Protein composition of rabbit alveolar surfactant subfractions

The goal of this investigation was to characterize the proteins in subfractions of alveolar surfactant obtained by lung lavage and separated by differential centrifugation. It was previously demonstrated that the material in the more sedimentable fraction, which was enriched in tubular-myelin and was surface-active may be a precursor to the less sedimentable, vesicular, inactive material [1]. Separation of the proteins by polyacrylamide gel electrophoresis showed that the more sedimentable subfractions and rabbit surfactant isolated by conventional methods contained proteins with molecular weights comparable to those previously reported for alveolar surface active material (approximately 36 000 and 10 000). The less sedimentable subfractions contained less of these proteins. Immunoblots with anti-dog surfactant apoprotein antibodies, which cross-react with rabbit proteins, supported these observations. Immunoblots also showed that all of the subfractions contained serum proteins and secretory IgA, with the less sedimentable subfractions containing more secretory IgA. These results suggested that changes in protein composition may accompany functional changes in surfactant in the alveoli.     

Changes in surfactant pools after a physiological increase in alveolar surfactant

We have used previously characterized models to investigate the reuptake of surfactant from the alveolus. In model 1, rats were swum in a water bath at 33 degrees C for 30 min, which increased tidal volume (VT) approximately 300% and frequency 60%; they were then allowed to rest for up to 4 h. In model 2, rats were exposed to 5% CO2-13% O2-82% N2 for 24 h, which increased both VT and frequency approximately 200%; these rats were then rested for up to 24 h. In both models we harvested a tissue fraction (lamellar bodies, lb) and two alveolar fractions--tubular myelin rich (alv-1) and tubular myelin poor (alv-2). Immediately after swimming, lb-dipalmitoylphosphatidylcholine (DPPClb) was 18% below the control of 0.94 +/- 0.037 (SE) mg/g wet lung (n = 24 rats; P less than 0.05); this returned to control by 2 h. Whereas DPPCalv-1 was constant at all time points, DPPCalv-2 was increased 50% above the control of 2.68 +/- 0.085 mg/g dry lung (n = 27 rats; P less than 0.001) immediately and up to 1 h after swimming. It returned to control levels between 2 and 3 h. After gas exposure, DPPC in lb, alv-1, and alv-2 was 33, 64, and 89%, respectively, above controls. All three fractions had normalized after 24 h. Our results demonstrate marked differences in the response of the surfactant system to acute and more prolonged stimuli. Of particular interest was the constancy of alv-1 with swimming, suggesting that it may be the controlled variable. However, the system appeared to be reset by prolonged hyperpnea, a process that may involve an increase in synthesis of surfactant.     

Structure and surface energy of the surfactant layer on the alveolar surface

The surface energy of the alveolar surfactant layer is determined in the scope of a modification of the structural model of Larsson et al. [(1999) J Disp Sci Technol 20:1-12], according to which this layer is built up of a lipid monolayer adsorbed at the hypophase/air interface and supported by a network of lipid bilayers immersed into the hypophase, i.e., the alveolar liquid. Formulae are derived for the dependence of the specific surface energy of the surfactant layer on the distance between the bilayers constituting the layer. It is shown that at equilibrium this energy can have values comparable with or less than 1 mJ/m2 needed for normal functioning of the alveolus during the respiration cycle. The specific surface energy of the surfactant layer with monolayer-bilayer structure can have such low values only if the layer is of optimal thickness and if the specific line energy of the monolayer-bilayer contact lines is negative and that of the bilayer-bilayer contact lines is positive. It is found that in dynamic regime the change in the specific surface energy of the alveolar surfactant layer with bilayer-monolayer structure is in qualitative agreement with that determined experimentally during lung inflation and deflation.     

Convertase activity in alveolar surfactant and lamellar bodies in fetal, newborn, and adult rabbits

Conversion ofheavy-aggregate alveolar surfactant (H) to a light-aggregate,nonsurface active form (L) is believed to involve the activity of anenzyme, namely, convertase. This conversion can bereproduced in vitro by the surface-area cycling technique. The purposeof the present study was to use this technique to investigate thedevelopmental aspects of convertase activity in fetal, newborn, andadult rabbits. H was isolated from alveolar lavage from term[31-day gestation (31d)] fetal rabbit pups, 1-, 4-, and7-day-old newborns, and adults, and the percent conversion to L wasdetermined. To assess lamellar bodies (LB) as a potential source ofactivity in this species, these structures were isolated from lungtissue of 27-day-gestation (27d) and 31d fetuses, 1-, 4-, and 7-day-oldnewborns, and adults and were cycled the same as for H. LB containedconsiderable activity at each developmental stage i.e., ~82% of a27d LB preparation converted to L after 3 h of cycling. In the adult,this value was 78%. Very little conversion of H was obtained fromfetal lung (i.e., <20% of the 31d fetal preparation converted to L),but, by postnatal day 4, this valuewas greatly increased (i.e., >80% conversion) and stayed elevated toadulthood. The activity for each H and LB fraction was temperature andconcentration dependent and diminished with storage at 4°C. Thesedata suggest the LB as the source of convertase activity in the rabbitand demonstrate dramatic developmental changes in this activity afterrelease of the LB contents to the alveoli.

     

The effects of substrate composition, quantity, and diversity on microbial activity

Variation in organic matter inputs caused by differences in plant community composition has been shown to affect microbial activity, although the mechanisms controlling these effects are not entirely understood. In this study we determine the effects of variation in substrate composition, quantity, and diversity on soil extracellular enzyme activity and respiration in laboratory microcosms. Microbial respiration responded predictably to substrate composition and quantity and was maximized by the addition of labile substrates and greater substrate quantity. However, there was no effect of substrate diversity on respiration. Substrate composition significantly affected enzyme activity. Phosphatase activity was maximized with addition of C and N together, supporting the common notion that addition of limiting resources increases investment in enzymes to acquire other limiting nutrients. Chitinase activity was maximized with the addition of chitin, suggesting that some enzymes may be stimulated by the addition of the substrate they degrade. In contrast, activities of glucosidase and peptidase were maximized by the addition of the products of these enzymes, glucose and alanine, respectively, for reasons that are unclear. Substrate diversity and quantity also stimulated enzyme activity for three and four of the six enzymes assayed, respectively. We found evidence of complementary (i.e., non-additive) effects of additions of different substrates on activity for three of the six enzymes assayed; for the remaining enzymes, effects of adding a greater diversity of substrates appeared to arise from the substrate-specific effects of those substrates included in the high-diversity treatment. Finally, in a comparison of measures of microbial respiration and enzyme activity, we found that labile C and nutrient-acquiring enzymes, not those involved in the degradation of recalcitrant compounds, were the best predictors of respiration rates. These results suggest that while composition, quantity, and diversity of inputs to microbial communities all affect microbial enzyme activity, the mechanisms controlling these relationships are unique for each particular enzyme.     

Changes in surface activity of surfactant and ultrastructure of the air-blood barrier in alcoholic intoxication in experimental animals

In the results of complex investigation of the lungs of 26 white rats, it was established, that there is the suppression of surface active properties of surfactant under influence of ethanol. In acute poisoning this suppression is associated with direct injury of surfactant with ethanol and inactivation of surfactant with serum proteins, which appear in the alveolar space because of the edema of air-haematic barrier. In prolonged influence the suppression of the surface activity of surfactant is due to the increase of its catabolism with alveolar macrophages.     

Influence of immobilization stress on the phospholipid composition of alveolar surfactant and lungs in rats

The influence of immobilization stress on the lipid composition of alveolar surfactant and lungs in rats immobilized for 12 and 24 hours, the effects of phospholipase A2, and lipid transfer activity in alveolar surfactant were investigated. The results indicate that alveolar surfactant phospholipids underwent more significant alterations compared to lung phospholipids. Furthermore, phospholipase A2 and lipid transfer activity were reduced in alveolar surfactant of immobilized rats. The reported data suggest that the lower lipid transfer activity might be responsible for the reduced phospholipids in the surfactant system.     

Structure and surface energy of the surfactant layer on the alveolar surface: inaccuracies and their correction

The article of Kashchiev and Exerowa (Eur Biophys J 30:34-41) is shown to incorporate a number of inaccuracies that fit the categories "historical", "anatomical", and "biophysical". These inaccuracies are corrected by reference to published research reports from 1978 to 1998. The monolayer-bilayer model proposed by Kashchiev and Exerowa may be thermodynamically correct in vitro, but has not been related to the structure of the alveolar surface in vivo, which is that of a foam ("the alveolar surface network").     

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.     

Cancer cachexia alters intracellular surfactant metabolism but not total alveolar surface area

Dyspnoea is frequently observed in cancer cachectic patients. Little is known whether this is accompanied by structural or functional alterations of the lung. We hypothesized that in analogy to calorie restriction cancer cachexia leads to loss of alveolar surface area and surfactant. Mice were subjected to subcutaneous injection of Lewis lung carcinoma cells (tumour group, TG) or saline (control group, CG). Twenty-one days later blood samples and the lungs were taken. Using design-based stereology, the alveolar surface area and the lamellar body (Lb) content were quantified. Messenger RNA expression of surfactant proteins, ABCA3 and various growth factors was investigated by quantitative RT-PCR. Intraalveolar surfactant subtype composition was analyzed by differential centrifugation. TG mice showed reduced body weight and anaemia but no reduction of lung volume or alveolar surface area. The volume of Lb was significantly reduced and mRNA levels of ABCA3 transporter tended to be lower in TG versus CG. Surfactant protein expression and the ratio between active and inactive intraalveolar surfactant subtypes were not altered in TG. Growth factor mRNA levels were not different between CG and TG lungs but the tumour expressed growth factor mRNA. Vascular endothelial growth factor was significantly enhanced in blood plasma. The present study demonstrates structural alterations of the lung associated with cancer cachexia. These include reduction of Lb content despite normal intraalveolar surfactant and alveolar surface area. The pulmonary phenotype of the cancer cachectic mouse differs from the calorie restricted mouse possibly due to growth factors released from the tumour tissue.     

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.     

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.     

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.