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
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ABSTRACT |
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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 (), 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
min was observed between the acidic POPG and the neutral
POPC systems with SP-B or SP-C with and without SP-A. However,
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
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
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
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INTRODUCTION |
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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 () of the interface and thus reduces the work of
breathing (7, 15, 19, 20, 25, 31, 33). Because films can
lower
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 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
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 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.
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METHODS |
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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 theQuasi-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 . The
min was achieved
when the bubble decreased in size without any further flattening on
increasing the chamber pressure. Overcompression of the bubble at low
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
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 min and
max among the
reconstituted surfactants. The compressibility of the film at a
of
15 mN/m [C15 = 1/A
(
A/
), where A is surface area and
A/
is the slope at a
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.
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RESULTS |
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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 , even with prolonged adsorption times. The
neutral PC system exhibited a slow initial adsorption phase followed by
a more rapid decline in
, which became apparent around 5 min. A
of ~40 mN/m was reached at ~120 min. The acidic PG system showed a
more rapid descent in
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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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 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,
max was lower than with the neutral POPC system.
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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 min.
Overcompression of the bubbles at low
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
max reached during
film expansion was higher. This was more evident with the neutral
system; the
max values attained during expansion
increased to ~60 mN/m vs. ~40 mN/m observed with the acidic
mixtures. Possibly because of the higher
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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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 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
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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Compression-expansion cycles.
The adsorbed films containing SP-C showed an improvement in surface
activity, particularly for the mixtures containing PG (Fig.
7). However, 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
max values achieved during expansion were lower,
remaining close to equilibrium
. 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
during the 4 min of delay between quasi-static cycles independent of the PL system.
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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 withIncluding 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.
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DISCUSSION |
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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 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
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 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
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 , 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 lowerIt 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 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 values near 0 mN/m. SP-C-based systems were only able to achieve
min
of 6-8 mN/m. This is similar to
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,
max during expansion was elevated with the
neutral PC system, and a larger compression ratio was required to reach
low
. 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
is increased above the equilibrium
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 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 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
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
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
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
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 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
SP-A-SP-C
SP-B
SP-A-SP-B
SP-A
> PL.
In contrast, rank order for surface activity as indicated by the
ability to attain low
values during quasi-static or dynamic cycling
was SP-A-SP-B > SP-B > SP-C
SP-A-SP-C
SP-A
PL. With the acidic system, rank order for adsorption was
SP-C
SP-A-SP-C > SP-B
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
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.
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ACKNOWLEDGEMENTS |
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We thank Dr. S-H. Yu for useful discussion and Dr. L. Stitt for assistance with the statistical analysis.
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FOOTNOTES |
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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.
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