Role of calcium ions the structure and function of pulmonary surfactant

Pulmonary surfactant isolated by centrifugation in buffers containing ions contains at least three different morphologic structures. The presence of one of these, tubular myelin, is dependent on calcium ions, since chelation of the calcium ions causes disruption of this structure. Addition of EDTA also decreases the ability of the surfactant to absorb rapidly to air-water interfaces and lower surface tension. Titration with calcium ions (2.5 or 5 mM) restores rapid surface adsorption and restores the tubular myelin structural forms. Magnesium ions cannot substitute for calcium ions in these processes. The reversibility of structure and function induced by calcium ions and EDTA is also accompanied by reversible isopycnic density shifts probably related to aggregation and disaggregation of the lipid-protein complex with calcium ions and EDTA, respectively.     

Fourier-transform infrared spectroscopy studies of lipid/protein interaction in pulmonary surfactant

The thermotropic behavior of intact bovine lung surfactant and its hydrophobic extract has been monitored via the temperature dependence of the 2850 cm-1 phospholipid acyl chain CH2 symmetric stretching frequencies in the IR spectrum. A broad, reversible, melting event was noted from about 15 to 40 degrees C in both the lipid extract and the native surfactant. Slight protein-induced disordering of the lipid acyl chains was evident. The melting event was confirmed by differential scanning calorimetry. The major surfactant protein, a 30-36-kDa class of glycoprotein (SP-A), has been isolated from bovine lung lavage and purified by affinity chromatography. SP-A was reconstituted into a binary lipid mixture of acyl chain perdeuterated dipalmitoylphosphatidylcholine/dipalmitoylphosphatidylglycerol (DPPC-d62/DPPG, 85:15 w/w), a ratio which approximates that in surfactant. Use of DPPC-d62 permitted the FT-IR determination of the effect of protein on the thermotropic behavior of individual phospholipids in the binary mixture. High levels of SP-A induced an ordering of the phospholipids, as shown by an increase in the transition temperature of DPPC-d62 compared to the lipid model. In contrast, a mixture of the other surfactant proteins induced a progressive disordering of the phospholipids and disruption of the cooperativity of the melting event. Transition widths of about 3 degrees, 9 degrees, and 27 degrees were noted for protein:lipid ratios of 0, 1:1, and 2:1 (w/w), respectively. Possible roles for the various proteins in surfactant function are discussed in light of these data.     

Interaction of the lipid and protein components of pulmonary surfactant Role of phosphatidylglycerol and calcium

We investigated the interaction of a major apolipoprotein of pulmonary surfactant with mixtures of lipids analogous to those found in natural surfactant. The apolipoprotein was extracted from canine surfactant and was purified to about 90% homogeneity. The apolipoprotein was mixed with liposomes of lipids in buffers containing 0.1 M sodium chloride and 3 mM calcium chloride at 22°C for 2 h or 37°C for 30 min. Two fractions were separated by centrifugation in sucrose density gradients at 15 000 rev./min. One was comprised of an aggregated, relatively high density recombinant lipoprotein which sedimented to a position toward the bottom of the centrifuge tube; the other remained at the top of the centrifuge tube and was mainly comprised of unbound lipid. The amount of lipid recovered as a sedimenting lipoprotein was dependent upon its composition. Those mixtures of lipids which contained dipalmitoyl phosphatidylglycerol formed sedimenting complexes which comprised 14% to 53% of the recovered lipid; those without phosphatidylglycerol formed such aggregates with less than 13% of the available lipid. Moreover, the lipid-to-protein stoichiometry of the recombinant was also dependent upon phosphatidylglycerol, and lipids containing this phospholipid displayed enhanced binding at a critical concentration of lipid which varied with temperature and composition. Calcium was required to form the sedimenting complex at 37°C. These results suggest that phosphatidylglycerol may be involved in the formation of a micelle-like complex, the stoichiometry of which is regulated over a narrow range of lipid concentration, and the structure of which involves calcium. The physiological advantage of forming this complex has not been determined. We found, however, that lipids containing phosphatidylglycerol absorbed more rapidly to an air/liquid interface than did those without. This rate of adsorption was further increased after interaction with the apolipoprotein.     

Partitioning of amphiphiles between coexisting ordered and disordered phases in two-phase lipid bilayer membranes

The partition coefficients (K(P)) of a series of single-chain and double-chain fluorescent amphiphiles, between solid ordered (P(beta') and L(beta)) and liquid disordered (L(alpha) of the type l(d)) lipid phases coexisting in the same lipid bilayer, was studied using steady-state fluorescence emission anisotropy. The single-chain amphiphiles were N-(7-nitrobenzoxa-2, 3-diazol-4-yl)-alkylamines, and the double-chain amphiphiles were N-(7-nitrobenzoxa-2, 3-diazol-4-yl)-phosphatidylethanolamines with chain lengths of 12-18 carbon atoms. Saturated 18-carbon alkyl/acyl chain compounds were also compared with Delta(9)-cis unsaturated chains of the same chain length. The fluorescence anisotropy of the probes was examined in lipid bilayers (multilamellar vesicles) prepared from an equimolar mixture of dilauroylphosphatidylcholine and distearoylphosphatidylcholine and studied as a function of temperature through the entire temperature range of coexistence of ordered gel phases and a disordered fluid phase in this system. The unsaturated chain amphiphiles partitioned exclusively into the fluid phase whenever this phase was present, as did the saturated chain amphiphiles with the shortest chains (C(12:0)), while K(P) ranges between 1 and 2, in favor of the L(beta) solid phase, for the amphiphiles with long saturated (C(18:0)) alkyl/acyl chains, with intermediate behavior for the intermediate chain lengths. All probes appeared to be totally excluded from P(beta') solid (gel) phases. The technique was also used to determine partitioning of some of the probes between coexisting liquid ordered (cholesterol-containing) (l(o)) and liquid disordered (l(d)) L(alpha) phases. In this case the ratio of signal amplitude to noise allowed us to obtain a qualitative, but not quantitative, measure of the phase partitioning of the probes. We conclude that the partitioning behavior of the probes examined between coexisting l(o) and l(d) phases is qualitatively similar to that observed between solid ordered and liquid disordered phases.     

The phase behavior of lipid monolayers containing pulmonary surfactant protein C studied by fluorescence light microscopy

Three compounds of the pulmonary surfactant – dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylglycerol (DPPG), and the surfactant associated protein C (SP-C) – were spread at the air-water interface of a Langmuir trough as a model system to mimic the properties of natural surfactant. Fluorescence microscopical images of the film formed at the interface were obtained during compression using a fluorescence dye bound covalently either to phosphatidylcholine or to SP-C. The images were quantified using statistical methods in respect to relative areas and relative fluorescence intensities of the domains found. In the early stage of compression, film pressure rose slightly and was accompanied by a phase separation which could be recognized in the images by the formation of bright and dark domains. On further compression, after a steep increase of film pressure, a plateau region of constant film pressure started abruptly. During compression in the plateau region, fluorescence intensity of the bright domain formed in the early stage of compression increased. The increasing fluorescence intensity, the non-Gaussian intensity distribution of the bright domain, and the small mean molecular area of the film in the plateau region gave rise to the assumption that multilayer structures were formed in the late stage of compression. The formation of the multilayer structures was fully reversible in repeated compression-expansion cycles including the plateau region of the phase diagram. The ability of lipid/SP-C mixtures to form reversible multilayer structures during compression may be relevant to stability in lungs during expiration and inhalation. Received: 13 February 1997 / Accepted: 22 May 1997     

Inhibition of pulmonary surfactant function by phospholipases

Previous studies have shown that respiratory failure associated with disorders such as acute pancreatitis correlates well with increased levels of phospholipase A2 (PLA2) in lung lavages and that intratracheal administration of PLA2 generates an acute lung injury. In addition, bacteria such as Pseudomonas have been shown to secrete phospholipase C (PLC). We studied the effects of these phospholipases on pulmonary surfactant activity using a pulsating bubble surfactometer. Concentrations greater than or equal to 0.1 unit/ml PLA2 destroyed surfactant biophysical activity, increasing surface tension at minimum bubble size from less than 1 to 15 mN/m. This surfactant inactivation was predominantly related to the effect of lysophosphatidylcholine on the surface film, although the fatty acids released with higher PLA2 concentrations also had a detrimental effect on surfactant function. Similarly, as little as 0.1 unit PLC increased the surface tension at minimal size of an oscillating bubble from less than 1 to 15 mN/m, an effect that could be mimicked by the addition of dipalmitin to surfactant in the absence of PLC. Moreover, lower, noninhibitory concentrations (0.01 unit/ml) of PLA2 and PLC increased the sensitivity of surfactant to other inhibitory agents, such as albumin. Thus, relatively low concentrations of PLC and PLA2 can cause severe breakdown of surfactant function and may contribute significantly to some forms of lung injury.     

ABCA3 as a lipid transporter in pulmonary surfactant biogenesis

ABCA3 protein is expressed predominantly at the limiting membrane of the lamellar bodies in alveolar type II cells, and mutations in the ABCA3 gene cause lethal respiratory distress in newborn infants. To investigate the function of ABCA3 protein, we generated Abca3-deficient mice by targeting Abca3. Full-term Abca3(-/-) newborn pups died within an hour after birth because of acute respiratory failure. Ultrastructural analysis revealed abnormally dense lamellar body-like organelles and no normal lamellar bodies in Abca3(-/-) alveolar type II cells. TLC and electrospray ionization mass spectrometry analyses of lipids in the pulmonary interstitium showed that phosphatidylcholine and phosphatidylglycerol, which contain palmitic acid and are abundant in normal surfactant lipids, were dramatically decreased in Abca3(-/-) lung. These findings indicate that ABCA3 plays an essential role in pulmonary surfactant lipid metabolism and lamellar body biogenesis, probably by transporting these lipids as substrates.     

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.     

Pulmonary surfactant: phase behavior and function

Pulmonary surfactant functions by first flowing rapidly into the alveolar air/water interface, but then resisting collapse from the surface when the adsorbed interfacial film is compressed during exhalation. Widely accepted models emphasize the importance of phase behavior in both processes. Recent studies show, however, that fluidity is a relatively minor determinant of adsorption and that solid films, which resist collapse, can form by kinetic processes unrelated to equilibrium phase behavior.     

Lateral phase separation in interfacial films of pulmonary surfactant.

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

X-ray diffraction study on ordered, disordered and reconstituted intercellular lipid lamellar structure in stratum corneum

From small angle X-ray diffraction for the stratum corneum of hairless mouse, it was obtained that in the normal stratum corneum, the 1st, 2nd and 3rd order diffraction peaks for the intercellular lipid lamellar structure appear at 13.8, 6.87 and 4.59 nm, respectively and also a broad hump for the 4th order reflection appears as observed by the previous researchers. In the damaged stratum corneum prepared by the treatment of sodium dodecyl sulfate, these small-angle diffraction peaks disappear and only the broad maxima remain around the 1st, 2nd and 3rd order diffraction peaks. These facts indicate that in the normal stratum the lamellar structure is ordered and in the damaged stratum corneum the lamellar structure is disordered. Furthermore, in the reconstituted lamellar structure obtained by immersing into the dilute suspension of the mixture of ceramide 3, cholesterol and stearic acid, the 1st, 2nd and 3rd order diffraction peaks reappear at 13.3, 6.67 and 4.44 nm, respectively. This fact indicates that the reorganization of the ordered lamellar structure takes place by adding the mixture to the damaged stratum corneum.     

The lipid transporter Mfsd2a maintains pulmonary surfactant homeostasis

Pulmonary surfactant is a lipoprotein complex essential for lung function, and insufficiency or altered surfactant composition is associated with major lung diseases, such as acute respiratory distress syndromes, idiopathic pulmonary fibrosis, and chronic obstructive pulmonary disease. Pulmonary surfactant is primarily composed of phosphatidylcholine (PC) in complex with specialized surfactant proteins and secreted by alveolar type 2 (AT2) cells. Surfactant homeostasis on the alveolar surface is balanced by the rates of synthesis and secretion with reuptake and recycling by AT2 cells, with some degradation by pulmonary macrophages and loss up the bronchial tree. However, whether phospholipid (PL) transporters exist in AT2 cells to mediate reuptake of surfactant PL remains to be identified. Here, we demonstrate that major facilitator superfamily domain containing 2a (Mfsd2a), a sodium-dependent lysophosphatidylcholine (LPC) transporter, is expressed at the apical surface of AT2 cells. A mouse model with inducible AT2 cell–specific deficiency of Mfsd2a exhibited AT2 cell hypertrophy with reduced total surfactant PL levels because of reductions in the most abundant surfactants, PC containing dipalmitic acid, and PC species containing the omega-3 fatty acid docosahexaenoic acid. These changes in surfactant levels and composition were mirrored by similar changes in the AT2 cell lipidome. Mechanistically, direct tracheal instillation of fluorescent LPC and PC probes indicated that Mfsd2a mediates the uptake of LPC generated by pulmonary phospholipase activity in the alveolar space. These studies reveal that Mfsd2a-mediated LPC uptake is quantitatively important in maintaining surfactant homeostasis and identify this lipid transporter as a physiological component of surfactant recycling.     

Altered function of pulmonary surfactant in fatty acid lung injury

To determine whether acute fatty acid lung injury impairs pulmonary surfactant function, we studied anesthetized ventilated rabbits given oleic acid (55 mg/kg iv, n = 11) or an equivalent volume of saline (n = 8). Measurements of pulmonary mechanics indicated a decrease in dynamic compliance within 5 min of injury and a decrease in lung volume that was disproportionately large at low pressures, consistent with diminished surfactant activity in vivo. Bronchoalveolar lavage fluid obtained 1 h after injury had significantly increased erythrocytes and total leukocytes, largely polymorphonuclear cells. The phospholipid content and composition of the cell-free fraction had only minor changes from those of controls, but the protein content was increased 35-fold. Measurements of lavage surface activity in vitro showed an increase in average minimum surface tension from 1.3 +/- 0.4 (SE) dyn/cm in controls to 20.2 +/- 3.9 dyn/cm in injured animals. The alterations in static pressure-volume curves and decrease in lavage surface activity suggest a severe alteration of surfactant function in this form of lung injury that occurs despite the presence of normal amounts of surfactant phospholipids.     

Effects of fixatives on function of pulmonary surfactant.

The structure of pulmonary surfactant films remains ill defined. Although plausible film fragments have been imaged by electron microscopy, questions about the significance of the findings and even about the true fixability of surfactant films by the usual fixatives glutaraldehyde (GA), osmium tetroxide (OsO(4)), and uranyl acetate (UA) have not been settled. We exposed functioning natural surfactant films to fixatives within a captive bubble surfactometer and analyzed the effect of fixatives on surfactant function. The capacity of surfactant to reach near-zero minimum surface tension on film compression was barely impaired after exposure to GA or OsO(4). Although neither GA nor OsO(4) prevented the surfactant from forming a surface active film, GA increased the equilibrium surface tension to above 30 mN/m, and both GA and OsO(4) decreased film stability as seen in the slowly rising minimum surface tension from 1 to ~5 mN/m in 10 min. In contrast, the effect of UA seriously impaired surface activity in that both adsorption and minimum surface tension were substantially increased. In conclusion, the fixatives tested in this study are not suitable to fix, i.e., to solidify, surfactant films. Evidently, however, OsO(4) and UA may serve as staining agents.