1 Medical Research Council Group in Fetal and Neonatal Health and Development, Departments of Biochemistry and Obstetrics and Gynaecology, University of Western Ontario, London N6A 5A5; 2 Transplant-Immunology Group, Robarts Research Institute, and Division of Cardiovascular-Thoracic Surgery, London Health Sciences Centre-University Campus, London N6A 5A5; and 3 Division of Respiratory Medicine, Department of Physiology, and Lawson Health Research Institute, St. Joseph's Health Centre, London, Ontario, Canada N6A 4V2
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ABSTRACT |
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The effects of surfactant treatment on surfactant subtype conversion after lung injury were examined. Dogs were subjected to hyperventilation for 8 h with or without surfactant treatment. Lungs were stored for 17 h, and the right lung was transplanted and reperfused for 6 h. Conversion of large aggregate (LA) surfactant to small aggregates was investigated using in vitro surface area cycling. LA from transplanted lungs (Transplant-LA) from the nontreated group converted more rapidly than Transplant-LA from the treated group. Transplant-LA from both groups converted more rapidly than LA from normal lungs. Calculations based on [3H]dipalmitoylphosphatidylcholine in the administered surfactant [bovine lipid extract surfactant (BLES)] showed that the endogenous component of Transplant-LA converted more rapidly than the exogenous component. This indicates exogenous BLES did not equilibrate completely with endogenous surfactant. LA from hyperventilated, stored donor right lungs and from the recipients' native lungs from the nontreated group converted more rapidly than corresponding LA in the BLES-treated group. Similar relative conversions were observed with exogenous components from all lungs. Relative conversion of endogenous component from Transplant-LA was more rapid than that from LA from donor's stored right lung or from the recipient's native right lung. Low levels of phenylmethylsulfonyl fluoride inhibited conversion of Transplant-LA to a greater extent than normal LA. LA from all experimental groups had similar protein levels. These studies show acute lung injury, transplant, ischemia-reperfusion, and surfactant treatment have major effects on surfactant subtype integrity.
carboxyesterase; convertase; dipalmitoylphosphatidylcholine; ischemia-reperfusion; surfactant subtype conversion
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INTRODUCTION |
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PULMONARY SURFACTANT is a lipid-protein complex that stabilizes the lung by reducing the surface tension at the alveolar air-liquid interface. Surfactant recovered by bronchoalveolar lavage consists of subfractions differing in morphological appearance and buoyant density (5, 30). The heavy subtype or surfactant large aggregate (LA) fraction, which contains lamellar bodies, tubular myelin, and large vesicles, is rich in the surfactant proteins [surfactant protein A (SP-A), SP-B, and SP-C] and is highly surface active. The light subtype or small aggregate (SA) subfraction, consisting of small vesicles, contains less surfactant protein and has poor surface activity (24, 28, 31).
The metabolic relation between the LA and SA subfractions has been studied in vivo by pulse-chase experiments (1, 17). These studies indicated that the larger, heavier subfractions (LA) are the metabolic precursors of the smaller, lighter surfactant subtypes (SA). This in vivo alveolar metabolism can be mimicked in vitro by the method of surface area cycling, as described by Gross and Narine (6). In this procedure, a tube containing resuspended large surfactant aggregates is rotated end over end, thereby changing the surface area, as occurs during breathing. The LA and SA can then be separated by differential centrifugation. SA formed during this process are morphologically similar to those obtained from lung lavage (6, 10, 29). Furthermore, SA formed via surface area cycling, like SA fractions obtained from lung lavage, do not reduce surface tension to low values.
Gross and Schultz (7) used surface area cycling to demonstrate that several serine protease inhibitors inhibit the conversion of the heavy subtype to SA. Pretreatment of purified surfactant LA with 10 mM diisopropylfluorophosphate (DFP) inhibited its ability to be converted to SA on subsequent cycling (4, 8). Purified surfactant was subsequently found to contain a single DFP-reactive protein of Mr 70-75. This DFP-binding protein, namely convertase, has been proposed by Gross (5) to be responsible for LA-to-SA conversion. Recent studies (2, 14) have indicated that convertase is a serine-dependent carboxyesterase. Other studies (11, 28) have suggested that the target for convertase appears to be SP-B. However, it appears likely in altered states of lung function, e.g., in lung injury, that other enzymes/proteases besides convertase may also be involved (15, 18, 22).
A previous investigation (23) by our group using a canine single lung transplantation model suggested a potential role for pulmonary surfactant in lung transplant. These studies demonstrated that treatment with bovine lipid extract surfactant (BLES) ameliorated many of the anticipated detrimental effects associated with prolonged ischemia and transplantation. These promising observations led to examination of the possibility that BLES treatment of the donor might protect the lung against mild lung injuries such as those produced by hyperventilation. Maintaining brain-damaged patients on respirators with high oxygen tensions and vigorous mechanical ventilation can compromise the clinical usefulness of their lungs. The model used involved hyperventilation for 8 h, followed by 16-18 h of ischemia, single left lung transplantation, and estimation of gaseous exchange during 6-h perfusion. The results demonstrated that pretreatment with BLES minimized lung injury during hyperventilation, resulting in a marked increase in oxygenation during reperfusion compared with the absence of donor treatment (19). Similar overall results were obtained in studies where BLES administration was delayed until hour 4 of hyperventilation.
We hypothesized that the previously observed beneficial effects of BLES treatment might extend to surfactant subtype conversion. Therefore, studies were conducted to examine the effects of mild lung injury due to hyperventilation plus transplant and surfactant therapy on surfactant subtype conversion in vitro. Most of the samples of LA used were obtained from the experimental animals referred to above (19, 23). It was observed that LA from BLES-treated animals converted to SA more slowly than LA from nontreated animals but more rapidly than LA from normal lungs. Unexpectedly, calculations based on radioactive dipalmitoylphosphatidylcholine (DPPC) in the administered surfactant suggested that the endogenous component of the LA from BLES-treated dogs converted more rapidly than the exogenous component. These studies indicate surfactant treatment has an effect on surfactant subtype conversion but that administered surfactant does not equilibrate completely with the endogenous pool.
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MATERIALS AND METHODS |
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Experimental model of lung injury and transplant.
The canine model used has been previously described (19,
21). A brief overview of the model is provided here. Conditioned dogs were divided into two weight-matched groups: donor and recipient (Fig. 1). The donors were subjected to
mild injury by hyperventilation at 45 ml/kg for 8 h (fraction of
inspired oxygen = 1, positive end-expiratory pressure = 5 cmH2O, tidal volume = 45 ml/kg, rate = 16 breaths/min). Random donor dogs were treated with
[3H]DPPC-labeled BLES (100 mg/kg) either when
hyperventilation was initiated (BLES instilled) or after 4 h (BLES
delayed instilled). After hyperventilation, the donor lungs were
excised and stored for another 16-18 h to induce ischemic
injury. The donor right lung was then lavaged with 0.9% saline. The
donor left lung was transplanted into the recipient, and the recipient
was ventilated at normal (20 ml/kg) tidal volume for another 6 h.
The transplanted lung (i.e., the treated, hyperventilated, and stored
donor left lung) and the recipient's native right lung were then
lavaged. The uninjured left lung, removed from the recipient before
transplantation, was also lavaged and served as a control normal lung.
LA were prepared from the lavage of each type of lung.
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Exogenous surfactant administration. The surfactant preparation used was BLES (BLES Biochemicals, London, Ontario, Canada) supplied at a concentration of 25 mg of phospholipid/ml. Radiolabeled surfactant was prepared as follows: initially, 0.2 ml BLES was extracted into chloroform according to the method of Bligh and Dyer (3). The upper aqueous phase was discarded, and 10 µCi 1,2-dipalmitoyl-L-3 phosphatidyl-[N-methyl-3H] choline (40-85 µCi/mol; Amersham Life Science, Oakville, Ontario, Canada) was added to the lower chloroform phase. The mixture was then dried under a stream of nitrogen. After the addition of 0.5 ml of 0.9% NaCl and a few glass beads to the dried material, the tube was capped and vortexed at maximum speed for 5 min. The resuspended surfactant was then removed and was added to a tube containing the bulk of the BLES. This tube was inverted several times and gently vortexed to obtain uniform mixing of the radioactive material. A treatment dose of 100 mg/kg of 3H-labeled BLES was instilled into the treatment groups at the appropriate times.
Experimental groups. Animals were randomly assigned to one of the following groups: 1) treatment of donors with instilled BLES immediately after the onset of high volume ventilation (instilled group, n = 5); 2) treatment by BLES instillation at hour 4 of ventilation (delayed instilled group, n = 5); and 3) no treatment to donor lungs (nontreated group, n = 6). Gas exchange studies on some of the dogs in the instilled group have been reported previously (19).
Preparation of LA from lung lavage.
Lungs were lavaged with 600-1,000 ml of saline three times. Lavage
was centrifuged at 150 g for 10 min at 4°C to remove cells and cellular debris. A LA pellet was obtained by centrifugation of the
150-g supernatant at 10,000 g for 60 min at 4°C. The
pellet (LA) was resuspended in saline (0.9%) at a concentration of
10-20 mg of phospholipid/ml and frozen in aliquots at 20°C.
Aliquots of the supernatant, which contains SA, were also stored at
20°C.
Surface area cycling.
LA were thawed and resuspended in conversion buffer (0.15 M NaCl, 10 mM
Tris, 1 mM CaCl2, 1 mM MgCl2 , and 0.1 mM EDTA;
pH 7.4) at a concentration of 0.25 mg of phospholipid/ml. Aliquots (2 ml) were placed in polystyrene tubes (Falcon 2058), capped, and
attached to a rotator (Roto-torque rotator). The tubes were cycled at
40 rev/min at 37°C so that the surface area changed from 1.1 to 9.0 cm2 twice each cycle (6, 28). Samples were
cycled for 30, 60, 180, and 360 min. Identical noncycled control
samples were kept at 37°C for 360 min. Some experiments utilized the
protease inhibitor phenylmethylsulfonyl fluoride (PMSF). PMSF was
dissolved in ethanol (500 mM) and added to samples at different
concentrations ranging from 0.25 to 10 mM before cycling. An equal
amount of ethanol was added to samples not receiving PMSF. In all
studies except those with PMSF, cycling was conducted within 2 wk after
transplantation. Control studies have demonstrated similar conversion
rates after 4-12 wk of storage at 20°C.
Separation of LA and SA. After cycling, LA and SA were separated by centrifugation at 40,000 g for 20 min at 4°C. Total phospholipid in the LA pellet and in the SA remaining in the supernatant was determined by lipid extraction (3) and phosphorous analysis (25). The treated lungs contained an exogenous surfactant (BLES administered) component and an endogenous surfactant component. SA formation from total LA was based on phosphorous assay. Conversion of the exogenous component was based on radioactive counts. The endogenous component conversion was calculated by subtracting the phosphorous content of the exogenous component from the total phospholipid determined by phosphorous assay. Protein contents were analyzed as described by Lowry et al. (16) with the addition of 0.1% SDS to dissolve lipids.
Data analysis. Results are expressed as means ± SE. Differences in means between conversion of LA from different lung types were tested by ANOVA followed by Bonferroni post hoc analysis. Differences corresponding to P < 0.05 were considered significant.
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RESULTS |
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The present study investigated the effects of mild acute lung injury induced through hyperventilation, ischemia, and transplantation with the integrity of recovered surfactant LA, as assessed by in vitro surface cycling. Weight-matched dogs were subjected to hyperventilation (45 ml/kg) for 8 h (19). Bronchoalveolar lavage samples obtained after an 8-h hyperventilation period exhibited increased SA-to-LA ratios and increased protein and leukocyte levels indicative of acute lung injury (data not shown). Instillation of BLES before hyperventilation or at hour 4 partially reversed the protein and leukocyte increases and abolished the decreases in gaseous exchange (19). This suggests that BLES treatment can moderate hyperventilation-induced mild lung injury. SA-to-LA ratios in recovered lavage were also reduced, but this effect could result from the presence of BLES, which is primarily in the LA state (19).
Relative physical integrity of the surfactant LA recovered from
transplant lungs from BLES instilled and nontreated donors was examined
by subjecting LA to in vitro surface cycling and measuring the
formation of surfactant SA using phospholipid phosphorous estimation.
LA from transplanted lungs (Transplant-LA) converted rapidly during the
initial 0.5 h and plateaued between 1 and 3 h (Fig.
2). LA from BLES instilled transplanted
lungs also exhibited rapid initial conversion but to a significantly
lower extent than Transplant-LA from nontreated animals. Conversion of
LA from BLES delayed instilled dogs overlapped with conversion of LA
from BLES instilled animals so the results (BLES treated) were pooled.
In all cases, formation of SA was significantly greater than conversion of LA from normal lungs at all time points examined. Furthermore, LA
from normal lungs also showed a more gradual increase in SA formation
such that conversion did not clearly plateau during the 6-h cycling
period.
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At least potentially, the slower, decreased formation of SA with LA
from BLES-treated lungs could be due to a slower conversion of the
treatment surfactant, BLES. Figure 3
compares conversion of BLES itself with the conversion of the
endogenous and exogenous surfactant components of LA from transplanted
lungs (based on [3H]DPPC, see below). Such a dilution
effect would require that the treatment BLES converts at a
significantly slower rate than the LA obtained from the treated
transplanted animals. However, BLES conversion was slightly more rapid
than conversion of Transplant-LA from treated lungs at each time point
examined (compare transplant treated, Fig. 2, with BLES, Fig. 3).
Although these differences were not significant, it appears unlikely
that the BLES present in Transplant-LA from treated lungs could account
for the observed lower conversion.
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Calculations based on the specific activity of [3H]DPPC in BLES allowed comparison of the rates of conversion of the endogenous and exogenous surfactant components of LA recovered from BLES-treated lungs. It should be appreciated that these calculations are based on the premise that conversion of the exogenous surfactant can be represented by transfer of [3H]DPPC from LA to SA. With these transplanted lungs, the recovered LA possessed similar endogenous (51.7 ± 2.1%, n = 10) and exogenous (48.3 ± 2.1%, n = 10) phospholipid pools. Surprisingly, SA formation from endogenous surfactant was greater than SA formation from the exogenous component (Fig. 3). These results suggest that the exogenous instilled BLES did not equilibrate fully with the endogenous surfactant despite being in the lungs for 25-30 h.
Conversion of LA from the donor's stored right lung (Donor Right-LA)
and from the recipient's own native right lung (Native Right-LA) were
also measured by in vitro cycling (Fig.
4). Conversion of Donor Right-LA and
Native Right-LA from nontreated dogs was significantly greater than
conversion of LA from corresponding lungs in the BLES-treated group.
Conversion of Donor Right-LA and Native Right-LA from the nontreated
and treated groups was also greater than that for LA from the normal
lungs. These results suggested that BLES treatment ameliorated, but did
not abolish, the effects of acute lung injury and reperfusion.
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Figure 5 compares surfactant subtype
conversion of exogenous (A and C) and endogenous
(B and D) surfactant components from donor right
and native right lungs with the conversion of Transplant-LA. Total
Transplant-LA (from BLES-treated lungs) converted more rapidly than
Donor Right-LA or Native Right-LA, but the differences were not
significant (data not shown). Figure 5A compares actual SA formation in micrograms of phospholipid from the exogenous LA component
of the three lung surfactants. Not surprisingly, very little absolute
conversion was observed with the exogenous component recovered from the
recipient's native lung. In contrast, the endogenous component from
Native Right-LA converted to a significantly greater extent than the
endogenous component from Transplant-LA or Donor Right-LA (Fig.
5B). The more rapid conversion of endogenous Native Right-LA
is likely related to the small proportion of exogenous surfactant in
the recipient's native lung, which accounted for 6.08 ± 1.5% of
the total Native Right-LA. However, the more rapid conversion could
also be related to the effects of transplantation, because total Native
Right-LA converted more rapidly than LA from normal organs (Fig. 4).
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Donor Right-LA contained 44.3 ± 2.4% exogenous surfactant, a level slightly lower than the 51.7 ± 2.1% in Transplant-LA. The effects of the different pool sizes were minimized by expressing the relative conversion of each component as a percentage of its amount in the cycled sample (Fig. 5, C and D). Interestingly, when plotted as a percentage of total (Fig. 5C), the exogenous components from all three LA surfactants converted in an identical manner. Comparison of the relative conversion of the endogenous components (Fig. 5D) revealed Transplant-LA converted significantly more rapidly than the endogenous components of Native Right-LA or Donor Right-LA except at the hour 6. The endogenous components from Transplant-LA, Donor Right-LA, and Native Right-LA converted significantly more rapidly than their corresponding exogenous components except for Donor Right-LA and Native Right-LA at 1 and 6 h. This observation suggests that the exogenous components of the transplanted, stored, and recipient's native lungs did not equilibrate completely with the endogenous surfactant.
In an attempt to clarify the nature of the altered integrity of
Transplant-LA from the nontreated group, the effect of PMSF, an active
serine-dependent enzyme inhibitor, on in vitro conversion of
Transplant-LA and LA from normal lungs was examined (Fig.
6). Low PMSF had little effect on
conversion, but ~60% inhibition was observed at high inhibitor
levels. A distinct difference in the inhibitory profile was observed
with Transplant-LA being significantly more susceptible at lower PMSF
concentration.
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Subtype conversion can be accelerated by serum proteins. Figure
7 shows LA from experimentally
manipulated lungs possessed high total protein levels, with no
significant difference between groups. Much lower total protein
remained associated with LA from control normal lungs.
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DISCUSSION |
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In vitro surface area cycling was used to examine the properties of surfactants recovered from BLES-treated and nontreated hyperventilated, stored, and transplanted reperfused lungs compared with surfactant from the recipients' normal left lungs (Fig. 1). Transplant-LA from nontreated animals converted rapidly and generated a high level of SA. Transplant-LA from BLES-treated lavage converted more slowly than Transplant-LA from nontreated animals but significantly faster than LA from normal lungs. Thus BLES instillation reversed part, but not all, of the effect of hyperventilation, ischemia, and reperfusion on LA integrity during cycling. These observations are consistent with improved lung function due to BLES treatment under these conditions (19).
Theoretically, the increased stability of LA from BLES-treated compared with untreated dogs could be due to the physical properties of instilled BLES. However, BLES itself converted rapidly (Fig. 3) with a rate slightly, although not significantly, higher than Transplant-LA from treated lungs. Donor Right-LA and Native Right-LA converted at a slightly lower (nonsignificant) rate than Transplant-LA. Consequently, it appears unlikely that the properties of BLES alone could explain the lower conversion observed with LA recovered after BLES treatment.
Surprisingly, calculations based on [3H]DPPC indicated that conversion of the endogenous component of Transplant-LA from treated lungs was greater than that of the exogenous component. This indicated that exogenous BLES did not equilibrate with the endogenous pool despite having been in the lungs during 8 or 4 h of hyperventilation, 17 h of cold storage, and 6 h of reperfusion. To our knowledge, a difference in the physical or metabolic processing of endogenous and exogenous surfactants has not been previously observed. Other investigators (12, 13) have concluded or assumed that once in the lung for a short period, DPPC and other surfactant components behave similarly to endogenous components. It should be noted that most of the previous work on subtype conversion has been conducted with neonatal or normal animals, not with acute lung injury or transplantation. For a number of reasons, mixing of exogenous surfactant with endogenous alveolar and tissue pools cannot occur instantaneously (12). However, the surfactant turnover times of 8-13 h estimated for normal rabbits and sheep are in the same range as the total nonischemic incubation periods employed in the present experiments. Hence, equilibration within the alveolar environment would be expected. Furthermore, a considerable amount of the administered [3H]DPPC should have been taken up and either metabolized or resecreted by type II cells.
The precise nature of the apparent derangement in surfactant processing is not known, but these observations imply the presence of a nonintegrated pool of exogenous surfactant within the lung during a considerable time frame. In addition, the slower in vitro conversion of [3H]DPPC in recovered LA compared with input BLES shows that the administered BLES has been modified within the lung. Modification of exogenous surfactant within the lung has previously been reported by Ikegami et al. (10), who reported that Survanta (also an organic lipid extract surfactant) recovered from preterm lambs possesses superior biophysical and physiological activity compared with administered Survanta. These authors concluded that incorporation of endogenous surfactant apoproteins, particularly SP-A, was responsible for the improved activity (for a review, see Ref. 12). SP-A incorporation could explain the slower conversion of [3H]DPPC observed here. Whether components of exogenous surfactant other than DPPC also exhibit different conversion rates than their endogenous counterparts must still be investigated.
Donor Right-LA and Native Right-LA from nontreated animals converted at a slower rate than Transplant-LA but more rapidly and to a significantly greater extent than LA from normal lungs. Somewhat slower conversion was observed with Donor Right-LA and Native Right-LA from BLES instilled animals, but conversion remained significantly greater than that of LA from normal lungs. Transplant-LA from treated lungs converted more rapidly than Donor Right-LA or Native Right-LA and significantly faster than LA from normal lungs at all time points. Although the differences were not significant, these results are consistent with a gradation in injury between transplanted and donor right or native right lungs. This suggestion was reinforced by the observation that the endogenous component of Transplant-LA converted more rapidly than the endogenous components of Donor Right-LA or Native Right-LA (Fig. 5D). These results indicate that whereas hyperventilation, storage, and ischemia produce acute lung injury, further injury arises during the 6-h reperfusion period. Previous studies (19-21, 29) have also indicated that reperfusion augments the injury arising through ischemia.
Conversion of LA recovered from the recipient's right lung was similar to the corresponding donor right lungs with both treatment and nontreatment groups. These results are consistent with previous observations showing increased permeability and depressed function of native right lungs after reperfusion (19, 23, 29). The basis of the detrimental effects on the recipient's native lung are not understood, but we (23) have previously suggested that these could result from cytokines released by the reperfused transplanted lung and/or from volutrauma arising from reduced compliance of the transplanted lung. BLES treatment also led to significant improvement in lung function and subtype conversion with the native right lungs.
The mechanisms by which hyperventilation, ischemia, and
transplant affect LA integrity must still be addressed. Studies
(5, 27, 28, 30) with isolated LA and reconstitution
studies have shown that the conversion of LA to SA requires rapid
adsorption to the air-water interface (e.g., surfactant lipids that do
not adsorb rapidly do not convert) and repeated alterations in surface area (e.g., LA in tubes positioned on the rotator so that they roll
rather than tumble end over end do not convert). Studies by Gross and
associates (4, 5, 14) have shown that inhibitors of
serine-active enzymes such as DFP, 1-antitrypsin, and
dihydrostatin markedly reduce in vitro conversion. Loss of SP-B from
canine surfactant during in vitro conversion was originally attributed to protease activity but appears to result from dissociation of SP-B
from surfactant lipids (11, 28). Convertase has recently been identified as a 70-kDa carboxyesterase (2, 14), but the manner by which the serine-dependent enzyme influences conversion is not known. SP-A contributes to LA integrity and protease activity on
this collectin, e.g., by neutrophil elastase, could contribute to the
observed effects of lung injury (5, 22, 30). Studies conducted using LA from the present series of animals revealed in vitro
conversion of LA from transplanted or normal lungs is markedly reduced
by the active serine-dependent enzyme inhibitor PMSF. Interestingly,
conversion of Transplant-LA was more susceptible to inhibition by PMSF
than conversion of normal LA. This is consistent with the possibility
that the enhanced conversion of Transplant-LA is due to differences in
the nature of the serine-dependent enzyme convertase, but other PMSF
inhibitable proteases could be involved. Serum proteins have also been
shown to affect in vitro conversion, although the mechanism is not
clear (9, 26). Protein levels were elevated to about the
same extent in LA isolated from all experimental lungs. Thus altered
serum levels could account, at least in part, for the more rapid
conversion relative to LA from normal lungs but could not explain
differences in conversion observed between Transplant-LA and LA from
the other experimental injured lungs.
In conclusion, it is evident from these and previous studies that BLES treatment greatly diminishes the deleterious effects of hyperventilation, ischemia, and reperfusion. BLES treatment resulted in lower SA-to-LA ratios in vivo and conversion rates in vitro. The reduced in vitro conversion rate could not be attributed to the physical properties of BLES, which, by itself, converts rapidly in vitro. Interestingly, [3H]DPPC from administered BLES converted more slowly than the endogenous component in Transplant-LA. This finding indicates that the exogenous surfactant did not equilibrate with endogenous surfactant within the lung. The slow conversion rate further demonstrates that the exogenous surfactant was modified within the lung. The basis for the differences in in vitro processing of endogenous and exogenous surfactants is not known. The endogenous component from Transplant-LA converted more rapidly than those from Donor Right-LA and Native Right-LA. However, the exogenous component of LA recovered from all experimental lungs converted with identical kinetics. This would suggest that exogenous surfactant is not as susceptible to the degree of injury as the endogenous components. The similar relative conversions observed with Donor Right-LA and Native Right-LA from BLES-treated groups were lower than with LA from nontreated groups but higher than LA from normal lungs. Taken together, these studies suggest BLES modulates but does not abolish lung injury under these circumstances.
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ACKNOWLEDGEMENTS |
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The superb technical assistance provided by Jennifer Duplan and Lynn Denning is gratefully acknowledged. We thank BLES Biochemicals (London, Ontario) for providing BLES. Marc Possmayer assisted with data processing and statistical analysis.
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FOOTNOTES |
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These studies were supported by a group grant from the Medical Research Council of Canada and operating grants from the Heart and Stroke Foundation of Canada and the Medical Research Council of Canada.
Present address of K. Inchley: Dept. of Microbiology and Immunology, Univ. of Western Ontario, London, Ontario, Canada N6A 5A5.
Address for reprint requests and other correspondence: F. Possmayer, Dept. of Obstetrics and Gynaecology, London Health Sciences Centre-UC, 339 Windermere Rd., London, Ontario, 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 6 April 2001; accepted in final form 24 September 2001.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Baritussio, A,
Bellina L,
Carraro R,
Rossi A,
Enzi G,
Magoon MW,
and
Mussini I.
Heterogeneity of alveolar surfactant in the rabbit: composition, morphology, and labelling of subfractions isolated by centrifugation of lung lavage.
Eur J Clin Invest
14:
24-29,
1984[ISI][Medline].
2.
Barr, F,
Clark H,
and
Hawgood S.
Identification of a putative surfactant convertase in rat lung as a secreted serine carboxylesterase.
Am J Physiol Lung Cell Mol Physiol
274:
L404-L410,
1998
3.
Bligh, EG,
and
Dyer WJ.
A rapid method of total lipid extraction and purification.
Can J Biochem Physiol
37:
911-917,
1959[ISI].
4.
Dhand, R,
Young J,
Teng A,
Krishnasamy S,
and
Gross NJ.
Is dipalmitoylphosphatidylcholine a substrate for convertase?
Am J Physiol Lung Cell Mol Physiol
278:
L19-L24,
2000
5.
Gross, NJ.
Extracellular metabolism of pulmonary surfactant: the role of a new serine protease.
Annu Rev Physiol
57:
135-150,
1995[ISI][Medline].
6.
Gross, NJ,
and
Narine KR.
Surfactant subtypes of mice: metabolic relationships and conversion in vitro.
J Appl Physiol
67:
414-421,
1989
7.
Gross, NJ,
and
Schultz RM.
Serine proteinase requirement for the extracellular metabolism of pulmonary surfactant.
Biochim Biophys Acta
1044:
222-230,
1990[ISI][Medline].
8.
Gross, NJ,
and
Schultz RM.
Requirements for extracellular metabolism of pulmonary surfactant: tentative identification of serine protease.
Am J Physiol Lung Cell Mol Physiol
262:
L446-L453,
1992
9.
Hall, SB,
Hyde RW,
and
Kahn MC.
Stabilization of lung surfactant particles against conversion by a cycling interface.
Am J Physiol Lung Cell Mol Physiol
272:
L335-L343,
1997
10.
Ikegami, M,
Ueda T,
Absolom D,
Baxter C,
Rider E,
and
Jobe AH.
Changes in exogenous surfactant in ventilated preterm lamb lungs.
Am Rev Respir Dis
148:
837-844,
1993[ISI][Medline].
11.
Inchley, K,
Cockshutt A,
Veldhuizen R,
and
Possmayer F.
Dissociation of surfactant protein B from canine surfactant large aggregates during formation of small surfactant aggregates by in vitro surface area cycling.
Biochim Biophys Acta
1440:
49-58,
1999[ISI][Medline].
12.
Jobe, AH.
Pathophysiology of respiratory distress syndrome and surfactant metabolism.
In: Fetal and Neonatal Physiology, edited by Polin RA,
and Fox WW.. Philadelphia, PA: Saunders, 1997, p. 1299-1313.
13.
Jobe, AH,
and
Ikegami M.
Surfactant metabolism.
Clin Perinatol
20:
683-696,
1993[ISI][Medline].
14.
Krishnasamy, S,
Gross NJ,
Teng AL,
Schultz RM,
and
Dhand R.
Lung "surfactant convertase" is a member of the carboxylesterase family.
Biochem Biophys Res Commun
235:
180-184,
1997[ISI][Medline].
15.
Lee, CT,
Fein AM,
Lippmann M,
Holtzman H,
Kimbel P,
and
Weinbaum G.
Elastolytic activity in pulmonary lavage fluid from patients with adult respiratory-distress syndrome.
N Engl J Med
304:
192-196,
1981[Abstract].
16.
Lowry, OH,
Rosebrough NJ,
Farr AL,
and
Randall RJ.
Protein measurement with the Folin reagent.
J Biol Chem
193:
265-275,
1951
17.
Magoon, MW,
Wright JR,
Baritussio A,
Williams MC,
Goerke J,
Benson BJ,
Hamilton RL,
and
Clements JA.
Subfractionation of lung surfactant. Implications for metabolism and surface activity.
Biochim Biophys Acta
750:
18-31,
1983[ISI][Medline].
18.
McGuire, WW,
Spragg RG,
Cohen AB,
and
Cochrane CG.
Studies on the pathogenesis of the adult respiratory distress syndrome.
J Clin Invest
69:
543-553,
1982[ISI][Medline].
19.
Novick, RJ,
Gilpin AA,
Gehman KE,
Ali IS,
Veldhuizen RA,
Duplan J,
Denning L,
Possmayer F,
Bjarneson D,
and
Lewis JF.
Mitigation of injury in canine lung grafts by exogenous surfactant therapy.
J Thorac Cardiovasc Surg
113:
342-353,
1997
20.
Novick, RJ,
MacDonald J,
Veldhuizen RA,
Wan F,
Duplan J,
Denning L,
Possmayer F,
Gilpin AA,
Yao LJ,
Bjarneson D,
and
Lewis JF.
Evaluation of surfactant treatment strategies after prolonged graft storage in lung transplantation.
Am J Respir Crit Care Med
154:
98-104,
1996[Abstract].
21.
Novick, RJ,
Veldhuizen RA,
Possmayer F,
Lee J,
Sandler D,
and
Lewis JF.
Exogenous surfactant therapy in thirty-eight hour lung graft preservation for transplantation.
J Thorac Cardiovasc Surg
108:
259-268,
1994
22.
Pison, U,
Tam EK,
Caughey GH,
and
Hawgood S.
Proteolytic inactivation of dog lung surfactant-associated proteins by neutrophil elastase.
Biochim Biophys Acta
992:
251-257,
1989[ISI][Medline].
23.
Possmayer, F,
Novick RJ,
Veldhuizen RAW,
Lee J,
Bjarneson D,
and
Lewis JF.
Potential role for pulmonary surfactant in lung transplantation.
In: Acute Respiratory Distress Syndrome: Cellular and Molecular Mechanisms and Clinical Management, edited by Matalon S,
and Sznajder JI.. New York: Plenum, 1998, p. 117-124.
24.
Putz, G,
Goerke J,
and
Clements JA.
Surface activity of rabbit pulmonary surfactant subfractions at different concentrations in a captive bubble.
J Appl Physiol
77:
597-605,
1994
25.
Rouser, G,
Fleischer S,
and
Yamamoto A.
Two dimensional thin layer chromatographic separation of polar lipids and determination of phospholipids by phosphorous analysis of spots.
Lipids
5:
494-496,
1970[ISI][Medline].
26.
Ueda, T,
Ikegami M,
and
Jobe A.
Surfactant subtypes: in vitro conversion, in vivo function, and effects of serum proteins.
Am J Respir Crit Care Med
149:
1254-1259,
1994[Abstract].
27.
Veldhuizen, RA,
Hearn SA,
Lewis JF,
and
Possmayer F.
Surface-area cycling of different surfactant preparations: SP-A and SP- B are essential for large-aggregate integrity.
Biochem J
300:
519-524,
1994[ISI][Medline].
28.
Veldhuizen, RA,
Inchley K,
Hearn SA,
Lewis JF,
and
Possmayer F.
Degradation of surfactant-associated protein B (SP-B) during in vitro conversion of large to small surfactant aggregates.
Biochem J
295:
141-147,
1993[ISI][Medline].
29.
Veldhuizen, RA,
Lee J,
Sandler D,
Hull W,
Whitsett JA,
Lewis J,
Possmayer F,
and
Novick RJ.
Alterations in pulmonary surfactant composition and activity after experimental lung transplantation.
Am Rev Respir Dis
148:
208-215,
1993[ISI][Medline].
30.
Veldhuizen, RA,
Yao LJ,
Hearn SA,
Possmayer F,
and
Lewis JF.
Surfactant-associated protein A is important for maintaining surfactant large-aggregate forms during surface-area cycling.
Biochem J
313:
835-840,
1996[ISI][Medline].
31.
Yamada, T,
Ikegami M,
and
Jobe AH.
Effects of surfactant subfractions on preterm rabbit lung function.
Pediatr Res
27:
592-598,
1990[Abstract].
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