Surface activity of the surfactant in experimental compression atelectasis

Surface surfactant activity was studied at different time periods of compression atelectasis, induced by hydrothorax in 35 guinea pigs. The animals were slaughtered 30 and 60 minutes or 3, 12 and 24 hours after hydrothorax. It has been demonstrated that experimental compression atelectasis is accompanied by surface activity lowering, associated with disorders in surfactant secretion into the alveolar lumen. The qualitative composition of surfactant phospholipids remains unchanged, which may play an essential role in the recovery of lung tissue aeration.     

The role of palmitic acid in pulmonary surfactant: enhancement of surface activity and prevention of inhibition by blood proteins

The surface activity of two surfactant preparations, Lipid Extract Surfactant (LES) and Survanta, was examined during adsorption and dynamic compression using a pulsating bubble surfactometer. At low surfactant phospholipid concentrations (1-2.5 mg/ml), Survanta reduces surface tension at minimum bubble radius faster than LES: however, with continued pulsation LES obtains a lower surface tension. Addition of surfactant-associated protein A (SP-A) to LES significantly reduces the time required to reduce surface tension. Survanta is completely unresponsive to the addition of SP-A in that no further reduction of surface tension is observed. Addition of various blood components has been previously shown to inactivate surfactants in vitro. Addition of fibrinogen to Survanta causes an increase in surface tension when measured in the absence of calcium. When assayed in the presence of calcium, inhibition by fibrinogen is not observed possibly due to aggregation of this protein. Albumin and alpha-globulin strongly inhibit Survanta at physiological serum concentrations both in the presence and absence of calcium. The surface activity of Survanta is also inhibited by lysophosphatidylcholine (lyso-PC). The role of palmitic acid in the surface activity of pulmonary surfactant was examined by adding palmitic acid to LES. At low phospholipid concentrations addition of palmitic acid (10% w/w of the surfactant phospholipid) greatly enhances the surface activity of LES. Maximal enhancement of surface activity and adsorption was observed at or above 7.5% added palmitic acid (w/w of surfactant lipid). LES supplemented with palmitic acid is more resistant to inhibition by fibrinogen, albumin, alpha-globulin and lyso-PC than LES alone, however, the counteraction of blood protein inhibition is not as pronounced as that observed with SP-A.     

Surface activity following natural surfactant treatment in premature lambs

Premature lambs with respiratory failure [CO2 partial pressure (PCO2) greater than 70 Torr] were treated with 50 mg/kg 3H-labeled natural surfactant by tracheal instillation. Minimum surface tensions of sequential samples suctioned from the airways fell from 25 +/- 3 dyn/cm before treatment to 8 +/- 5 dyn/cm after treatment and again rose to 32 +/- 2 dyn/cm at death. Minimum surface tensions of alveolar wash samples taken at death were 27 +/- 4 dyn/cm, whereas surfactant fractions reisolated from the alveolar washes lowered surface tension to under 10 dyn/cm. The alveolar washes, surfactant reisolated from the alveolar washes, and natural surfactant had similar phospholipid compositions; however, the alveolar washes contained about 40 times more protein per micromole phosphatidylcholine. The natural surfactant used for treatment apparently was inactivated by an inhibitor of surfactant function. After intravenous injections of [14C]palmitic acid, labeled saturated phosphatidylcholine appeared on the airways, indicating endogenous synthesis and secretion. However, the specific activity of the 3H-labeled saturated phosphatidylcholine in the natural surfactant used for treatment decreased by only 30 +/- 4% in the alveolar wash; thus the treatment dose was not diluted to a large extent by endogenous pools.     

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.     

Proteolipid in bovine lung surfactant: its role in surfactant function

The chemical and biophysical properties of the proteins in the lipid extracts of lung surfactant have not clearly been determined. These proteins were isolated from lung surfactant lipids by Sephadex LH-20 chromatography and purified with silicic acid chromatography followed by dialysis against organic solvents. The proteolipid thus obtained had a protein to phospholipid ratio of 3 to 1 (w/w). The proteolipid apoprotein had a nominal molecular weight of ca. 5 kDa. We evaluated the functional role of this proteolipid by combining it with proteolipid-depleted surfactant lipids or synthetic dipalmitoylphosphatidylcholine (DPPC) and then measuring with a pulsating bubble surfactometer. The proteolipid and DPPC recombinant reproduced the surface activity of natural lung surfactant. We conclude that this 5 kDa proteolipid apoprotein is a functionally important constituent of lung surfactant.     

Structure of pulmonary surfactant membranes and films: the role of proteins and lipid-protein interactions

The pulmonary surfactant system constitutes an excellent example of how dynamic membrane polymorphism governs some biological functions through specific lipid-lipid, lipid-protein and protein-protein interactions assembled in highly differentiated cells. Lipid-protein surfactant complexes are assembled in alveolar pneumocytes in the form of tightly packed membranes, which are stored in specialized organelles called lamellar bodies (LB). Upon secretion of LBs, surfactant develops a membrane-based network that covers rapidly and efficiently the whole respiratory surface. This membrane-based surface layer is organized in a way that permits efficient gas exchange while optimizing the encounter of many different molecules and cells at the epithelial surface, in a cross-talk essential to keep the whole organism safe from potential pathogenic invaders. The present review summarizes what is known about the structure of the different forms of surfactant, with special emphasis on current models of the molecular organization of surfactant membrane components. The architecture and the behaviour shown by surfactant structures in vivo are interpreted, to some extent, from the interactions and the properties exhibited by different surfactant models as they have been studied in vitro, particularly addressing the possible role played by surfactant proteins. However, the limitations in structural complexity and biophysical performance of surfactant preparations reconstituted in vitro will be highlighted in particular, to allow for a proper evaluation of the significance of the experimental model systems used so far to study structure-function relationships in surfactant, and to define future challenges in the design and production of more efficient clinical surfactants.     

Inhibition of surfactant activity by Pneumocystis carinii organisms and components in vitro

This study examines the direct inhibitory effects of Pneumocystis carinii (Pc) organisms and chemical components on the surface activity and composition of whole calf lung surfactant (WLS) and calf lung surfactant extract (CLSE) in vitro. Incubation of WLS suspensions with intact Pc organisms (10(7) per milligram of surfactant phospholipid) did not significantly alter total phospholipid levels or surfactant protein A content. Incubation with intact Pc organisms also did not impair dynamic surface tension lowering in suspensions of WLS or centrifuged large surfactant aggregates on a bubble surfactometer (37 degrees C, 20 cycles/min, 0.5 and 2.5 mg phospholipid/ml). However, exposure of WLS or CLSE to disrupted (sonicated) Pc organisms led to severe detriments in activity, with minimum surface tensions of 17-19 mN/m vs. <1 mN/m for surfactants alone. Extracted hydrophobic chemical components from Pc (98.8% lipids, 0.1 mM) reduced the surface activity of WLS and CLSE similarly to sonicated Pc organisms, whereas extracted hydrophilic chemical components from Pc (primarily proteins) had only minor effects on surface tension lowering. These results indicate that in addition to surfactant dysfunction induced by inflammatory lung injury and edema-derived inhibitors in Pc pneumonia, disrupted Pc organisms in the alveolar lumen also have the potential to directly inhibit endogenous and exogenous lung surfactants in affected patients.     

A biophysical mechanism by which plasma proteins inhibit lung surfactant activity

These in vitro experiments study a potential mechanism by which plasma proteins, found in the alveoli during pulmonary edema and hemorrhage, may act to inhibit the surface activity of pulmonary surfactant. The results indicate that the inhibition of the adsorption facility and surface tension lowering ability of a calf lung surfactant extract (CLSE) by albumin, hemoglobin, or fibrinogen may be completely abolished by centrifugation of the protein-surfactant mixture at 12,500 x g. Furthermore, albumin, hemoglobin and fibrinogen (1.25 mg/ml) were shown to inhibit the adsorption of high concentrations of CLSE (0.32 mg/ml), normally unaffected by the addition of exogenous proteins, when the CLSE was injected into the subphase under a preformed protein surface film. Similarly, injection of large amounts of these proteins (2.5 mg/ml) into the subphase beneath a preformed CLSE surface film was without effect, even though the CLSE concentration was only 0.06 mg/ml, a surfactant concentration which is normally inhibited by even small amounts of exogenous protein. Taken together, the data suggest that some proteins may inhibit surfactant function by preventing the surfactant phospholipids from adsorbing to the air-liquid interface, possibly by a competition between the proteins and CLSE phospholipids for space at the air-liquid interface rather than direct molecular interactions between proteins and surfactant.     

The role of ribosomal proteins in in vitro ribosome-membrane interactions]

The in vitro binding of total ribosomal proteins with rough endoplasmic membranes, from which 70% of ribosomes are eliminated by EDTA (ME) is studied. It is found that in conditions of specific interaction of ribosomes with membranes about 75% of total ribosomal proteins are bound with ME. Membranes, heterogenous in their content (different protein/lipid ratio), became homogenous in their buyoant density after the binding with proteins. The ability of membrane-ribosomal protein complex to bind ribosomes is not decreased, as it can be expected, but is considerablly increased, thus indicating on a non-specific character of ribosome binding. Ribosomal subunits lacking about half of structural protein are capable to bind with ribosome-binding membrane receptors and with some additional sites. This binding is also non-specific, because the binding efficiency of large and small subunits is the same.     

Immunoregulatory functions of surfactant proteins

Because the lungs function as the body's gas-exchange organ, they are inevitably exposed to air that is contaminated with pathogens, allergens and pollutants. Host-defence mechanisms within the lungs must facilitate clearance of inhaled pathogens and particles while minimizing an inflammatory response that could damage the thin, delicate gas-exchanging epithelium. Pulmonary surfactant is a complex of lipids and proteins that enhances pathogen clearance and regulates adaptive and innate immune-cell functions. In this article, I review the structure and functions of the surfactant proteins SP-A and SP-D in regulating host immune defence and in modulating inflammatory responses.     

The role of surfactant in asthma

The study of genetic polymorphisms has touched every aspect of pulmonary and critical care medicine. We review recent progress made using genetic polymorphisms to define pathophysiology, to identify persons at risk for pulmonary disease and to predict treatment response. Several pitfalls are commonly encountered in studying genetic polymorphisms, and this article points out criteria that should be applied to design high-quality genetic polymorphism studies.     

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.     

Pulmonary surfactant and inflammation in septic adult mice: role of surfactant protein A.

Surfactant alterations, alveolar cytokine changes, and the role of surfactant protein (SP)-A in septic mice were investigated. Sepsis was induced via cecal ligation and perforation (CLP). Septic and sham mice were euthanized at 0, 3, 6, 9, 12, 15, and 18 h after surgery. Mice deficient in SP-A and mice that overexpressed SP-A were euthanized 18 h after surgery. In wild-type, sham-operated mice, surfactant pool sizes were similar at all time points, whereas in the CLP groups there was a significant decrease in small-aggregate surfactant pool sizes beginning 6 h after CLP. Interleukin-6 concentrations in bronchoalveolar lavage fluid from septic animals increased from 6 to 18 h after surgery. Identical surfactant alterations and concentrations of cytokines were observed in septic mice that were SP-A deficient or that overexpressed SP-A. In conclusion, alterations of pulmonary surfactant and alveolar cytokines occur simultaneously, 6 h after a systemic insult. In addition, we did not detect a role for SP-A in regulating surfactant phospholipid pool sizes or pulmonary inflammation in septic mice.     

Effects of hydrophobic surfactant proteins on collapse of pulmonary surfactant monolayers

To determine if hydrophobic surfactant proteins affect the stability of pulmonary surfactant monolayers at an air/water interface, the studies reported here compared the kinetics of collapse for the complete set of lipids in calf surfactant with and without the proteins. Monomolecular films spread at the surface of captive bubbles were compressed at 37°C to surface pressures above 46 mN/m, at which collapse first occurred. The rate of area-compression required to maintain a constant surface pressure was measured to directly determine the rate of collapse. For films with and without the proteins, higher surface pressures initially produced faster collapse, but the rates then reached a maximum and decreased to values <0.04 min⁻¹ above 53 mN/m. The maximum rate for the lipids with the proteins (1.22 ± 0.28 min⁻¹) was almost twice the value for the lipids alone (0.71 ± 0.15 min⁻¹). Because small increments in surface pressure produced large shifts in the rate close to the fastest collapse, compressions at a series of constant speeds also established the threshold rate required to achieve high surface pressure as an indirect indication of the fastest collapse. Both samples produced a sharply defined threshold that occurred at slightly faster compression with the proteins present, supporting the conclusion of the direct measurements that the proteins produce a faster maximum rate of collapse. Our results indicate that at 47-53 mN/m, the hydrophobic surfactant proteins destabilize the compressed monolayers and tend to limit access to the higher surface pressures at which the lipid films become metastable.     

The proteins of human lung surfactant

Human pulmonary surfactant was purified from bronchoalveolar lavage of patients. The proteins present in surfactant were analyzed by SDS-polyacrylamide gel electrophoresis into serum and non-serum components. One non-serum surfactant protein (Mr = 43 000) was then identified in the 100 000 X g supernatant of a lung homogenate on the basis of phospholipid binding. This lung protein was purified and partially characterized. The presence of 3-methyl histidine and reaction in Western blot analysis with antibody against chicken muscle actin both strongly suggested that the 43 000 Da protein of human surfactant is indeed cytoplasmic actin. It is proposed that this surfactant protein is involved in the secretion and not necessarily in the function of surfactant.     

Surface activity and film formation from the surface associated material of artificial surfactant preparations

Surfactant proteins B and C (SP-B and SP-C) are present in natural derived surfactant preparations used for treatment of respiratory distress syndrome. Herein the surface activity of an SP-C analogue (SP-C(LKS)), a hybrid peptide between SP-C and bacteriorhodopsin (SP-C/BR) and a model peptide (KL(4)) was studied with a captive bubble surfactometer (CBS). The peptides were mixed with either 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)/phosphatidylglycerol (PG) (7:3, by weight) or DPPC/PG/palmitic acid (68:22:9, by weight) at a concentration of 1 mg/ml in HEPES buffer, pH 6.9 and a polypeptide/lipid weight ratio of 0.02--0.03. In some lipid/peptide preparations also 2% of SP-B was included. Adsorption, monitored as surface tension vs. time for 10 min after bubble formation did not show discernible differences for the whole set of preparations. Equilibrium surface tensions of approximately 25 mN/m were reached after 5--10 min for all preparations, although those with SP-C/BR appeared not to reach end point of adsorption within 10 min. Area compression needed to reach minimum surface tension of 0.5--2.0 mN/m was least for the KL(4) preparation, about 13% in the first cycle. 3% SP-C(LKS) in DPPC:PG (7:3, by weight) reached minimum surface tension upon 27% compression in the first cycle. If DPPC:PG:PA (68:22:9, by weight) was used instead only 16% area compression was needed and 14% if also 2% SP-B was included. 3% SP-C(LKS) in DPPC:PG (7:3, by weight)+2% SP-B needed 34% compression to reach minimum surface tension. The replenishment of material from a surface associated surfactant reservoir was estimated with subphase depletion experiments. With the 2% KL(4) preparation incorporation of excess material took place at a surface tension of 25--35 mN/m during stepwise bubble expansion and excess material equivalent to 4.3 monolayers was found. When 2% SP-B was added to 3% SP-C(LKS) in DPPC:PG (7:3, by weight) the number of excess monolayers increased from 1.5 to 3.6 and the incorporation took place at 30--40 mN/m. When SP-B was added to 3% SP-C(LKS) in DPPC:PG:PA (68:22:9, by weight) the number of excess monolayers increased from 0.5 to 3.4 and incorporation took place at 40--50 mN/m. With 2% SP-C/BR incorporation took place at 40--45 mN/m, frequent instability clicks were observed and excess material of approximately 1.1 monolayer was estimated.