respiratory distress syndrome.     

Component-specific surface and physiological activity in bovine-derived lung surfactants

Composition, surface activity and effects on pressure-volume (P-V) mechanics are examined for lavaged calf lung surfactant (LS) and the clinical exogenous surfactants Infasurf and Survanta. Lavaged LS and Infasurf had closely-matching compositions of phospholipids and neutral lipids. Survanta had higher levels of free fatty acids and triglycerides consistent with its content of added synthetic palmitic acid and tripalmitin. Infasurf and Survanta both contained less total protein than LS because of extraction with hydrophobic solvents, but the total protein content relative to phospholipid in Survanta was about 45% lower than in Infasurf. This difference was primarily due to surfactant protein (SP)-B, which was present by ELISA at a mean weight percent relative to phospholipid of 1.04% in LS, 0.90% in Infasurf, and 0.044% in Survanta. Studies on component fractions separated by gel permeation chromatography showed that SP-B was a major contributor to the adsorption, dynamic surface activity, and P-V mechanical effects of Infasurf, which approached whole LS in magnitude. Survanta had lower adsorption, higher minimum surface tension, and a smaller effect on surfactant-deficient P-V mechanics consistent with minimal contributions from SP-B. Addition of 0.05% by weight of purified bovine SP-B to Survanta did not improve surface or physiological activity, but added 0.7% SP-B improved adsorption, dynamic surface tension lowering, and P-V activity to levels similar to Infasurf. The SP-B content of lung surfactants appears to be a crucial factor in their surface activity and efficacy in improving surfactant-deficient pulmonary P-V mechanics.     

Mimicking SP-C palmitoylation on a peptoid-based SP-B analogue markedly improves surface activity

Hydrophobic lung surfactant proteins B and C (SP-B and SP-C) are critical for normal respiration in vertebrates, and each comprises specific structural attributes that enable the surface-tension-reducing ability of the lipid-protein mixture in lung surfactant. The difficulty in obtaining pure SP-B and SP-C on a large scale has hindered efforts to develop a non-animal-derived surfactant replacement therapy for respiratory distress. Although peptide-based SP-C mimics exhibit similar activity to the natural protein, helical peptide-based mimics of SP-B benefit from dimeric structures. To determine if in vitro surface activity improvements in a mixed lipid film could be garnered without creating a dimerized structural motif, a helical and cationic peptoid-based SP-B mimic was modified by SP-C-like N-terminus alkylation with octadecylamine. “Hybridized” mono- and dialkylated peptoids significantly decreased the maximum surface tension of the lipid film during cycling on the pulsating bubble surfactometer relative to the unalkylated variant. Peptoids were localized in the fluid phase of giant unilamellar vesicle lipid bilayers, as has been described for SP-B and SP-C. Using Langmuir-Wilhelmy surface balance epifluorescence imaging (FM) and atomic force microscopy (AFM), only lipid-alkylated peptoid films revealed micro- and nanostructures closely resembling films containing SP-B. AFM images of lipid-alkylated peptoid films showed gel condensed-phase domains surrounded by a distinct phase containing “nanosilo” structures believed to enhance re-spreading of submonolayer material. N-terminus alkylation may be a simple, effective method for increasing lipid affinity and surface activity of single-helix SP-B mimics.     

Effect of the hydrophobic surfactant proteins on the surface activity of spread films in the captive bubble surfactometer

The main function of pulmonary surfactant, a mixture of lipids and proteins, is to reduce the surface tension at the air/liquid interface of the lung. The hydrophobic surfactant proteins SP-B and SP-C are required for this process. When testing their activity in spread films in a captive bubble surfactometer, both SP-B and SP-C showed concentration dependence for lipid insertion as well as for lipid film refinement. Higher activity in DPPC refinement of the monolayer was observed for SP-B compared with SP-C. Further differences between both proteins were found, when subphase phospholipid vesicles, able to create a monolayer-attached lipid reservoir, were omitted. SP-C containing monolayers showed gradually increasing minimum surface tensions upon cycling, indicating that a lipid reservoir is required to prevent loss of material from the monolayer. Despite reversible cycling dynamics, SP-B containing monolayers failed to reach near-zero minimum surface tensions, indicating that the reservoir is required for stable films.     

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

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

Lipid oxidation and signalling in programmed cell death

The pulmonary surfactant lines as a complex monolayer of lipids and proteins the alveolar epithelial surface. The monolayer dynamically adapts the surface tension of this interface to the varying surface areas during inhalation and exhalation. Its presence in the alveoli is thus a prerequisite for a proper lung function. The lipid moiety represents about 90% of the surfactant and contains mainly dipalmitoylphosphatidylcholine (DPPC) and phosphatidylglycerol (PG). The surfactant proteins involved in the surface tension adaption are called SP-A, SP-B and SP-C. The aim of the present investigation is to analyse the properties of monolayer films made from pure SP-C and from mixtures of DPPC, DPPG and SP-C in order to mimic the surfactant monolayer with minimal compositional requirement. Pressure-area diagrams were taken. Ellipsometric measurements at the air-water interface of a Langmuir film balance allowed measurement of the changes in monolayer thickness upon compression. Isotherms of pure SP-C monolayers exhibit a plateau between 22 and 25 mN/m. A further plateau is reached at higher compression. Structures of the monolayer formed during compression are reversible during expansion. Together with ellipsometric data which show a stepwise increase in film thickness (coverage) during compression, we conclude that pure SP-C films rearrange reversibly into multilayers of homogenous thickness.

Lipid monolayers collapse locally and irreversibly if films are compressed to approximately 0–4 nm²/molecule. In contrast, mixed DPPG/SP-C monolayers with less than 5 mol% protein collapse in a controlled and reversible way. The pressure-area diagrams exhibit a plateau at 20 mN/m, indicating partial demixing of SP-C and DPPG. The thickness isotherm obtained by ellipsometry indicates a transformation into multilayer structures. In DPPC/DPPG/SP-C mixtures again a reversible collapse was observed but without a drastic increase in surface layer thickness which may be due to the formation of protrusion under the surface. Thus lipid monolayers containing small amounts of SP-C may mimic the lung surfactant.     

Effects of cholesterol on surface activity and surface topography of spread surfactant films

Pulmonary surfactant forms a monolayer of lipids and proteins at the alveolar air/liquid interface. Although cholesterol is a natural component of surfactant, its function in surface dynamics is unclear. To further elucidate the role of cholesterol in surfactant, we used a captive bubble surfactometer (CBS) to measure surface activity of spread films containing dipalmitoylphosphatidylcholine/1-palmitoyl-2-oleoylphosphatidylcholine/1-palmitoyl-2-oleoylphosphatidylglycerol (DPPC/POPC/POPG, 50/30/20 molar percentages), surfactant protein B (SP-B, 0.75 mol %), and/or surfactant protein C (SP-C, 3 mol %) with up to 20 mol % cholesterol. A cholesterol concentration of 10 mol % was optimal for reaching and maintaining low surface tensions in SP-B-containing films but led to an increase in maximum surface tension in films containing SP-C. No effect of cholesterol on surface activity was found in films containing both SP-B and SP-C. Atomic force microscopy (AFM) was used, for the first time, to visualize the effect of cholesterol on topography of SP-B- and/or SP-C-containing films compressed to a surface tension of 22 mN/m. The protrusions found in the presence of cholesterol were homogeneously dispersed over the film, whereas in the absence of cholesterol the protrusions tended to be more clustered into network structures. A more homogeneous dispersion of surfactant lipid components may facilitate lipid insertion into the surfactant monolayer. Our data provide additional evidence that natural surfactant, containing SP-B and SP-C, is superior to surfactants lacking one of the components, and furthermore, this raises the possibility that the cholesterol found in surfactant of warm-blooded mammals does not have a function in surface activity.     

Inhibition of mixtures of surfactant lipids and synthetic sequences of surfactant proteins SP-B and SP-C

The respiratory distress syndrome of premature infants is caused by both surfactant deficiency and surfactant inhibition by capillary-alveolar leakage of serum factors. Dispersions of a standard surfactant lipid mixture, with and without various synthetic peptides, modeled on human surfactant proteins SP-B (residues 1-25, 49-66, 1-78) and SP-C (residues 1-10), were evaluated for inhibition by serum and by plasma constituents using a pulsating bubble surfactometer. Inhibition was derived from the changes in surface properties of these mixtures after addition of human serum or plasma constituents. Modified bovine surfactant (TA) containing native SP-B and SP-C was used as a control. In the absence of serum inhibitors, mixtures with synthetic peptides gave results similar to surfactant TA. However, inhibition was more evident in the dispersions with synthetic peptides when compared with surfactant TA. The peptide/phospholipid mixture with the entire sequence of SP-B and the first 10 residues of SP-C were more resistant to inhibition than mixtures with synthetic peptides containing fewer domains. Addition of calcium reduced the inhibitory effects of serum both in mixtures containing synthetic peptides and in surfactant TA. Therefore, synthetic SP-B and SP-C peptides in surfactant lipids, in cooperation with calcium, permit resistance to inhibition by several plasma constituents that probably inactivate surfactant by a variety of different mechanisms.     

Surfactant protein B inhibits secretory phospholipase A2 hydrolysis of surfactant phospholipids

Hydrolysis of surfactant phospholipids (PL) by secretory phospholipases A(2) (sPLA(2)) contributes to surfactant damage in inflammatory airway diseases such as acute lung injury/acute respiratory distress syndrome. We and others have reported that each sPLA(2) exhibits specificity in hydrolyzing different PLs in pulmonary surfactant and that the presence of hydrophilic surfactant protein A (SP-A) alters sPLA(2)-mediated hydrolysis. This report tests the hypothesis that hydrophobic SP-B also inhibits sPLA(2)-mediated surfactant hydrolysis. Three surfactant preparations were used containing varied amounts of SP-B and radiolabeled tracers of phosphatidylcholine (PC) or phosphatidylglycerol (PG): 1) washed ovine surfactant (OS) (pre- and postorganic extraction) compared with Survanta (protein poor), 2) Survanta supplemented with purified bovine SP-B (1-5%, wt/wt), and 3) a mixture of dipalmitoylphosphatidylcholine (DPPC), 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC), and 1-palmitoyl-2-oleoyl-phosphatidylglycerol (POPG) (DPPC:POPC:POPG, 40:40:20) prepared as vesicles and monomolecular films in the presence or absence of SP-B. Hydrolysis of PG and PC by Group IB sPLA(2) (PLA2G1A) was significantly lower in the extracted OS, which contains SP-B, compared with Survanta (P = 0.005), which is SP-B poor. Hydrolysis of PG and PC in nonextracted OS, which contains all SPs, was lower than both Survanta and extracted OS. When Survanta was supplemented with 1% SP-B, PG and PC hydrolysis by PLA2G1B was significantly lower (P < 0.001) than in Survanta alone. When supplemented into pure lipid vesicles and monomolecular films composed of PG and PC mixtures, SP-B also inhibited hydrolysis by both PLA2G1B and Group IIA sPLA2 (PLA2G2A). In films, PLA2G1B hydrolyzed surfactant PL monolayers at surface pressures ≤30 mN/m (P < 0.01), and SP-B lowered the surface pressure range at which hydrolysis can occur. These results suggest the hydrophobic SP, SP-B, protects alveolar surfactant PL from hydrolysis mediated by multiple sPLA(2) in both vesicles (alveolar subphase) and monomolecular films (air-liquid interface).     

Protein-lipid interactions and surface activity in the pulmonary surfactant system

Pulmonary surfactant is a lipid-protein complex, synthesized and secreted by the respiratory epithelium of lungs to the alveolar spaces, whose main function is to reduce the surface tension at the air-liquid interface to minimize the work of breathing. The activity of surfactant at the alveoli involves three main processes: (i) transfer of surface active molecules from the aqueous hypophase into the interface, (ii) surface tension reduction to values close to 0 mN/m during compression at expiration and (iii) re-extension of the surface active film upon expansion at inspiration. Phospholipids are the main surface active components of pulmonary surfactant, but the dynamic behaviour of phospholipids along the breathing cycle requires the necessary participation of some specific surfactant associated proteins. The present review summarizes the current knowledge on the structure, disposition and lipid-protein interactions of the hydrophobic surfactant proteins SP-B and SP-C, the two main actors participating in the surface properties of pulmonary surfactant. Some of the methodologies currently used to evaluate the surface activity of the proteins in lipid-protein surfactant preparations are also revised. Working models for the potential molecular mechanism of SP-B and SP-C are finally discussed. SP-B might act in surfactant as a sort of amphipathic tag, directing the lipid-protein complexes to insert and re-insert very efficiently into the air-liquid interface along successive breathing cycles. SP-C could be essential to maintain association of lipid-protein complexes with the interface at the highest compressed states, at the end of exhalation. The understanding of the mechanisms of action of these proteins is critical to approach the design and development of new clinical surfactant preparations for therapeutical applications.     

Combined and independent action of proteins SP-B and SP-C in the surface behavior and mechanical stability of pulmonary surfactant films

The hydrophobic proteins SP-B and SP-C are essential for pulmonary surfactant function, even though they are a relatively minor component (<2% of surfactant dry mass). Despite countless studies, their specific differential action and their possible concerted role to optimize the surface properties of surfactant films have not been completely elucidated. Under conditions kept as physiologically relevant as possible, we tested the surface activity and mechanical stability of several surfactant films of varying protein composition in vitro using a captive bubble surfactometer and a novel (to our knowledge) stability test. We found that in the naturally derived surfactant lipid mixtures, surfactant protein SP-B promoted film formation and reextension to lower surface tensions than SP-C, and in particular played a vital role in sustaining film stability at the most compressed states, whereas SP-C produced no stabilization. Preparations containing both proteins together revealed a slight combined effect in enhancing film formation. These results provide a qualitative and quantitative framework for the development of future synthetic therapeutic surfactants, and illustrate the crucial need to include SP-B or an efficient SP-B analog for optimal function.     

Multilayers at the surface of solutions of exogenous lung surfactant: Direct observation by neutron reflection

Pharmacy-grade exogenous lung surfactant preparations of bovine and porcine origin, dispersed in physiological electrolyte solution have been studied. The organization and dynamics at the air/water interface at physiological temperature was analysed by neutron reflection. The results show that a well-defined surface phase is formed, consisting of a multilayer structure of lipid/protein bilayers alternating with aqueous layers, with a repetition period of about 70 Å and correlation depths of 3 to > 25 bilayers, depending on electrolyte composition and time. The experimental surfactant concentration of 0.15% (w/w) is far below that used in therapeutic application of exogenous surfactants and it is therefore likely that similar multilayer structures are also formed at the alveolar surface in the clinical situation during surfactant substitution therapy. Lung surfactant preparations in dry form swell in aqueous solution towards a limit of about 60% (w/w) of water, forming a lamellar liquid-crystalline phase above about 34 °C, which disperses into lamellar bodies at higher water concentrations. The lamellar spacings in the surface multilayers at the air/water interface are smaller than those in the saturated limit even though they are in contact with much greater water concentrations. The surface multilayers are laterally disordered in a way that is consistent with fragments of Lα-phase lamellae. The near surface layers of the multilayer structure have a significant protein content (only SP-B and SP-C are present in the preparations). The results demonstrate that a multilayer structure can be formed in exogenous surfactant even at very low concentrations and indicate that multilayers need to be incorporated into present interpretations of in vitro studies of similar lung surfactant preparations, which are largely based on monolayer models.     

Surfactant protein interactions with neutral and acidic phospholipid films

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

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.     

Effects of pulmonary surfactant proteins SP-B and SP-C and calcium ions on the surface properties of hydrophobic fractions of lung surfactant

This study focused on two hydrophobic fractions (HF-A and HF-B) isolated from porcine lung surfactant (LS) that had similar phospholipid composition, but HF-A consisted of the hydrophobic LS specific proteins (SP-B and SP-C), in contrast to HF-B. Monolayers spread in a Langmuir trough were formed at the air/water interface of both fractions and the rate of adsorption-desorption and the respreading potential of the LS constituents was studied during six consecutive compression/decompression cycles of the monolayers. By drawing a comparison between the behavior of HF-A and HF-B monolayers on the subphase of 150 mm NaCl, either with or without additional Ca²⁺, we estimated the role of hydrophobic LS proteins and Ca²⁺ ions for LS surface activity. The results demonstrated much higher ability of the HF-A sample, compared to HF-B, to maintain lower surface tension (γ) during monolayer compression and its better respreading capacity during decompression. For instance, at a surface concentration corresponding to 80 Ų per phospholipid molecule, the HF-A monolayers showed a much lower γ _max value (surface tension at 100% of the trough area), being ca. 31.0 mN/m, compared to the HF-B monolayers (γ _max? 62.0 mN/m). The surface tension after compression to 20% of the initial area (γ _min) reached ca. 7.0 and 19.0 mN/m in the HF-A and HF-B monolayers, respectively. Better respreading of the HF-A monolayers compared to the HF-B monolayers was due to the faster adsorption and spreading of LS phospholipids during decompression, facilitated by the hydrophobic proteins. As the phospholipid composition of both fractions was similar, we showed that the hydrophobic surfactant proteins were responsible also for the prevention of the irreversible loss of material from the surface during monolayer compression/decompression. The effects observed demonstrated also that the hydrophobic surfactant proteins were the stronger determinant, compared with Ca²⁺ ions, for the surface tension decrease and respreading of the monolayers during film compression/decompression. For instance, when the HF-A monolayers were spread on a subphase with an additional 5 mm Ca²⁺ ion content, no significant changes were detected in the γ _min and γ _max values between the first and sixth cycle, compared to the monolayers spread on a subphase of 150 mm NaCl only. However, in the absence of positively charged SP-B and SP-C (HF-B sample) in highly compressed monolayers, Ca²⁺ ions were able to cause the effects shown by SP-B and SP-C, although to a less extent. The role of the electrostatic and hydrophobic interactions is discussed for the better respreading of LS components in the presence of LS proteins and Ca²⁺ ions.     

The role of surfactant proteins in DPPC enrichment of surface films

A pressure-driven captive bubble surfactometer was used to determine the role of surfactant proteins in refinement of the surface film. The advantage of this apparatus is that surface films can be spread at the interface of an air bubble with a different lipid/protein composition than the subphase vesicles. Using different combinations of subphase vesicles and spread surface films a clear correlation between dipalmitoylphosphatidylcholine (DPPC) content and minimum surface tension was observed. Spread phospholipid films containing 50% DPPC over a subphase containing 50% DPPC vesicles did not form stable surface films with a low minimum surface tension. Addition of surfactant protein B (SP-B) to the surface film led to a progressive decrease in minimum surface tension toward 1 mN/m upon cycling, indicating an enrichment in DPPC. Surfactant protein C (SP-C) had no such detectable refining effect on the film. Surfactant protein A (SP-A) had a positive effect on refinement when it was present in the subphase. However, this effect was only observed when SP-A was combined with SP-B and incubated with subphase vesicles before addition to the air bubble containing sample chamber. Comparison of spread films with adsorbed films indicated that refinement induced by SP-B occurs by selective removal of non-DPPC lipids upon cycling. SP-A, combined with SP-B, induces a selective adsorption of DPPC from subphase vesicles into the surface film. This is achieved by formation of large lipid structures which might resemble tubular myelin.     

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)     

Deficiency of SP-B reveals protective role of SP-C during oxygen lung injury.

Although the surface properties of surfactant protein (SP)-B and SP-C are similar, the contributions that either protein may make to lung function have not been identified in vivo. Mutations in SP-B cause lethal respiratory failure at birth; however, SP-B null mice are deficient in both SP-B and SP-C. To identify potential contributions of SP-C to lung function in vivo, the following transgenic mice were generated and exposed to 95% O(2) for 3 days: (SP-B(+/+),SP-C(+/+)), (SP-B(+/+), SP-C(-/-)), (SP-B(+/-),SP-C(+/+)), (SP-B(+/-),SP-C(+/-)), and (SP-B(+/-),SP-C(-/-)). Hyperoxia altered pressure-volume curves in mice that were heterozygous for SP-B, and these values were further decreased in (SP-B(+/-),SP-C(-/-)) mice. Likewise, alveolar interleukin (IL)-6 and IL-1 beta were maximally increased by O(2) exposure of (SP-B(+/-),SP-C(-/-)) mice compared with the other genotypes. Lung hysteresivity was lower in the (SP-B(+/-),SP-C(-/-)) mice. Surfactant isolated from (SP-B(+/+),SP-C(-/-)) and (SP-B(+/-),SP-C(-/-)) mice failed to stabilize the surface tension of microbubbles, showing that SP-C plays a role in stabilization or recruitment of phospholipid films at low bubble radius. Genetically decreased levels of SP-B combined with superimposed O(2)-induced injury reveals the distinct contribution of SP-C to pulmonary function in vivo.     

cDNA cloning of ovine pulmonary SP-A, SP-B, and SP-C: isolation of two different sequences for SP-B

Pulmonary surfactant promotes alveolar stability by lowering the surface tension at the air-liquid interface in the peripheral air spaces. The three surfactant proteins SP-A, SP-B, and SP-C contribute to dynamic surface properties involved during respiration. We have cloned and sequenced the complete cDNAs for ovine SP-A and SP-C and two distinct forms of ovine SP-B cDNAs. The nucleotide sequence of ovine SP-A cDNA consists of 1,901 bp and encodes a protein of 248 amino acids. Ovine SP-C cDNA contains 809 bp, predicting a protein of 190 amino acids. Ovine SP-B is encoded by two mRNA species, which differ by a 69-bp in-frame deletion in the region coding for the active airway protein. The larger SP-B cDNA comprises 1,660 bp, encoding a putative protein of 374 amino acids. With the sequences reported, a more complete analysis of surfactant regulation and the determination of their physiological function in vivo will be enabled.     

Naturally derived commercial surfactants differ in composition of surfactant lipids and in surface viscosity

Pulmonary surfactant biophysical properties are best described by surface tension and surface viscosity. Besides lecithin, surfactant contains a variety of minor lipids, such as plasmalogens, polyunsaturated fatty acid-containing phospholipids (PUFA-PL), and cholesterol. Plasmalogens and cholesterol improve surface properties of lipid mixtures significantly. High PUFA-PL and plasmalogen content in tracheal aspirate of preterm infants reduces the risk of developing chronic lung disease. Different preparations are available for exogenous surfactant substitution; however, little is known about lipid composition and surface viscosity. Thus lipid composition and surface properties (measured by oscillating drop surfactometer) of three commercial surfactant preparations (Alveofact, Curosurf, Survanta) were compared. Lipid composition exhibited strong differences: Survanta had the highest proportion of disaturated PL and total neutral lipids and the lowest proportion of PUFA-PL. Highest plasmalogen and PUFA-PL concentrations were found in Curosurf (3.8 +/- 0.1 vs. 26 +/- 1 mol%) compared with Alveofact (0.9 +/- 0.3 vs. 11 +/- 1) and Survanta (1.5 +/- 0.2 vs. 6 +/- 1). In Survanta samples, viscosity increased >8 x 10(-6) kg/s at surface tension of 30 mN/m. Curosurf showed only slightly increased surface viscosity below surface tensions of 25 mN/m, and viscosity did not reach 5 x 10(-6) kg/s. By adding defined PL to Survanta, we obtained a Curosurf-like lipid mixture (without plasmalogens) that exhibited biophysical properties like Curosurf. Different lipid compositions could explain some of the differences in surface viscosity. Therefore, PL pattern and minor surfactant lipids are important for biophysical activity and should be considered when designing synthetic surfactant preparations.