State of the surface activity of pulmonary surfactant in rats at different periods after left-sided pneumonectomy

The surface activity of the seven-fold washing of the right lung was measured on the modified Wilhelmy's balance after the leftside pneumonectomy in rats. It appeared to be normal (gamma min-23--24 dynes/cm) up to the 5th day, and at the remote postoperative periods. The intracellular edema of the air-blood barrier components and the release of the edema fluid into the alveolar lumen in the "vesicle" composition failed to influence the surface properties of the lung surfactant. A sharp increase of the alveolar dimensions on the 5th--7th postoperative day was followed by an increase of the surface-active properties of the lung washings (gamma-min-11--15 dynes/cm) and by the intensified secretion of the material of the osmiophilic lamellar bodies from the alveolar cells of the 2nd type into the alveolar lumen. The cytological mechanisms providing the intensified production of the surfactant in the hypertrophic alveoli are activation of the lipid synthesis in the alveolar cells of the 2nd type, their hypertrophy, and also the appearance of binuclear cells.     

Surface activity in situ, in vivo, and in the captive bubble surfactometer

For studies of the mechanical effects of lung surfactants, the captive bubble surfactometer (CBS) combines the advantages of the continuous film of Pattle's bubbles with the feasibility of the Langmuir-Wilhelmy balance to produce surface tension-area hysteresis loops. The CBS allows the compression of films to very low and stable surface tensions of 1-2 mN/m. Such low and stable surface tensions are in line with results obtained from pressure-volume studies on excised lungs. In addition, the CBS is useful to test other essential physical properties of the surfactant system, including: (1) rapid film formation (within seconds) through adsorption from the hypophase; (2) low film compressibility with a fall in surface tension to very low (<2 mN/m) values during surface compression; and (3) effective replenishment of the surface film on expansion by the incorporation of surfactant material from material associated with the surface (the surface associated surfactant reservoir). Morphological observations of films fixed in situ or in vitro reveal frequently their multilayered structure, which is consistent with the concept of the surface reservoir. The deviation of the bubbles from a Laplacian shape at very low surface tension and the morphological observations suggest that the surfactant film cannot be considered a simple monolayer.     

Surface forces in lungs. I. Alveolar surface tension-lung volume relationships

Alveolar surface tension (gamma)-lung volume relationships were obtained for quasi-static and dynamic lung pressure-volume (PV) histories from measurements of PV curves of liquid- and air-filled excised rabbit lungs. PV relationships were measured at room temperature in lungs filled with test liquids with constant liquid-liquid interfacial tensions with alveolar surface-active materials; and air-filled lungs before and after the normal alveolar surface film was covered with test liquids with constant values of liquid- and air-liquid interfacial tensions. Interfacial tensions of test liquids were measured in a surface balance on monolayers of dipalmitoyl phosphatidylcholine. Values of gamma for the normal air-filled lung were obtained either from points of intersection between PV curves with the normal and test liquid interface or from a general relationship between gamma and the component of recoil pressure due to surface tension derived from the data. In contrast to previous analyses that have used PV measurements, this approach does not depend on assumptions about lung microstructural geometry. Surface tension-volume relationships for the normal air-filled lung show a prominent hysteresis with surface tension ranging from near 0 at low volumes during lung deflation to transiently high values near 40 dyn/cm during inflation; value of equilibrium surface tension (gamma EQ) near 28 dyn/cm; and characteristic transitions in surface film compressibility and associated transitions in film kinetic behavior in nonequilibrium film states where gamma deviates from gamma EQ. These features are consistent with the behavior predicted from current models of alveolar surface film behavior.     

Surface properties of rat pulmonary surfactant studied with the captive bubble method: adsorption, hysteresis, stability.

Surface tension-area relations from pulmonary surfactant were obtained with a new apparatus that contains a leak free captive bubble of controllable size. Rat pulmonary surfactant was studied at phospholipid concentrations of 50, 200 and 400 micrograms/ml. At the highest concentration, adsorption was rapid, reaching surface tensions below 30 mN/m within 1 s, while at the lowest concentration, approximately 3 min were required. Upon a first quasi static or dynamic compression, stable surface tensions below 1 mN/m could be obtained by a film area reduction of approximately 50%. After three to four cycles the surface tension-area relations became stationary, and the tension fell from 25-30 to approximately 1 mN/m for a film area reduction of less than 20%. Hysteresis became negligible, provided the films were not collapsed by further area reduction. Under these conditions, the films could be cycled for more than 20 min without any noticeable loss in surface activity. After only three to four consecutive cycles, surfactant films exhibited the low surface tensions, collapse rates and compressibilities characteristic of alveolar surfaces in situ. Remarkably, surface tension and area are interrelated in the captive bubble which may promote low and stable surface tensions. If the surface tension of the captive bubble suddenly increases ('click') because of mechanical vibration or unstable surfactant, the bubble shape changes from flat to more spherical. The associated isovolumetric decrease in surface area prevents the surface tension from rising as much as it would have in a constant-area situation. This feedback mechanism may also have a favorable effect in stabilizing alveolar surface tension at low lung volumes.     

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

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

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.     

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.     

Arguments against and alternatives for an extracellular surfactant layer in the alveoli of mammalian lung

It is generally believed that lung alveoli contain an extracellular aqueous layer of surfactant material, which is allegedly required to prevent alveolar collapse at small lung volume; the surfactant's major constituent is a fully saturated phospholipid, referred to as dipalmitoyl lecithin or DPL. I herein demonstrate that the surfactant hypothesis of alveolar stability is fundamentally wrong. Although DPL is synthesized inside type II epithelial cells and stored in the typical inclusion bodies therein and lowers surface tension to zero in the surface balance, there is no evidence to the effect that type II cells secrete the DPL surfactant into the aqueous intra-alveolar layer which is shown by electron microscopy in support of the surfactant theory. To the contrary, all the evidence indicates that, when seen, such an extracellular layer is an artifact. This is probably upon the damage glutaraldehyde inflicts onto alveolar structures during fixation of air-inflated lung tissue. Furthermore, several cogent arguments invalidate the belief that an extracellular layer of DPL and serum proteins is present in the alveoli of normal lung. In light of these arguments, a surface tension role of DPL in alveolar stability is excluded. Three hypotheses for an alternative role of DPL in respiration mechanics are proposed. They are: (a) alveolar clearance by viscolytic and surfactant action (bubble or foam formation) on the aqueous systems which are present in lung alveoli during edema and in prenatal life and which would otherwise be impervious to air; (b) homeostasis of blood palmitate in normal lung; (c) modulation of the elasticity of terminal lung tissue by the intact inclusion bodies and parts thereof inside type II cells in normal lung.     

Pulmonary surfactant protein-A (SP-A) restores the surface properties of surfactant after oxidation by a mechanism that requires the Cys6 interchain disulfide bond and the phospholipid binding domain

Reactive oxygen species produced by activated leuko-cytes in the alveolar epithelial lining fluid have been implicated in the inactivation of pulmonary surfactant and the impairment of lung function. Oxidation of bovine lipid extract surfactant (BLES), a therapeutic surfactant, with hypochlorous acid (H-BLES) or the Fenton reaction (F-BLES) led to temporary increases in conjugated dienes and formation of malondialdehyde and 4-hydroxy-2-nonenal. Electrospray ionization mass spectrometry revealed the appearance of lipid hydroperoxides, peroxides, lysophospholipids, and free fatty acids. Captive bubble tensiometer studies of H-BLES demonstrated prolonged adsorption times, film instability at low surface tensions during film compression, and reduced respreadability during film expansion. F-BLES exhibited prolonged adsorption times, a marked effect on increasing compressibility during compression, and a lesser effect on reducing respreadability on expansion. Addition of native bovine or rat surfactant-associated protein A (SP-A) reversed the effects of oxidation on surfactant biophysical properties. Studies using mutant recombinant rat SP-As indicated that an intact carbohydrate recognition domain and disulfide-dependent oligomeric assembly are critical for these effects, but the collagen-like region is not required. We conclude that SP-A can reverse the detrimental effects of surfactant oxidation on the biophysical properties of surfactant, by a mechanism that is dependent on interchain disulfide bond formation and the C-terminal domains of the protein.     

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.     

The role of the gel <=> liquid-crystalline phase transition in the lung surfactant cycle

Lipid polymorphism plays an important role in the lung surfactant cycle. A better understanding of the influence of phase transitions on the formation of a lipid film from dispersions of vesicles will help to describe the mechanism of action of lung surfactant. The surface pressure (or tension) of dispersions of DPPC, DMPC, and DPPE unilamellar vesicles was studied as a function of temperature. These aggregates rapidly fuse with a clean air-water interface when the system is at their phase transition temperature (Tm), showing a direct correlation between phase transition and film formation. Based on these results, an explanation on how fluid aggregates in the alveolar subphase can form a rigid monolayer at the alveolar interface is proposed.     

Surface properties of sulfur- and ether-linked phosphonolipids with and without purified hydrophobic lung surfactant proteins

Two novel C16:0 sulfur-linked phosphonolipids (S-lipid and SO(2)-lipid) and two ether-linked phosphonolipids (C16:0 DEPN-8 and C16:1 UnDEPN-8) were studied for surface behavior alone and in mixtures with purified bovine lung surfactant proteins (SP)-B and/or SP-C. Synthetic C16:0 phosphonolipids all had improved adsorption and film respreading compared to dipalmitoyl phosphatidylcholine, and SO(2)-lipid and DEPN-8 reached maximum surface pressures of 72mN/m (minimum surface tensions of <1mN/m) in compressed films on the Wilhelmy balance (23 degrees C). Dispersions of DEPN-8 (0.5mg/ml) and SO(2)-lipid (2.5mg/ml) also reached minimum surface tensions of <1mN/m on a pulsating bubble surfactometer (37 degrees C, 20cycles/min, 50% area compression). Synthetic lung surfactants containing DEPN-8 or SO(2)-lipid+0.75% SP-B+0.75% SP-C had dynamic surface activity on the bubble equal to that of calf lung surfactant extract (CLSE). Surfactants containing DEPN-8 or SO(2)-lipid plus 1.5% SP-B also had very high surface activity, but less than when both apoproteins were present together. Adding 10wt.% of UnDEPN-8 to synthetic lung surfactants did not improve dynamic surface activity. Surfactants containing DEPN-8 or SO(2)-lipid plus 0.75% SP-B/0.75% SP-C were chemically and biophysically resistant to phospholipase A(2) (PLA(2)), while CLSE was severely inhibited by PLA(2). The high activity and inhibition resistance of synthetic surfactants containing DEPN-8 or SO(2)-lipid plus SP-B/SP-C are promising for future applications in treating surfactant dysfunction in inflammatory lung injury.     

A captive bubble method reproduces the in situ behavior of lung surfactant monolayers

We tested a new captive bubble surface tensiometer with films adsorbed from aqueous suspensions of rabbit lung surfactant and a bovine lung surfactant lipid extract and with films of dipalmitoyl-sn-3-glycerophosphorylcholine (DPPC) spread from solvents. The lack of tubes penetrating the bubble surface eliminated potential leakage pathways for the surface film, which was compressed by increasing external pressure. Surface tensions and areas were calculated directly from bubble shapes without the need of pressure measurements. After only one to two compressions, the rabbit surfactant films exhibited the low surface tension, collapse rates, and compressibilities characteristic of the alveolar surface in situ and approached the behavior of spread DPPC films. The bubble "clicking" phenomenon described earlier by Pattle (Proc. R. Soc. Lond. B Biol. Sci. 148: 217-240, 1958) was also reproduced, but only with the bovine extract, which did not perform as well as the rabbit surfactant in surface tests. These findings suggest that surfactant apoprotein SP-A, which was probably present in the rabbit but not the bovine preparations, enhances both adsorption and stability of pulmonary surfactant monolayers.     

Surfactant-spreading and surface-compression disturbance on a thin viscous film

Spreading of a new surfactant in the presence of a pre-existing surfactant distribution is investigated both experimentally and theoretically for a thin viscous substrate. The experiments are designed to provide a better understanding of the fundamental interfacial and fluid dynamics for spreading of surfactants instilled into the lung. Quantitative measurements of spreading rates were conducted using a fluorescent new surfactant that was excited by argon laser light as it spread on an air-glycerin interface in a petri dish. It is found that pre-existing surfactant impedes surfactant spreading. However, fluorescent microspheres used as surface markers show that pre-existing surfactant facilitates the propagation of a surface-compression disturbance, which travels faster than the leading edge of the new surfactant. The experimental results compare well with the theory developed using lubrication approximations. An effective diffusivity of the thin film system is found to be Deff = (E*gamma)/(mu/H), which indicates that the surface-compression disturbance propagates faster for larger background surfactant concentration, gamma, larger constant slope of the sigma*-gamma* relation, -E*, and smaller viscous resistance, mu/H. Note that sigma* and gamma* are the dimensional surface tension and concentration, respectively, mu is fluid viscosity, and H is the unperturbed film thickness.     

A conformation transition of lung surfactant lipids probably involved in respiration.

X-ray scattering and freeze-fracture electron microscopy of a lung surfactant extract show the existence of a complex lamellar phase, L gamma, over a wide range of concentrations and temperatures. This lamellar phase, which consists of two bilayer motifs comprised of monolayers with stiff chains alternating with monolayers with disordered chains, allows us to propose a structural model of a collapse phase at the alveolar pulmonary interface. This model accounts for the increase in surface pressure during the compression as well as the easy respreading upon expansion of the interface during the respiratory cycle.     

Tidal volume effects on surfactant treatment responses with the initiation of ventilation in preterm lambs

Wada, Kazuko, Alan H. Jobe, and Machiko Ikegami. Tidalvolume effects on surfactant treatment responses with the initiation ofventilation in preterm lambs. J. Appl.Physiol. 83(4): 1054-1061, 1997.We hypothesizedthat initiation of ventilation in preterm lambs with high volumes wouldcause lung injury and decrease the subsequent response to surfactanttreatment. Preterm lambs were randomized to ventilation for 30 minafter birth with 5 ml/kg (VT5),10 ml/kg (VT10), or 20 ml/kg(VT20) tidal volumes and then ventilated with ~10 ml/kg tidal volumes to achieve arterialPCO₂ values of ~50 Torr to 6 h ofage. VT20 lambs had lowercompliances, lower ventilatory efficiencies, higher recoveries ofprotein, and lower recoveries of surfactant in alveolar lavages and in surfactant that had decreased compliances when tested in preterm rabbits than VT5 orVT10 lambs. Other lambsrandomized to treatment with surfactant at birth and ventilation with6, 12, or 20 ml/kg tidal volumes for 30 min had no indicators of lunginjury. An initial tidal volume of 20 ml/kg decreased the subsequentresponse to surfactant treatment, an effect that was prevented withsurfactant treatment at birth.

     

Effect of budesonide and salbutamol on surfactant properties.

The objective of this study was to evaluate the in vitro effect of budesonide and salbutamol on the surfactant biophysical properties. The surface-tension properties of two bovine lipid extracts [bovine lipid extract surfactant (BLES) and Survanta] and a rat lung lavage natural surfactant were evaluated in vitro by the captive bubble surfactometer. Measurements were obtained before and after the addition of a low and high concentration of budesonide and salbutamol. Whereas salbutamol had no significant effect, budesonide markedly reduced the surface-tension-lowering properties of all surfactant preparations. Surfactant adsorption (decrease in surface tension vs. time) was significantly reduced (P < 0.01) at a high budesonide concentration with BLES, both concentrations with Survanta, and a low concentration with natural surfactant. At both concentrations, budesonide reduced (P < 0.01) Survanta film stability (minimal surface vs. time at minimum bubble volume), whereas no changes were seen with BLES. The minimal surface tension obtained for all surfactant preparations was significantly higher (P < 0.01), and the percentage of film area compression required to reach minimum surface tension was significantly lower after the addition of budesonide. In conclusion, budesonide, at concentrations used therapeutically, adversely affects the surface-tension-lowering properties of surfactant. We speculate that it may have the same adverse effect on the human surfactant.     

Interaction between chitosan and bovine lung extract surfactants

The interaction between a cationic polyelectrolyte, chitosan, and an exogenous bovine lung extract surfactant (BLES) was studied using dynamic compression/expansion cycles of dilute BLES preparations in a Constrained Sessile Drop (CSD) device equipped with an environmental chamber conditioned at 37 degrees C and 100% R.H. air. Under these conditions, dilute BLES preparations tend to produce variable and relatively high minimum surface tensions. Upon addition of "low" chitosan to BLES ratios, the minimum surface tension of BLES-chitosan preparations were consistently low (i.e. <5 mJ/m2), and the resulting surfactant monolayers (adsorbed at the air-water interface) were highly elastic and stable. However, the use of "high" chitosan to BLES ratios induced the collapse of the surfactant monolayer at high minimum surface tensions (i.e. >15 mJ/m2). The zeta potential of the lung surfactant aggregates in the subphase suggests that chitosan binds to the anionic lipids (phosphatidyl glycerols) in BLES, and that this binding is ultimately responsible for the changes in the surface activity (elasticity and stability) of these surfactant-polyelectrolyte mixtures. Furthermore the transition from "low" to "high" chitosan to BLES ratios correlates with the flocculation and de-flocculation of surfactant aggregates in the subphase. It is proposed that the aggregation/segregation of "patches" of anionic lipids in the surfactant monolayer produced at different chitosan to BLES ratios explains the enhancing/inhibitory effects of chitosan. These observations highlight the importance of electrostatic interactions in lung surfactant systems.     

Alveolar sphingolipids generated in response to TNF-alpha modifies surfactant biophysical activity.

Sphingolipids represent a diverse group of bioactive lipid species that are generated intracellularly in response to tumor necrosis factor-alpha (TNF-alpha) and are implicated as potential mediators of acute lung injury. The purpose of these studies was to determine whether there was an extracellular, TNF-alpha-regulated pool of sphingolipids in the alveolus that modulates the surface tension lowering capacity of pulmonary surfactant. Intratracheal instillation of TNF-alpha in adult rats led to a twofold increase in the amount of surfactant-associated ceramide and tended to decrease levels of sphingomyelin without significantly altering sphingosine or sphinganine content. TNF-alpha induction of alveolar ceramide was associated with nearly an 80% increase in acid sphingomyelinase activity recovered in cell-free alveolar lavage. Ceramide administered in a dose-dependent manner potently antagonized the surface tension lowering effects of natural surfactant in vitro. Intratracheal TNF-alpha and ceramide treatment of rats significantly increased lung permeability, as was evidenced by extravasation of Evans blue dye into alveolar lavage and lung tissue. Thus these studies are the first to demonstrate the existence of a cytokine-regulated alveolar pool of sphingomyelin hydrolysis products that impairs the biophysical properties of the alveolar surfactant film. The results also suggest the presence of a secretory alveolar sphingomylinase that is TNF-alpha responsive and mediates effects of the cytokine on alveolar sphingolipid metabolism.     

The molecular mechanism of monolayer-bilayer transformations of lung surfactant from molecular dynamics simulations

The aqueous lining of the lung surface exposed to the air is covered by lung surfactant, a film consisting of lipid and protein components. The main function of lung surfactant is to reduce the surface tension of the air-water interface to the low values necessary for breathing. This function requires the exchange of material between the lipid monolayer at the interface and lipid reservoirs under dynamic compression and expansion of the interface during the breathing cycle. We simulated the reversible exchange of material between the monolayer and lipid reservoirs under compression and expansion of the interface. We used a mixture of dipalmitoyl-phosphatidylcholine, palmitoyl-oleoyl-phosphatidylglycerol, cholesterol, and surfactant-associated protein C as a functional analog of mammalian lung surfactant. In our simulations, the monolayer collapses into the water subphase on compression and forms bilayer folds. On monolayer reexpansion, the material is transferred from the folds back to the interface. The simulations indicate that the connectivity of the bilayer aggregates to the monolayer is necessary for the reversibility of the monolayer-bilayer transformation. The simulations also show that bilayer aggregates are unstable in the air subphase and stable in the water subphase.