Surfactant protein interactions with neutral and acidic phospholipid films

Karina Rodriguez-Capote1,2, Kaushik Nag1, Samuel Schürch3, and Fred Possmayer1

1 Departments of Obstetrics and Gynaecology and Biochemistry, University of Western Ontario, London, Ontario N6A 5A5; 3 Departments of Physiology and Biophysics, University of Calgary, Calgary, Alberta, Canada T2N 4N1; and 2 Departamento de Bioquimica, Instituto Superior de Ciencias Médicas de la Habana-Instituto de Ciencias Básicas y Preclínicas Victoria de Giron, Havana, Cuba


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 µg/ml with physiological levels of the surfactant protein (SP) A SP-B, and SP-C alone and in combination at 37°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.

acute lung injury; acute respiratory distress syndrome; captive bubble tensiometer; dipalmitoylphosphatidylcholine; lipid monolayer; phosphatidylglycerol; phosphatidylcholine; respiratory distress syndrome; surface area cycling; surface tension; synergism


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

PULMONARY SURFACTANT IS ESSENTIAL for normal lung function. By forming highly surface active films at the air-alveolar fluid interface, surfactant reduces the surface tension (gamma ) of the interface and thus reduces the work of breathing (7, 15, 19, 20, 25, 31, 33). Because films can lower gamma  to near 0 mN/m during lateral compression, they prevent alveolar collapse at end expiration. Surfactant also prevents alveolar edema, and some components at least have an important role in lung defense. Surfactant dysfunction is an important factor contributing to the pathophysiology of several diseases including neonatal respiratory distress syndrome and adult respiratory distress syndrome. Although some variation exists among species and with certain disease states, surfactant is composed mainly of phospholipids (PL; ~85%), and small amounts of surfactant-associated protein (SP) A, SP-B, SP-C, and SP-D (20, 33). The major surfactant PL are dipalmitoylphosphatidylcholine (DPPC; 35-50%), unsaturated phosphatidylcholine (PC; 25-35%) and the acidic PL phosphatidylglycerol (PG; 8-15%).

It is generally accepted that DPPC is primarily responsible for the ability of lung surfactant films to attain low gamma  during film compression (7, 19, 20, 25, 31, 33). However, the contribution of lipid components of surfactant other than DPPC remains vague. Recent studies by our laboratory, conducted with a captive bubble tensiometer (CBT), indicated that preparations composed of DPPC-1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPG)-SP-B (70:30:1) (14) were more effective in lowering gamma  to low values during the initial and subsequent film compressions than equivalent mixtures containing PC instead of PG.

The present investigations extended the former studies to include examination of the relative effectiveness of PG or PC on surfactant film formation and the ability to achieve low gamma  in the presence of SP-C. The effects of SP-A on the surface activity of preparations containing PC-PG and SP-B-SP-C were also examined. Furthermore, to gain insight into the role of individual surfactant components and their interactions under limiting conditions where adsorption is slow, surfactant concentration was lowered to 50 µg/ml, 5% of the previously used concentration. The relative levels of the hydrophobic apoproteins were maintained at the physiological levels observed with natural surfactant.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Materials

DPPC was purchased from Sigma (St. Louis, MO) and 1-palmitoyl-2-oleoyl-sn-glycero-3 phospho-rac-(1-glycerol)-sodium salt (POPG-Na) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) were obtained from Avanti Polar Lipids (Birmingham, AL). Bovine serum albumin was obtained from Sigma. Other chemicals were of the highest reagent quality available.

Protein Isolation

Pulmonary protein A was purified by chromatography on mannose columns as described by Cockshutt et al. (5). The purity of the protein was tested by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE; 12% gel) under reducing conditions.

Pulmonary SP-B and SP-C were isolated from bovine lipid extract surfactant (BLES) (BLES Pharmaceuticals, London, ON, Canada) by a modification of the LH-20/LH-60 method of Curstedt et al. (6) as previously described (21). The purity of the proteins was assessed by SDS-PAGE using 18% gels (39). Protein concentrations were determined by a modification of the Lowry et al. (12, 21) method. Correction factors of 2.0 for SP-B and 3.0 for SP-C relative to bovine serum albumin were adopted on the basis of amino acid analysis as indicated previously (43). SP-C palmitoylation was quantified by the triethylamine-iodoacetamide procedure (22). PL, estimated by phosphorus assay (23), could not be detected in the SP-B preparation but contributed ~3.9% of total PL in the reconstituted SP-C samples.

Sample Preparation

The lipids DPPC, POPG, and POPC were dissolved in chloroform-methanol (3:1) and mixed in ratios of DPPC to POPG or DPPC to POPC (70:30) with and without 1% by weight SP-B or 2% SP-C, also in organic solvent. These levels were adopted on the basis of previous studies estimating total and relative levels of SP-B and SP-C in lipid extracts of bovine surfactant using amino acid analysis and NH2-terminal quantification through dansylation (39). The lipids and the lipid-protein mixtures were dried under a stream of nitrogen in Teflon tubes and then reconstituted in the following buffer (in mM): 0.150 NaCl, 2 Tris · HCl, and 1.5 CaCl2 at a pH 7.0 with and without SP-A (1% wt/wt) to a final concentration of 50 µg/ml. Initial studies in which the reconstituted samples were sonicated at 0-4°C using a Branson ultrasonic sonicator equipped with a microprobe revealed that under the conditions used adsorption due to SP-B could be markedly reduced. Consequently, for the studies reported here, the samples were vortexed and shaken on a Burrel unit wrist shaker with glass beads for 1 h at room temperature until the suspension became completely homogeneous. Samples were incubated at 37°C for at least 1 h before studies on surface activity were initiated (40-42).

Captive Bubble Tensiometry

A custom designed CBT was employed to study the properties of the surface films by measuring adsorption and estimating the gamma  values of dynamically or quasi-statically contracting or expanding bubbles (24). After the CBT chamber was filled with the desired lipid or lipid-protein suspension and the temperature equilibrated at 37 ± 1°C, an air bubble ~8 mm in diameter was introduced into the suspension. The change in bubble shape was recorded to monitor the adsorption of the materials to the bubble air-saline interface. After adsorption, the bubble chamber was sealed, and quasi-static or dynamic compression-expansion of the bubble was performed. Images documenting changes in bubble area were recorded during each individual experiment, and bubble shapes were analyzed using custom-designed software (24).

Quasi-static experiments were conducted by increasing the pressure in the sample chamber stepwise by slowly turning the plunger 5-10° (~10% of bubble volume per step) and waiting 10 s after each step. We conducted five quasi-static cycles, with an intercycle delay of 4 min to allow the system to reequilibrate. As the pressure increased, the bubble volume and surface area decreased, compressing the adsorbed film at the air-water interface. The bubble progressively flattened, indicating a lower gamma . The gamma min was achieved when the bubble decreased in size without any further flattening on increasing the chamber pressure. Overcompression of the bubble at low gamma  values was avoided wherever possible. In separate experiments, dynamic modes were conducted by cycling the bubble at 30 cycles/min. With use of the experience gained with the quasi-static studies, the surface area of the bubble was reduced until the film reached gamma min and then expanded to 100-120% of original area and recycled between these two limits.

All experiments were performed at least three times with individual freshly prepared samples. Standard deviation of the mean was obtained from n = 3 or greater sets of data, using separate samples. Surface tensions of adsorbed PL films at 120 min were compared by t-test. Statistical analysis by ANOVA and Tukey's highly significant difference test for multiple comparisons was used to detect differences in gamma min and gamma max among the reconstituted surfactants. The compressibility of the film at a gamma  of 15 mN/m [C15 = 1/A (delta A/delta gamma ), where A is surface area and delta A/delta gamma is the slope at a gamma  of 15 mN/m] for each surfactant was compared against a baseline value of 0.005 (mN/m)-1, compressibility of pure DPPC films (20, 24), using a t-test. Significance was concluded when P < 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Studies With SP-A and SP-B

Adsorption. Adsorption isotherms for DPPC-POPC and DPPC-POPG with and without SP-A and SP-B are shown in Fig. 1. In the absence of proteins, film formation was very slow and neither system attained equilibrium gamma , even with prolonged adsorption times. The neutral PC system exhibited a slow initial adsorption phase followed by a more rapid decline in gamma , which became apparent around 5 min. A gamma  of ~40 mN/m was reached at ~120 min. The acidic PG system showed a more rapid descent in gamma  within 1 min, followed by a slower decrease. The two distinct phases observed with the neutral system could not be clearly distinguished with the acidic system. The ~30 mN/m observed at 120 min was significantly lower (P < 0.005, n = 3) than that observed with the neutral PC system.


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Fig. 1.   Adsorption isotherms of dipalmitoylphosphatidylcholine (DPPC)-1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) (A) and DPPC-1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPG) (B) in the absence and presence of surfactant protein (SP) A, SP-B, or SP-A-SP-B combined. Isotherms were obtained by monitoring adsorption of 50 µg/ml of lipid or lipid-protein mixtures in the captive bubble tensiometer (CBT) at 37°C. Data are means ± SE of 3 independent experiments.

Addition of SP-A (1% relative to the weight of PL) resulted in a further decrease in gamma . This effect was more pronounced with the neutral system. However, in both systems, the adsorption isotherms appeared to consist of two phases: an initial slow stage, followed by a more accelerated second phase at ~15 min, until the equilibrium gamma  (~23 mN/m) was achieved.

When SP-B (1% wt/wt) was added, a more rapid decrease in gamma  relative to PL alone was observed until equilibrium with both systems. In the PL-SP-B adsorption isotherms, the two adsorption phases were not as evident as with PL alone or with the PL-SP-A systems. However, a small secondary phase at ~10 min was still evident with the neutral system. With the acidic system in the presence of SP-B, only a slight deviation was perceptible, with the individual curves between 7 and 12 min, and the shape of the curve approached a rectangular hyperbola. Further addition of SP-A to the neutral PL-SP-B system resulted in a rapid initial decrease in gamma  ("first adsorption phase"), followed by another decline after ~5 min as the gamma  reduction became slower ("second adsorption phase"). In contrast, the presence of both SP-A and SP-B in the acidic PL system resulted in a slight retardation in the initial rate of gamma  reduction, although the curve described an almost perfect rectangular hyperbola. With both systems, the films reached equilibrium gamma  at approximately the same time in the presence of either SP-B or SP-A-SP-B. In all cases, the acidic PG-protein mixtures attained equilibrium prior to the corresponding neutral PC-protein system.

Quasi-static compression-expansion cycles. Once the systems had equilibrated, the films were subjected to five quasi-static compression-expansion cycles (see METHODS). Isotherms were constructed for the first, third, and fifth compression and expansion cycles [Fig. 2, A (DPPC-POPC) and B (DPPC-POPG)]. Lipid mixtures in the absence of proteins needed large compression ratios (more than 60% surface area reduction) and only achieved a gamma min of ~15 mN/m. With both systems, the slopes of the isotherms decreased around 20-30 mN/m, producing a plateau. Such plateaus can be indicative of the squeeze-out of the more fluid lipids (1, 24, 25). Despite the apparent squeeze-out of fluid lipids, minima of only ~15 mN/m were attained. A very small improvement in terms of partial elimination of the squeeze-out plateau was observed between the first and fifth cycles with the acidic POPG system. The acidic system also exhibited less hysteresis, indicating possible respreading of the film during film reexpansion. In keeping with this suggestion, gamma max was lower than with the neutral POPC system.


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Fig. 2.   Quasi-static compression ()-expansion (open circle ) isotherms of DPPC-POPC (A) and DPPC-POPG (B) in the absence and presence of SP-A, SP-B, or SP-A-SP-B combined. Isotherms were constructed by averaging 3 independent experiments for the 1st, 3rd, and 5th compression-expansion cycles. The compressibility at 15 mN/m (C15) for the individual systems are presented in the rectangular boxes. N/A, not applicable.

Although addition of SP-A allowed the film to achieve equilibrium gamma  during adsorption, gamma min values of only ~10-12 mN/m were attained during film compression, indicating little improvement over PL alone. Broad horizontal isotherms, indicative of squeeze-out plateaus, were generated with both systems containing SP-A in all cycles. Hysteresis was greater compared with PL alone. With the acidic system, gamma max increased during expansion and remained elevated at the initiation of compression, indicating film deterioration.

The compressibility at 15 mN/m (C15) can be used to describe film properties below the squeeze-out plateau (25). Relatively high values (poor surface activity) of ~0.035 (POPC) and 0.065 (POPG) were observed for PL alone compared with the C15 of 0.005 of pure DPPC films.

The addition of SP-B to PL enhanced surface activity so that both systems could be compressed to near 0 mN/m. Similar to SP-A containing samples, broad squeeze-out plateaus initiating around 20 mN/m were observed, followed by a more rapid decrease to near 0 mN/m. With the neutral system, ~80% surface area reduction was required to achieve low gamma , and a slight decrease in the length of the squeeze-out plateau was noted on cycling. In contrast, with the acidic system, low gamma  was attained with ~40% surface area reduction and a shorter squeeze-out plateau was observed even during the first cycle. However, due to the increased gamma max during expansion, there was little improvement in the overall surface area reduction required with successive cycles. C15 remained relatively high for the neutral POPC system. A considerably improved compressibility was observed with the acidic system such that the C15 values for the third and fifth cycles were statistically indistinguishable from DPPC.

Addition of SP-A to the SP-B-containing mixtures resulted in a clear improvement in surface activity for both films. There was shortening in the length of the squeeze-out plateaus. Consequently, with the initial cycle, only ~60% (neutral PC system) and ~30% (acidic PG system) film compression was sufficient to achieve gamma min near 0 mN/m. A further decrease in required surface area reduction on cycling was manifested with both systems. These arose first from an additional shortening of the squeeze-out plateaus and second from the lower gamma max attained during film expansion compared with the PL-SP-B samples. The acidic PG system appeared to be more surface active at each cycle due principally to the shorter squeeze-out plateau observed at the first cycle. It is important to note that these films, unlike PL-SP-B, were able to decrease the gamma  close to equilibrium gamma  during the 4 min of delay between quasi-static cycles independently of the PL system. Compressibility was improved with the neutral system over SP-B alone. Compressibility was even lower with the POPG system. C15 values for the third and fifth cycles were similar to DPPC films.

Because the surface film compressions started at different initial gamma  values, the percent area reductions required to achieve near 0 mN/m are not directly comparable. Consequently, to obtain a suitable assessment of surface activity, the percent area reduction required to achieve gamma  near 0 from 20 mN/m (just below the equilibrium gamma ) were calculated for each sample. Figure 3, A (neutral system) and B (acidic system), displays the percentage of surface area compression required for the films to attain a gamma min near 0 from 20 mN/m during quasi-static cycling. Extrapolated compressions of over 100% were estimated for the PL alone and with SP-A, films that could not attain low gamma min even with maximum possible compression.


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Fig. 3.   Percentage of surface area compression required for the films to attain a minimum surface tension (gamma min) near 0 from 20 mN/m during quasi-static cycling. Extrapolated compressions of over 100% were estimated for the phospholipid (PL) alone and with SP-A because these films could not attain low gamma min even at 90% of compression. Comparisons among the neutral or acidic surfactant systems were conducted using Tukey's studentized range (highly significant difference) test. Significant differences between cycles were observed for SP-A-SP-B in the neutral system (A) and SP-B and SP-A-SP-B in the acidic system (B). In addition, these analyses demonstrated significant differences between the 2 PL systems involving homologous preparations in the corresponding cycle, except for PL alone or PL-SP-A. Letters, comparisons among surfactant mixtures within a cycle. Means with the same letter are not significantly different.

Addition of SP-B improved surface activity during compression with both systems but this was superior with the acidic system. Less than 40% surface area reduction was required during the first cycle and consecutive cycles required ~20%. The small area reduction observed here with DPPC-POPG-SP-B mixtures (Fig. 3B) suggests that after the third cycle the film is highly enriched in DPPC. Addition of SP-A resulted in a further enhancement of the surface activity with both systems. The presence of both proteins dramatically decreased the percent compression necessary to achieve gamma  near 0 mN/m and also showed significant improvement with cycling. With the acidic PG system, the area reduction required approached ~15%, which is similar to that reported for pure DPPC films (20, 24, 25). Comparison of the neutral PC and acidic PG systems by ANOVA revealed a number of differences between homologous mixtures during corresponding cycles. Except for PL alone or with SP-A, PG-containing samples required significantly lower compression ratios for any given cycle. As noted above, C15 for the acidic system for SP-B and SP-A-SP-B approached that of DPPC.

Dynamic compression-expansion cycles. Dynamic cycling of surfactant films in the CBT mimics more closely normal respiratory compression and expansion within the alveolar airspace. For these experiments, adsorbed films were cycled at a rate of 30 cycles/min between 100 and 120% of the original bubble surface area and the area required to achieve gamma min. Overcompression of the bubbles at low gamma  was avoided. The data were averaged from three separate experimental samples, and isotherms for cycles 1, 10, and 21 are shown in Fig. 4, A (neutral system) and B (acidic system). In general, the overall conclusions obtained from dynamic cycling were similar to those obtained in quasi-static cycling. However, the gamma max reached during film expansion was higher. This was more evident with the neutral system; the gamma max values attained during expansion increased to ~60 mN/m vs. ~40 mN/m observed with the acidic mixtures. Possibly because of the higher gamma max, the neutral system showed little improvement in compression ratio during cycling. In contrast, the acidic system demonstrated marked improvement during cycling in the presence of the SP-A-SP-B combination (Fig. 4B).


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Fig. 4.   Dynamic compression ()-expansion (open circle ) isotherms of DPPC-POPC (A) and DPPC-POPG (B) in the absence and presence of SP-A, SP-B, or SP-A-SP-B combined. Isotherms were constructed by averaging 3 independent experiments for the 1st, 10th, and 21st dynamic cycles. Compressibilities at 15 mN/m (C15) for individual systems are presented in the boxes.

The gamma min values were similar to those observed quasi-statically. The percent area reductions required to attain gamma  near 0 from 20 mN/m were lower with the acidic system for all the PL mixtures containing SP-B or SP-A-SP-B (Fig. 5). C15 values were higher than those observed during quasi-static cycling with PL alone and with SP-A but were similar or even lower with SP-B and SP-A-SP-B, consistent with excellent surface activity under a more physiological condition. POPG-based surfactants containing SP-B and SP-A-SP-B possessed C15 values that were statistically similar to DPPC alone.


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Fig. 5.   Percentage surface area compression required for the films to attain a gamma min near 0 from 20 mN/m during dynamic cycling. Extrapolated compressions of over 100% were estimated for the PL alone and with SP-A because these films could not attain low gamma min even at 90% compression. Comparisons among surfactants were conducted using Tukey's studentized range (highly significant difference) test. Significant differences between cycles were observed for SP-A-SP-B in the neutral system (A) and SP-B and SP-A-SP-B in the acidic system (B) PL films. In addition, these analyses demonstrated significant differences between the 2 PL systems involving homologous preparations in the corresponding cycle, except for PL alone or PL-SP-A. Letters, comparisons among surfactant mixtures within a cycle. Means with the same letter are not significantly different.

Studies With SP-C and SP-A-SP-C

Adsorption. The presence of SP-C enhanced adsorption with both PL systems (Fig. 6). Similar to the PL-SP-B mixtures, equilibrium gamma  was attained more rapidly with the acidic system. Further addition of SP-A slightly retarded adsorption with both PL systems during initial adsorption, although equilibrium gamma  was attained at similar times to the absence of SP-A with both systems. Initial and secondary adsorption phases were observed with the neutral system but were not evident with the acidic system.


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Fig. 6.   Adsorption isotherms of DPPC-POPC (A) and DPPC-POPG (B) with SP-C or SP-A-SP-C combined. Isotherms were obtained by monitoring adsorption in the CBT of 50 µg/ml of lipid-protein mixtures at 37°C. Data are means ± SE of 3 independent experiments.

Compression-expansion cycles. The adsorbed films containing SP-C showed an improvement in surface activity, particularly for the mixtures containing PG (Fig. 7). However, gamma min of only ~6-8 mN/m was achieved during film compression, and it tended to be higher with the acidic POPG system. This contrasts with SP-B-containing mixtures, which readily reached values near 0 mN/m. PL-SP-C combinations also differed from those with SP-B in that the gamma max values achieved during expansion were lower, remaining close to equilibrium gamma . There was no evidence of squeeze-out plateaus with the acidic system. The surface tension-surface area isotherms were almost linear. However, little improvement was observed during consecutive quasi-static cycles. These films, similar to PL-SP-A-SP-B, were able to reestablish equilibrium gamma  during the 4 min of delay between quasi-static cycles independent of the PL system.


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Fig. 7.   Quasi-static compression ()-expansion (open circle ) isotherms of DPPC-POPC (A) and DPPC-POPG (B) in the presence of SP-C or SP-A-SP-C combined. Isotherms were created by averaging 3 independent experiments for the 1st, 3rd, and 5th compression-expansion cycles. C15 values for each system are presented in the boxes.

Addition of SP-A to PL-SP-C samples resulted in a reduction of the surface activity. This was indicated by the appearance of a broad squeeze-out plateau, which was considerably longer for the acidic system. These results demonstrate the absence of advantageous interactions between SP-C and PG compared with PC and the lack of synergism between SP-C and SP-A, which was evident between SP-B and SP-A for both PL systems.

Expression of the results as percent compression required to reach low gamma  from 20 mN/m (Fig. 2) also corroborated that SP-C was not as effective as SP-B in lowering gamma min of adsorbed films. Little improvement was observed on consecutive quasi-static (Fig. 3) or dynamic (Fig. 5) cycles. Further addition of SP-A provided no significant improvement with POPC, and with the acidic system, the film deteriorated.

Overall, dynamic C15 values in the presence of SP-C and SP-A-SP-C were similar to those observed with quasi-static cycling. This may reflect the ability of SP-C-based samples to respread rapidly. The exception was SP-A-SP-C with the acidic system, which exhibited higher compressibility (poor surface activity) with prolonged dynamic cycling (Fig. 8).


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Fig. 8.   Dynamic compression ()-expansion (open circle ) isotherms of DPPC-POPC (A) and DPPC-POPG (B) in the presence of SP-C or SP-A-SP-C combined. Isotherms were created by averaging 3 independent experiments for the 1st, 10th, and 21st compression-expansion cycles. C15 values are presented in the boxes.

Studies With SP-B-SP-C and SP-A-SP-B-SP-C

Additional studies were conducted using PL-SP-B-SP-C combinations. Quasi-static studies conducted with SP-B-SP-C generated compression-expansion curves with gamma min values similar to the SP-B curves shown, whereas gamma max values were low as with the SP-C-based surfactants with both systems (data not shown). These studies indicated some additive effects with both hydrophobic proteins. As for SP-B and SP-A-SP-B, C15 values for the PG system were similar to pure DPPC during the third and fifth quasi-static and the tenth and twenty-first dynamic compressions (data not shown).

Including SP-A at 1 or 5% did not lead to further improvement during quasi-static cycling. In addition, similar overall conclusions were obtained when such samples were examined under dynamic conditions.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Attempts at determining the mechanisms by which pulmonary surfactant achieves low tensions at the air-water interface of the alveoli during expiration have been hampered by the complex nature of this biological material. We have attempted to define specific roles for individual components through reconstitution experiments in which SP-B or SP-C was added to DPPC-POPC or DPPC-POPG (70:30) mixtures. In addition, the interactions between these low molecular weight, hydrophobic proteins and SP-A were examined with the above lipid systems. The CBT was employed for these studies because of previous experience, which showed that this apparatus can retain complex films at high surface pressures (low gamma  values) at 37°C without apparent loss of surface active material from the interface. The ability of the CBT to maintain film stability at low gamma  values at 37°C is presumably related to the bubble resting against a hydrophilic agar plug that does not have any potential escape routes for "creep" or "leakage." This contrasts with studies using the Langmuir-Wilhelmy balance (8, 24) and the pulsating bubble surfactometer (42), where surface material can be lost, presumably to the Teflon walls and barrier or to the plastic capillary.

Adsorption Studies

In agreement with previous studies (15, 19, 20, 25, 35), pure PL molecules showed slow adsorption. With the neutral lipid system, two distinct adsorption phases were apparent: a slow initial phase and a secondary, more rapid phase at ~5 min. A secondary adsorption phase was also observed with SP-A at ~10 min. Although less distinct, there was evidence of secondary adsorption phases for the neutral lipid system with SP-A, SP-B, SP-A-SP-B, SP-C and SP-C-SP-A.

With the acidic PG system, little or no evidence for a secondary adsorption phase was noted either with pure phospholipids or in the presence of SP-B, and these curves approached rectangular hyperbolas. However, secondary phases could be clearly detected with SP-A alone (~15 min) and with SP-A-SP-B (~5 min). The existence of two distinct phases in adsorption has been suggested previously by Walters et al. (35), who examined the ability of SP-B plus SP-C to promote adsorption of DPPC, DPPC-DPPG, neutral surfactant lipids (lacking anionic lipids), and total surfactant lipids. Their results suggested that the initial reduction in gamma  during adsorption depends strongly on anionic phospholipids, which are more effective in juxtaposition of vesicles to the surface due to favorable electrostatic interactions with the interface. These initial interactions are relatively independent of lipid fluidity. Later stages of adsorption were less dependent on charge but were more effective with fluid lipids. These later interactions involved a cooperative association of PL vesicles with material already adsorbed at the interface, in agreement with former studies by Oosterlaken-Dijksterhuis et al. (16, 17), who demonstrated that spread monolayers containing SP-B or SP-C facilitated insertion of lipids from subphase vesicles into the monolayer in a calcium-dependent manner. The reported ability of anionic lipids to facilitate initial interactions with the air-water interface is consistent with the apparent lack of a lag phase and a lower final gamma  with the acidic PG system relative to the neutral lipid system. Furthermore, in each of the other combinations examined, the acidic system achieved equilibrium earlier than the corresponding neutral PC system counterpart.

Including 1% SP-A led to the appearance of a clearly observed secondary adsorption phase with all combinations except DPPC-POPG-SP-B (Fig. 1B) and DPPC-POPG-SP-C (Fig. 6B). It is possible that the lack of a distinct secondary adsorption phase relates to a more rapid initial adsorption phase with these SP-B- and SP-C-based samples. However, it should be noted that adsorption with this PL system is very much retarded compared with adsorption rates observed with BLES or natural surfactant, which is considerably more fluid than the present mixtures. SP-A can aggregate lipid vesicles (13), and this might contribute to the slower initial adsorption with DPPC-PG-SP-A-SP-B (Fig. 2B) and DPPC-PC-SP-A-SP-C (Fig. 6A). However, the nature of the secondary adsorption phase observed with SP-A and why it has not been reported previously are not understood and will require further investigation. The fact that this phenomenon was observed with both neutral and acidic systems in the presence and absence of SP-B and SP-C suggests an interaction with DPPC may be involved. The results reported here are surprising in that SP-A did not accelerate the ability of the lipid mixtures containing SP-B to attain equilibrium gamma , as previously observed by our group and others (3, 5, 10, 13, 20, 34, 40, 42). The SP-A preparation used for these studies greatly accelerates film formation with BLES under conditions similar to those used here. Therefore, the lack of an effect on adsorption with the SP-B-based system may be related to the low fluidity of the DPPC-POPC and DPPC-POPG combinations employed.

With the neutral PL mixture, SP-A did not affect time to equilibrium with either the SP-B- or SP-C-based systems. In all cases, times to equilibrium were shorter with the acidic PL mixtures. SP-C-based systems attained equilibrium slightly more rapidly than the SP-B systems, and as with the neutral PL system, SP-A did not influence time to equilibrium with either hydrophobic protein. These results differ from previous Wilhelmy balance and pulsating bubble studies using DPPC-egg PG, where SP-C was considerably more effective in promoting adsorption than SP-B and SP-A had a major effect on adsorption in the presence of SP-B (40, 42). In addition, studies employing total lung surfactant PL and acidic lipid-depleted PL from calf lung surfactant extracts indicated SP-B was as or more effective in promoting adsorption than SP-C (36). These differences would indicate fluidity can influence the adsorptive effectiveness of reconstituted surfactants. The present conditions, which result in the absence of a significant effect of SP-A on the time to reach equilibrium in the presence of SP-B or SP-C, were deliberately chosen to establish whether the rate of equilibrium had an effect on film properties during compression, as discussed in the next section.

Film Compression

In addition to accelerating surface film formation, surfactant apoproteins alter the properties of surface films so that they are more surface active (i.e., they attain lower gamma  values during the lateral compression generated by surface area reduction) (7, 15, 19, 20, 25). Whereas the PL-SP-A and PL-SP-B mixtures both attain equilibrium gamma  values, only the SP-B-based surfactant can attain low tensions. Likewise, although it might be anticipated that pure lipid films compressed to equilibrium would have similar structures to SP-B-based films adsorbed to equilibrium, the former have extremely limited surface activity (Fig. 2). This implies a fundamental difference in the structures of these films at equilibrium, leading to a difference in their ability to form nonmonolayer structures during compression. SP-C-based films are surface active but, unlike SP-B-containing films, cannot reduce gamma  below 6-8 mN/m under the same conditions, also implying a structural difference.

It may be constructive to consider these observations in terms of the surface-associated surfactant reservoir proposed by Schürch (7, 19, 20, 25, 27), which considers the surface film to be composed of an interfacial monolayer plus material attached to that monolayer. Subphase washout experiments have demonstrated that this reservoir can provide surface-active material to the interface during overexpansion of adsorbed films at equilibrium gamma  and can organize surfactant material during overcompression at low tensions near 0 mN/m, so that the collapse phase material reinserts into the surface monolayer during reexpansion. The surface-associated reservoir thus corresponds to functional material associated with the monolayer. It has become clear that SP-B and SP-C can contribute to the formation and function of this surfactant reservoir. We hypothesize that the reservoir is formed during adsorption and that the rate of film formation and the chemical composition of the film determine the size and the efficacy of this surface-associated reservoir.

These and other results (32) indicate that the surfactant reservoir formed in the presence of SP-B can provide a compartment or pool, which can accept non-DPPC lipids from the surface monolayer and can supply DPPC-enriched lipids during film expansion. The results presented here indicate that SP-B is more effective than SP-C in forming a functional reservoir capable of attaining gamma  values near 0 mN/m. SP-C-based systems were only able to achieve gamma min of 6-8 mN/m. This is similar to gamma min reported during dynamic compression of Survanta, a lipid extract surfactant which is largely SP-C based (4, 9). In addition to achieving lower tensions on the initial compression, the SP-B-based mixtures showed improved surface activity during quasi-static and dynamic cycling (Figs. 2B, 3B, and 4B). Although improvement was observed with both the neutral PC and the acidic PG systems, surface activity remained superior with the acidic system. In each case, gamma max during expansion was elevated with the neutral PC system, and a larger compression ratio was required to reach low gamma . Thus there appears to be a specific interaction between SP-B and PG. Whether this interaction is limited to the more rapid film formation during the initial adsorption involves a more efficient squeeze-out of fluid lipid during compression or is influenced by adsorption of fresh surfactant material during film expansion when gamma  is increased above the equilibrium gamma  cannot be determined definitively from the present data. However, the relatively slow adsorption rates observed (Fig. 1, A and B) and the disappearance of hysteresis during cycling would indicate that reorganization of the surface film during compression-expansion cycles provides the major basis for the improvement in surface activity. This interpretation is consistent with previous studies by Ingenito et al. (11), who demonstrated the acidic PL in calf lung surfactant extracts contributed to the reduction in film compression ratio to attain low tensions during dynamic cycling of SP-B plus SP-C-containing samples on a pulsating bubble. These authors also observed inclusion of the acidic PL negated the inhibitory effects of neutral lipids during dynamic cycling.

Although addition of SP-A alone enhanced film formation with both the neutral PC and the acidic PG systems, little improvement in the ability to reduce gamma  during compression was noted. If anything, SP-A alone appears to broaden the squeeze-out plateau at ~20 mN/m with both systems (Fig. 2B) and delay adsorption during dynamic cycling. Thus although under the conditions used, SP-A-based films achieve equilibrium, they exhibit very poor surface activity relative to SP-B- or SP-C-based films during quasi-static or dynamic cycling. This demonstrates that the ability to achieve equilibrium is not sufficient to promote surface activity during compression. Including SP-A with SP-B led to a distinct improvement even during the initial compression from equilibrium. Improvement was observed with both the neutral PC- and the acidic PG SP-B-based systems. No improvement was observed with the SP-C systems. Rather, SP-A interfered with the surface activity due to SP-C alone in the initial and subsequent cycles. These data are compatible with a SP-A-SP-B interaction, leading to an improved reservoir compared with SP-B alone, both during film formation and during cycling regardless of the fluid PL component. There appears to be no beneficial interaction between SP-A and either SP-C or PG with the given systems. These conclusions are consistent with previous pulsating bubble studies using acidic PL systems, which did not observe synergistic effects between SP-A and SP-C (10, 40).

It is important to note that under the conditions employed, SP-A had a miniscule effect on the rate of attaining equilibrium in the presence of SP-B. This would indicate that the improved surface activity of SP-A-SP-B films observed in the initial and subsequent compressions relates to a cooperative effect on reservoir formation, as opposed to the enhancement in the rate of film formation reported previously for SP-B-based systems (2, 5, 10, 26, 34, 40). Recent studies have shown that fluorescent SP-A tends to localize very near fluorescent SP-B but not near fluorescent SP-C (29). SP-A present in the subphase specifically localizes under gel phase domains generated by compressing porcine lipid extract surfactants but not DPPC films (38; Nag K, Petersen N, and Possmayer F, unpublished results). Novel structures observed during compression of DPPC-POPG-SP-B films over subphase SP-A are not seen when SP-C is substituted for SP-B (29). Although some of these latter studies have been conducted at room temperature rather than at 37°C, they support a special relationship between SP-A and SP-B but not SP-C. This potential relationship is reinforced by the observation that SP-A and SP-B, along with DPPC, PG, and calcium, suffice for the in vitro formation of tubular myelin. Tubular myelin appears to be a major source of the surface film in vivo (28, 37).

The SP-C-based surfactant preparations examined here were only able to reduce gamma  to 6-8 mN/m, even though film formation to equilibrium was more rapid than with the corresponding SP-B-based surfactants. Thus surfactant apoprotein composition has a more important role in generating an effective reservoir than the rate of film formation. SP-C-based reconstituted surfactants show a limited ability to attain low tensions with Wilhelmy balance and pulsating bubble studies (18, 36). However, SP-C-based surfactants tended to display lower gamma max values during quasi-static and dynamic cycling, an observation consistent with the previous reports from this and other laboratories (21, 30). The ability to limit gamma  during inspiration may be important in the lung in vivo. Also, SP-C-based surfactants display a superior ability to reinsert lipids forced out of the interfacial monolayer into a reservoir during overcompression at very low tensions (21, 27). It should be mentioned that DPPC-egg PG-SP-C mixtures prepared with the SP-C used for the present studies achieved gamma min <=  3 mN/m when tested at 1 mg/ml. This indicates that surfactant fluidity and concentration (discussed below) can influence reservoir formation. This ability of SP-C-based surfactants to achieve low gamma min under appropriate conditions agrees with previous studies conducted with the CBT (21, 30).

The present studies were conducted at the relatively low concentration of 50 µg/ml PL. It has long been appreciated that increasing surfactant bulk concentration markedly improves interfacial properties (3, 26, 27). For example, BLES adsorbed to equilibrium at 50 µg/ml requires a surface area reduction of 60% to attain low tensions, whereas 30% is sufficient with films adsorbed at 1 mg/ml. Increasing bulk concentrations of the DPPC-POPC-SP-B or DPPC-POPG-SP-B mixtures used for the present studies to 1 mg/ml led to a marked reduction in the length of the squeeze-out plateau and compressibility at C15 (data not shown). This concentration effect explains apparent discrepancies between the present and previous studies employing higher concentrations of DPPC-POPC and DPPC-POPG with and without SP-B (14). Because the effect of bubble concentration remains after subphase washout (26, 27), these effects are consistent with formation of a larger or an improved reservoir with higher bulk concentrations or the presence of SP-A during film formation (25, 27). Except for the SP-A addition experiments reported here (Fig. 2), improved surface activity associated with SP-A addition or increased bulk concentrations consistently correlates with enhanced adsorption rate. It can be added that the improved surface activity of egg PG over POPG-containing samples discussed in the previous paragraph was accompanied by accelerated adsorption. It is clear that the relationship between adsorption rates and surface activity during film compression is complex and requires further study.

In summary, we have examined the ability of surfactant apoproteins, alone and together, to facilitate film formation and the attainment of very low gamma  values during film compression with low concentrations (50 µg/ml) of DPPC-PC and DPPC-PG (70:30) using a CBT at 37°C. Rank order for adsorption with the neutral PL system was SP-C approx  SP-A-SP-C approx  SP-B approx  SP-A-SP-B SP-A > PL. In contrast, rank order for surface activity as indicated by the ability to attain low gamma  values during quasi-static or dynamic cycling was SP-A-SP-B > SP-B > SP-C approx  SP-A-SP-C SP-A approx  PL. With the acidic system, rank order for adsorption was SP-C approx  SP-A-SP-C > SP-B approx  SP-A-SP-B > SP-A > PL. Rank order for first compression surface activity during cycling was SP-A-SP-B > SP-B SP-C SP-A-SP-C SP-A PL. The results show that there is no direct correlation between the rate of film formation and surface activity during compression. Under the conditions used, SP-A had no effect on film formation rate with either SP-B- or SP-C-based systems. However, as reported previously, this colectin markedly improved surface activity with the SP-B-PG system. We found that SP-A also enhanced surface activity with the neutral PL samples containing SP-B. The improvement due to SP-A was more evident with the acidic PG system, confirming SP-B-PG interactions reported previously. SP-A hampered the ability of SP-C-based surfactants to attain low tensions with the acidic system. These results further emphasize the previously identified synergism between SP-A and SP-B. The present investigations also confirm apparent interactions between SP-B and acidic lipids. Such interactions were not evident with SP-C-based surfactants. The results are consistent with the previously suggested ability of SP-B to promote selective adsorption of DPPC and the squeeze-out of fluid, particularly acidic fluid lipids. SP-C can also promote squeeze-out of fluid lipids during compression and contributes to respreading to maintain low gamma max during surface area expansion. A modest synergy between SP-B and SP-C was observed, particularly during dynamic cycling. Although SP-A enhances film formation with the present PL systems, by itself it has little effect on surface activity during compression. However, SP-A enhances the surface activity of SP-B- but not SP-C-based surfactants.


    ACKNOWLEDGEMENTS

We thank Dr. S-H. Yu for useful discussion and Dr. L. Stitt for assistance with the statistical analysis.


    FOOTNOTES

These studies were supported by grants from the Ontario Thoracic Society and Group Grant M268C7 from the Medical Research Council (MRC) of Canada. K. Nag was the recipient of a Canadian Lung Association-MRC Fellowship and S. Schürch is an Alberta Heritage Senior Scientist.

The CBT used for these studies was constructed with funds from an equipment grant from the MRC (Canada) and support from the Academic Development Fund of the University of Western Ontario

Address for reprint requests and other correspondence: F. Possmayer, Dept. of Obstetrics and Gynaecology, Univ. of Western Ontario, London Health Sciences Centre, 339 Windermere Road, London, ON, Canada N6A 5A5 (E-mail: fpossmay{at}uwo.ca).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 23 August 2000; accepted in final form 9 February 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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