1 Medical Service, A serine-active enzyme, "surfactant
convertase," is required for the conversion of surfactant from the
tubular myelin (TM) form to the small vesicular (SV) form. This
transformation involves at least two steps, the conversion of TM to a
surface-active film at the air-fluid interface and the reorientation of
the film into the surface-inactive SV form; we asked if convertase was
required for the first of these steps. Rat and mouse TMs were
pretreated with diisopropyl fluorophosphate (DFP) to inactivate
endogenous convertase activity or with vehicle and then were analyzed
for their ability to lower surface tension in vitro as an index of the
conversion of TM to a surface film. DFP pretreatment did not alter the
ability of TM preparations to lower surface tension, as assessed by
pulsating bubble, and it did not affect the behavior of TM in a surface
balance. In an experiment designed to test the ability of TM to feed a
surface film to exhaustion, TMs that had been pretreated with DFP or
vehicle performed similarly. These experiments show that convertase
activity is not required for the conversion of TM to a monolayer and
suggest, instead, that convertase acts at a post surface film stage.
surfactant subtype; surfactant conversion
PULMONARY SURFACTANT is secreted from alveolar type II
cells as lamellar bodies (LB) and evolves sequentially from this form through the tubular myelin (TM) form of surfactant, a surface film, and
a small vesicular (SV) form (1, 9, 12, 16, 17). The mechanisms that
control surfactant extracellular metabolism through these subtypes are
largely unknown. Although some of these steps appear to proceed
spontaneously under appropriate ionic conditions (14), a newly
described serine-active enzyme, "surfactant convertase," appears
to be required for the conversion of TM through SV (7, 10, 11). The
molecular actions of this enzyme and its substrate are unknown. The
present study examines its role in surfactant biophysics.
The conversion of TM, which is surface active, to SV, which is much
less surface active, can be reproduced in vitro by a procedure that we
call "surface cycling" (9). Such conversion requires not only the
presence of convertase activity but also the presence of an air-liquid
interface (11). The latter requirement suggests that the conversion of
TM to SV probably involves at least two steps,
1) conversion of TM to the surface
film and 2) conversion of the film
to SV, and is consistent with the postulated sequence of surfactant
subtype evolution referenced above. The essential role of convertase in
the overall conversion of TM to SV has been derived from in vitro
experiments (9, 10) in which these two steps are not distinguishable.
The aim of the present experiments was to determine if convertase
activity was required for the first of these steps, the conversion of
TM to the surface film, as inferred by the ability of purified TM to
express surface activity in the presence or absence of convertase. We
studied the rate of film formation using a pulsating bubble, the
minimum surface tension ( All reagents were obtained from Sigma Chemical (St. Louis, MO), and
solvents were obtained from Fisher Scientific (Fair Lawn, NJ).
Animals and radiolabeling.
All experiments were performed on mice or rats. Female CF#1 mice, ~15
g body wt, were obtained from Carworth Farms (Portage, MI); female
Sprague-Dawley rats, ~150 g, were obtained from Charles River
Laboratories (Wilmington, MA). For experiments in which radiolabeled
surfactant was used, groups of three to six mice received 20 µCi of
[3H]choline (NEN,
Boston, MA) by intraperitoneal injection 18 h before death.
TM preparation.
TM was obtained as described previously (8, 10) from bronchoalveolar
lavage of rats or mice that had received lethal injections of
pentobarbital sodium. [The method described here yields a mixture
of tubular myelin and myelin-like membranes, as seen by electron
microscopy (8). For convenience, we call this "TM" or "the TM
form" of surfactant throughout this paper.] The lungs were
lavaged pertracheally with lavage buffer (AF: 0.15 M NaCl,
2 mM CaCl2, and 5 mM
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, pH 7.4 at 37°C) that was flushed into and out of the lungs three times with five changes. After lavage, the animals with lungs in
situ were placed in a 37°C incubator, and a small amount of AF
buffer containing 10 µM isoproterenol (as a surfactant secretogogue) was instilled into the lungs. This was flushed into and out of the
lungs at 10-min intervals and was removed after 30 min. This process
was repeated one time. All lavages, the initial bronchoalveolar lavages
and the material obtained from incubated lungs, were combined and
divided into two halves. One half was incubated with diisopropyl fluorophosphate (DFP), final concentration 10 mM, at 23°C for 30 min to inhibit endogenous convertase activity. The other half was
similarly incubated but without DFP. Treated and untreated preparations
were separately centrifuged through a step of 0.1 M sucrose in AF
buffer onto a cushion of 0.9 M sucrose in AF buffer in a SW28 rotor at
25,000 revolutions/min (rpm) for 2 h at 4°C. The material that
accumulated at the 0.1/0.9 M interface was aspirated, diluted with
5-10 volumes of AF buffer, and sedimented in the SW28 rotor at
25,000 rpm for 2 h. The pellet, called TM preparation, was resuspended
in a small volume of AF buffer, dispersed by a single gentle pass
through a glass-Teflon Potter homogenizer (size AA; AH Thomas,
Philadelphia, PA), and analyzed for surface activity immediately or
frozen for later analysis.
ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References
min)
obtained using a pulsating bubble and a modified Kimray-Greenfield surfactometer, and the rate at which a subphase of TM was able to feed
surfactant to the surface, in each case comparing purified TM with and
without active convertase. We found no differences in surface
biophysics in any of these experiments, from which we infer that
convertase is not required for the conversion of TM to a surface-active
film. This result suggests that convertase is more likely to act at a
post surface film stage in the extracellular metabolism of surfactant.
METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References
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Fig. 1.
Validation of a tubular myelin (TM) preparation. Continuous sucrose
gradients were centrifuged to equilibrium, and the 3 phospholipid
profiles were separated for viewing.
Gradient A is an aliquot of TM that was not
cycled and shows that most of the phospholipid resolved around a broad
peak buoyant density of 1.051 g/ml, which is typical of TM. A minor
peak is present at 1.061 g/ml, which is typical of the lamellar body
(LB) form. Gradient B is an aliquot of the same
preparation that had been cycled for 2 h in vitro and shows that more
than one-half of the phospholipid is now present at a peak of buoyant
density (1.025 g/ml), which is typical of the "light" or small
vesicular (SV) subtype. The rest remained at the buoyant density of TM.
Gradient C is of an aliquot of TM that had been
pretreated with diisopropyl fluorophosphate (DFP) and subsequently
cycled for 2 h. Nearly all of the phospholipid remains at the buoyant
density of TM, with only ~15% being found at the density of SV
subtype. The LB shoulder at 1.061 g/ml seen in
gradient A has disappeared, which is consistent
with our previous finding that the conversion of LB to TM is not
dependent on the action of convertase (9, 10).
Surface tension analysis.
Two methods were employed, a modified Kimray-Greenfield surfactometer
method and a pulsating bubble method. The modified Kimray-Greenfield surfactometer (calibrated before each use with weights) was placed in a
37°C incubator that was lined with wet paper to provide a humid
environment. The trough was filled with 50 ml of AF buffer as subphase,
and the surface was "cleaned" by repeated aspiration of the
surface by suction of the surface via a Pasteur pipette positioned at
the surface, replenishing the volume aspirated on each occasion. The
"cleanness" of the surface was validated by continuous
measurement of the surface tension while the surface area was
compressed to 15% of the initial surface area without a decrease in
the surface tension. When the surface was clean, 250 µg of
phospholipid TM preparation, pretreated with either DFP or vehicle,
were applied below the surface of the trough subphase. This was stirred
with a magnetic stirring bar for 10 min. The surface was then slowly
compressed to 15% of its initial area and then reexpanded to initial
surface area while surface tension was measured continuously; the cycle
time was 180 s. From the surface tension-surface area plots so
obtained, we derived the following data: maximum surface tension
(max),
min, and area of hysteresis,
which was obtained by cutting the compression-expansion loop from the
surface area-surface tension chart and weighing it.
Surface feeding experiment. The ability of TM surfactant preparations to feed a surface film to exhaustion was determined in the Kimray-Greenfield surfactometer. TM was prepared from mice that had received [3H]choline. In each experiment, an aliquot of 154 µg of TM surfactant phospholipid (a 10-fold excess of that required to provide a monomolecular film to the 57-cm2 surface, assuming an average molecular area at the surface of 48 A2), either pretreated with DFP as above or not, was delivered below the surface while the subphase was continuously stirred by a magnetic stirring bar. The surface was aged for 10 min with continuous stirring of the subphase to allow a surface film to form and then was compressed to 15% of initial surface area with a Teflon dam while surface tension was continuously recorded. At 15% initial surface area, the surface was rapidly aspirated by suction through a Pasteur pipette positioned just above the surface, aspirating 2.0-3.5 ml into a trap close to the pipette tip. The volume of the aspirate was measured (by calibrations on the trap), the pipette was flushed by immediate aspiration of a further 1 ml of water into the trap, and a volume of fresh, warmed, cleaned subphase buffer equal to the volume aspirated from the trough was replaced in the trough. The total radioactivity [in disintegrations/min (dpm)] of each aspirate was measured. The subphase was continuously stirred again for 10 min while the cycle of surface compression, surface aspiration, and replacement of subphase buffer was repeated. These cycles were repeated until surface tension measurements showed negligible residual surface activity, which occurred consistently after 15-18 cycles of spreading and aspiration of the surface film.
The amount of surface-associated phospholipid that was removed with each round of surface spreading and aspiration was calculated as follows. The specific activity of the TM preparation that was used was determined (in dpm/µg phospholipid). The activity of the subphase (in dpm/ml) was measured at the start and after the last cycle; this declined with each cycle of aspiration due to the fact that the subphase was replenished with fresh buffer after each aspiration. We initially calculated the activity of the subphase after each cycle of aspiration and replenishment (a datum that would be required later in the calculation) by the dilutional factor cycle by cycle. However, we found that it was easier and, for practical purposes, just as accurate to interpolate the exponential decline in subphase activity from the initial and final measurements. The volume of the surface film that was aspirated was assumed to be negligible. The absolute radioactivity of the surface itself that was aspirated with each cycle was the total radioactivity in each aspirate minus the activity of the subphase removed with the surface; this later was the product of the total volume of the aspirate and the activity (in dpm/ml) of the aspirate at that cycle number. From the radioactivity of the surface itself, i.e., corrected for the amount of subphase removed with the surface, and the specific activity of TM (assumed to be constant), we calculated the amount of phospholipid that was removed as a surface film with each cycle of film spreading, aging, and aspiration. This is equal to the amount of surface phospholipid delivered to the surface with each round. Preliminary experiments were performed to determine the optimal time of aging of the surface (as determined by the fall in surface activity and the amount of phospholipid that could be aspirated from the surface); both reached stability at ~10 min. Preliminary experiments also showed that the amount of surface phospholipid that could be aspirated from the surface was independent of the initial amount delivered to the subphase, provided this was greater than ~75 µg of phospholipid as TM. We used two times this amount.Statistical analysis. Means, SDs, SEs, and Student's t comparisons were performed on a hand-held calculator.
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RESULTS |
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Surface balance study. Three paired experiments were performed. In each experiment, mouse TM preparations that had been pretreated with DFP to inactivate endogenous convertase activity were compared with TM preparations from the same batch that had not been pretreated with DFP. Each batch of TM was analyzed in duplicate, entering the mean of each duplicate as a single datum. The results (see Table 1) show that there were no differences between TM containing active convertase and DFP-inactivated convertase with respect to any of the measured parameters.
|
Pulsating bubble surfactometry. We examined paired untreated and DFP-pretreated TM samples in triplicate (5 experiments). The means from each triplicate determination were entered as a single datum for statistical purposes. The results are shown in Fig. 2. Similar initial surface tensions were observed on the first pulsation after 10 s of adsorption with an Rmin of 37.5 ± 4.0 (SE) mN/m (active) and 34.6 ± 4.2 mN/m (inactive), which were not significantly different. Essentially identical curves were observed for the two groups of TM preparations at Rmax during 100 pulsations, reflecting similar abilities to adsorb to the surface during bubble expansion. In addition, similar rates of decline of surface tension at Rmin were observed over the 100 pulsations, showing that the surface tensions achieved through dynamic compressions during a 50% reduction in bubble surface area were similar. The Rmin values after 100 pulsations were 11.1 ± 3.6 (active) and 8.1 ± 2.4 mN/m (inactive), which were not significantly different.
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Feeding of TM to the surface to exhaustion.
Three identical paired experiments were performed with separate
[3H]choline-labeled
mouse TM preparations. Again, each pair was derived from the same group
of animals, one half of whose TM had been pretreated with DFP and the
other not. Each paired experiment yielded similar results. The results
of one experiment are shown in Fig. 3. TM
with active convertase is shown in Fig.
3A, and TM with inactive convertase in
shown in Fig. 3B, but only the compression phase of each surface tension-surface area cycle is shown.
In both, surface tension declined to a minimum value at ~20-25%
of the starting surface area during the first six
compression-aspiration cycles. Immediately the surface was aspirated,
the surface tension returned to a high value (~60-70 mN/m, data
not shown). During cycles
7-10,
min was reached only at
successively smaller surface areas, and during
cycles
11-15,
the
min rose progressively, so that by about cycle
16-18
there was little reduction in surface tension even at minimum surface
area. All samples behaved similarly, regardless of whether they
contained active or DFP-inactivated convertase.
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DISCUSSION |
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As stated in the introduction, the action of a serine-active enzyme, surfactant convertase, appears to be required for the conversion of surfactant from the TM form to the SV form (7, 10, 11). However, this transition probably involves at least two steps, the conversion of TM to a surface film and the conversion of this film or fragments extruded by it to the SV subtype. It is therefore of interest to determine at which of these two steps convertase is required. The aim of the present experiments was to determine if convertase was required in the first of these steps by the analysis of the ability of purified TM to express typical surface-active properties in the presence or absence of active convertase.
To perform these studies, we purified surfactant predominantly in the TM form, usually containing a component of the LB form, as validated by the buoyant density of the phospholipid profile on continuous sucrose gradients centrifuged to equilibrium (8, 9). As stated in METHODS, the material that we call TM for convenience is heterogeneous in that it consists of some TM plus myelin-like concentric membranes (8, 10), as well as some LB form. Such preparations contain convertase activity in amounts sufficient to mediate conversion of TM to SV on cycling in vitro, as shown previously (11), and again in the present experiments (Fig. 1). To explore the role of convertase in the expression of surface activity by TM, it was necessary to inactivate endogenous convertase activity. We did this by dividing the surfactant lavaged from rats or mice into two equal portions and treating one with DFP to inactivate its convertase; the other was identically handled but was not exposed to DFP. This yielded paired samples of TM that would be comparable apart from DFP pretreatment. Before use, we validated each paired TM preparation to show that both were almost entirely of the buoyant density typical of TM before cycling and that its TM was capable of converting to the buoyant density of SV on cycling in vitro unless pretreated with DFP (Fig. 1).
As can be seen from the results, the quality of the surfactant
preparations was questionable in that the
min values obtained were higher
and the rate of film formation was slower than with ideal preparations
(e.g., Refs. 6 and 13). In part, this may have been due to the need to
incubate the surfactants (with DFP or vehicle) before assay. As they
nevertheless retain some surface activity, we consider them adequate to
answer the question whether convertase is required for the expression
of surface activity.
In each of the three groups of experiments we performed, we were unable
to detect any differences in the surface-active behavior of TM that
contained active or inactivated convertase. The rate of film formation
was indistinguishable as measured by the rate of surface tension
lowering in a pulsating bubble (Fig. 2). The min achieved by purified TM was
also indistinguishable whether measured in a modified Kimray-Greenfield
surfactometer (Table 1) or by a pulsating bubble technique (Fig. 2).
Because the results of the above two experiments depend on the ability of quite small amounts of TM to reduce surface tension and because lavaged surfactant contains endogenous convertase, it is conceivable that small amounts of TM could have been "primed" by convertase activity before its exposure to DFP. This might enable TM, even after DFP treatment, to show surface activity even if convertase were, in fact, necessary to feed TM to the surface film.
To exclude this possibility, we devised an additional experiment that would call for a relatively large proportion of the TM in a preparation to be delivered to the surface. The rationale was that if the action of convertase is necessary to convert TM to a surface-active form, TM preparations that lacked active convertase would fail to lower surface tension sooner than those that contained active convertase and also that the cumulative amount of phospholipid that could be aspirated as a surface film would be less. This was not the case (Fig. 4). We calculate that ~35% of the TM applied to the trough was delivered to the surface over the course of 15-20 rounds of TM surfactant spreading and aspiration whether active convertase was present or not. The reason why this figure, ~35%, was not closer to 100% is explained as being due to the fact that most of the TM that was not recovered from the surface itself was coincidentally removed as subphase along with the surface during successive aspirations, ~40% of the total applied to the trough in each case. Thus it was not available for delivery to the surface for reasons related to the experimental technique.
Only ~4 µg of phospholipid were recovered from the surface with each cycle of surface spreading and aspiration. This is less than the 15.4 µg of phospholipid we calculate that the available surface area can accommodate as a monolayer. As the surface tension at maximal area after surface spreading was relatively high (64 mN/m), it is unlikely that a complete surface film was formed. Some "squeeze out" of the surface film may have occurred during the compression that preceded each aspiration. Also, it is possible that not all of the surface was recovered by each aspiration. However, none of these explanations would affect the conclusion that feeding of surfactant to the surface was equivalent whether active or inactive convertase was present.
The results of all of these experiments are consistent and suggest that convertase is not required for the expression of surface activity by TM, tending to exclude a role for convertase in mediating the conversion of TM to a surface-active form of surfactant. This conclusion is also consistent with data from others that show that synthetic TM reconstituted de novo from purified phospholipids and surfactant apoproteins and therefore, presumably devoid of convertase activity, is able to express surface activity (2, 15). By implication, the results of the present experiments and those of previous reports suggest that convertase acts at a post surface film stage, perhaps promoting the reorientation of fragments squeezed out of the film into the SV form.
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ACKNOWLEDGEMENTS |
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We acknowledge the excellent technical support of James D'Anza, Vija Bublys, and Andelle Teng.
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FOOTNOTES |
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This work was supported by grants from Veterans Affairs Research (to N. J. Gross and R. Dhand), from the National Heart, Lung, and Blood Institute (HL-45782-01 to N. J. Gross), and from the Medical Research Council of Canada (M268C7 to F. Possmayer).
Address for reprint requests: N. J. Gross, PO Box 1485, Hines, IL 60141.
Received 12 March 1997; accepted in final form 16 July 1997.
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