Department of Molecular Pathology, Chest Disease Research
Institute, Kyoto University, Kyoto 606, Japan
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INTRODUCTION |
SURFACTANT-ASSOCIATED PROTEINS (SP) are important to
the expression of surface activity of pulmonary surfactants (17), lipid metabolism (7), and pulmonary defense (20). In particular, hydrophobic
proteins, such as SP-B and SP-C, are considered vital to the surface
activity of surfactants (6, 31). These proteins are secreted with
lipids from alveolar type II cells and promote surface adsorption of
lipid molecules at the air-water interface. Although some of these
proteins are recycled by alveolar type II cells (3, 16), some are
ingested and metabolized by alveolar macrophages (4, 30). The metabolic
fate of such proteins is not fully understood.
In pulmonary alveolar proteinosis (PAP), a large amount of dimeric form
of SP-C ([SP-C]2)
accumulates in the lung (18, 26). Fused-membrane structures, unique
structures found in insoluble material of bronchoalveolar lavage fluid
(BALF) and in macrophages in PAP lungs (9), are composed of dimeric and
monomeric forms of SP-C (19). We hypothesized that
[SP-C]2 accumulated in
the patients' lungs because of its undigestability by macrophages.
To demonstrate differences in the mode of removal between SP-C and
[SP-C]2, we examined
the fate of these proteins in the mouse lung after intratracheal
instillation of liposomes containing the proteins obtained from PAP
patients. In this experiment, we focused on the interconversion of
[SP-C]2 to its
monomeric form and the participation of glutathione (GSH), a major
reducing substance, in the conversion.
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MATERIALS AND METHODS |
Lipids and reagents.
L-
-Dipalmitoylphosphatidylcholine
(DPPC) and
L--phosphatidylglycerol
from egg were purchased from Sigma Chemical (St. Louis, MO).
Succinimidyl-6- (7-nitrobenz-2-oxa-1,3-diazol-4-yl)-amino hexanoic acid
(NBD) was purchased from Molecular Probes (Eugene, OR).
Reduced and oxidized glutathione (GSH and GSSG); GSH reductase; 5,5
-dinitro-bis(2-nitrobenzoic acid); NADPH; and lactate
dehydrogenase (LDH) assay kit (LDH CII-test Wako) were purchased from
Wako Pure Chemical Industries (Osaka, Japan).
L-Buthionine-(S,R)-sulfoximine (BSO), diamide (DA),
N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES), and organic solvents were obtained from Nacalai Tesque
(Kyoto, Japan).
Isolation of SP-C and
[SP-C]2.
SP-C and [SP-C]2 were
isolated from BALF obtained from patients with PAP who received
therapeutic lung lavage in the Chest Disease Research Institute
Hospital, Kyoto University. BALF was centrifuged at 16,000 g for 30 min at 4°C. The
precipitate was extracted with chloroform-methanol according to the
method of Folch and colleagues (8). After organic solvents were
evaporated, the residue was dissolved in chloroform-methanol-0.1 M HCl
(1:1:0.1) and applied to a Sephadex LH60 column (Pharmacia
Biotechnology, Uppsala, Sweden) (6).
Hydrophobic proteins eluted by the same solvent were examined by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (12.5%)
according to the method of Weber and Osborn (28) and were separately
pooled. Protein concentration was determined by the method of Thompson
and Morrison (21), and the purity of proteins was examined by SDS-PAGE
and by amino acid composition analysis (19). Proteins were stored in
the elution solvent at 
20°C until used.
Preparation of fluorescence-labeled proteins.
Proteins were dried with a rotary evaporator,
redissolved in chloroform-methanol (2:1), and reacted with
NBD after adjusting the pH to 7 with HEPES buffer (pH 8.5)
according to the method of Horowitz and colleagues (12). Unreacted
reagent was removed by Sephadex LH60 column chromatography, and labeled
SP-C and [SP-C]2 (referred to as NBD-SP-C and
NBD-[SP-C]2,
respectively) were stored at 20°C. The surface activity of
lipid-protein complexes examined by a pulsating bubble surfactometer
(Electronetics, Amherst, NY) did not differ between
labeled and unlabeled proteins.
Preparation of liposomes.
Mixtures of DPPC and egg
L--phosphatidylglycerol (4:1)
in chloroform were combined with NBD-SP-C (1 and 2% by weight) or NBD-[SP-C]2 (2% by
weight), and solvents were dried under nitrogen and in
vacuo. Liposomes were prepared by sonication after
addition of phosphate-buffered saline (pH 7.4) [10 mg
phospholipids (PL)/ml] and were then centrifuged at 100,000 g for 40 min at 4°C.
Precipitate was suspended in sterile physiological saline (10 mg PL/ml)
and stored at 20°C. In some experiments, liposomes
containing NBD-[SP-C]2 were incubated with 10 mM GSH at 37°C for 2 h and centrifuged at
100,000 g for 40 min at 4°C
(referred to as GSH-treated
NBD-[SP-C]2). The
precipitate was suspended again in saline at a concentration of 10 mg
PL/ml.
Determination of PL, GSH, and LDH in the lung wash.
Specific-pathogen-free female BALB/c mice were
purchased from Shimizu Animal Laboratory (Kyoto, Japan) and were used
at the age of 9-11 wk in the following experiments. To establish
the optimal sampling conditions for determination of GSH level in alveoli, intra-alveolar PL, GSH, and LDH content was determined by
sequential lung lavage in normal mice. Mice were killed by exsanguination from the aorta under pentobarbital sodium anesthesia (60 mg/kg), and lungs were perfused from the pulmonary artery with saline
containing heparin (10 U/ml). The trachea was cannulated with a
19-gauge needle. Lungs were lavaged with 1 ml of saline, flushed in and
out of the lungs three times, and the final wash was recovered. This
procedure was repeated six times, and each lavage fluid was centrifuged
at 150 g for 10 min at 4°C to
remove cells. The supernatant was further centrifuged at 20,000 g for 40 min at 4°C. The white
precipitate (white layer) was used for the determination of total PL
according to the method of Bartlett (2) after extraction with
chloroform-methanol. The total GSH in the supernatant was
determined according to the method of Tietze (22), and LDH was
determined with a commercial kit.
Disappearance of intratracheally instilled labeled proteins.
Mice were anesthetized with pentobarbital sodium (60 mg/kg), and the trachea of each was exposed by making an incision in the neck. Animals were placed in the dorsal position on a surgical board set at an oblique angle of ~80°. The liposome suspension (total volume of 0.1 ml for 1 animal), containing either NBD-SP-C, NBD-[SP-C]2, or
GSH-treated
NBD-[SP-C]2, was
instilled into the trachea through a 27-gauge needle by dividing the
suspension into five aliquots. After instillation was performed,
control mice were killed at time
0 by cutting the abdominal aorta. After their incisions
were sutured, other mice were kept in an animal room (24 ± 1°C,
humidity 45 ± 5%) with free intake of food and water. They were
killed at various time intervals after instillation. Lungs were lavaged
seven times with 1 ml of saline as described above. Combined lavage
fluid and excised lungs, homogenized with 1 ml of saline, were
extracted with chloroform-methanol. Solvents were evaporated, and the
residue was dissolved in 5 ml of chloroform. A portion of the extract
was used to determine total recovery of fluorescence-labeled proteins
with a fluorescence spectrophotometer (F-2000; Hitachi, Tokyo, Japan).
The recovery was expressed as a percentage of the total amount of
fluorescence instilled into the lung. Efficiency of extraction of
NBD-SP-C added to lavage fluid and to tissues was 93 and 80%,
respectively. The rest of the lipid and protein extract was separated
on a Sephadex LH60 column to examine the interconversion of
NBD-[SP-C]2
to NBD-SP-C and vice
versa. Percentage of conversion was calculated from
areas corresponding to original NBD-proteins and converted proteins on
chromatograms (see Fig. 3) which were measured by an image analyzer
(JIM-5000; JEOL, Tokyo, Japan). Characterization of converted proteins
was done by SDS-PAGE under reducing and nonreducing conditions.
Inhibition of in vivo conversion of
NBD-[SP-C]2 to NBD-SP-C by BSO
and DA.
To determine the effect of GSH in the conversion of
[SP-C]2 to SP-C, BSO
(18 mg, corresponding to ~3 mmol/kg) was injected subcutaneously and
DA was injected intraperitoneally at 4 and 2 h, respectively, before
intratracheal instillation of liposomes containing
NBD-[SP-C]2
(see Fig. 5). Animals killed 4 h after instillation received an
additional DA injection 2 h before being killed. Those killed at 8 h
after instillation received additional BSO (9 mg) 3 h before being
killed and two additional DA injections at 3 and 6 h before they were
killed. We examined two different doses of DA (0.3 and 2 mg, corresponding to 0.07 and 0.5 mmol/kg). Animals were killed as
described above, and lungs were homogenized with 1 ml of saline
containing 0.5 mM DA to inhibit conversion by free GSH during
homogenization. The total recovery and the amount of SP-C formed were
determined as described above.
Total GSH concentration in lavage fluids as well as GSH and GSSG
content were determined in the lung homogenate of the animals (without
instillation of liposomes) treated with a higher dose of DA. GSH
concentration was determined as described above, except that only the
first lung wash was obtained. After lavage was performed, lungs were
homogenized with 2 ml of 5% trichloroacetic acid in 0.01 M HCl and centrifuged at 17,000 g for
15 min at 2°C. The supernatant was used for the determination of
GSH and GSSG concentrations.
In vitro conversion of
[SP-C]2 to SP-C by GSH.
Liposomes containing 500 µg of PL and 10 µg of
NBD-[SP-C]2 were
incubated with various concentrations of GSH for 4 h or for various
periods with 0.4 and 5 mM GSH at 4 and 37°C in 0.5 ml of reaction
buffer [0.15 M KCl in 0.1 M
tris(hydroxymethyl)aminomethane-HCl containing 1 mM EDTA, pH
8.1] (10). Inhibition by DA in this system was
also performed. After liposomes were incubated, proteins were extracted
with chloroform-methanol and then analyzed as described above. To
examine the effect of GSH on unlabeled proteins, we prepared liposomes
from total lipid extracts of BALF from patients (6 mg PL, 0.35 mg SP-B,
0.23 mg [SP-C]2, and
0.24 mg SP-C). Liposomes were incubated with 10 mM GSH at
37°C for 2 h in 3 ml of reaction buffer, and proteins were examined
with SDS-PAGE after separation from lipids by Sephadex LH60 column
chromatography. The relative abundance of
[SP-C]2 and SP-C was
determined after SDS-PAGE by staining with Coomassie brilliant blue and
by densitometry (Densitron; JOOKOO, Kawasaki, Japan).
Statistical analysis.
Commercially available computer software was used to
analyze differences of the means between groups by nonpaired Student's t-test (StatView; Abacus Concepts,
Berkeley, CA) and to obtain disappearance curves and half-lives of
fluorescence-labeled proteins (CA-Cricket Graph III; Computer
Associates International, Hauppauge, NY).
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RESULTS |
Content of PL, GSH, and LDH in mouse lung was evaluated
in sequential lung washings. As shown in Fig.
1, total amount of PL in seven washings
decreased exponentially, and the first wash contained about one-half of
the total PL recovered by seven washings. GSH content, however,
increased after the second wash and decreased gradually after the
fourth wash. We decided to use the first wash to determine
intra-alveolar GSH content in the following experiments.
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Fig. 1.
Content of total phospholipids (PL;
A), and total glutathione (GSH)
and lactate dehydrogenase (LDH; B) in 7 sequential lung lavage fluids in normal BALB/c mice. PL was determined in white layer
obtained by centrifugation of each lavage fluid at 20,000 g for 40 min after cells were removed.
GSH and LDH were determined in final supernatant. Vertical bars, SD of
means of 3 animals.
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Figure 2 shows the recovery of proteins
after intratracheal instillation of liposomes containing 1 and 2%
NBD-SP-C and 2% NBD-[SP-C]2 relative
to the total amount of instilled proteins. These three regression
curves were statistically significant
(r2 = 0.917, 0.977, and 0.961 for NBD-[SP-C]2, 1% NBD-SP-C,
and 2% NBD-SP-C, respectively). Similar disappearance curves and
biological half-lives were obtained with liposomes with different
contents of SP-C (17.7 h for liposomes containing 1% SP-C, with 95%
confidence range of 16.7-18.8 h; 18.8 h for those containing 2%,
with 95% confidence range of 15.8-20.1 h). At
time 0, recovery of
NBD-[SP-C]2 was lower
than recovery of NBD-SP-C, but the difference was not significant.
NBD-[SP-C]2 was
removed from lungs more slowly (biological half-life 30.1 h, with 95%
confidence range of 25.1-33.4 h) than NBD-SP-C was removed. By
using regression analysis, we found that the difference in the
regression slopes between
NBD-[SP-C]2 and 1% SP-C was
statistically significant (P < 0.001) but that the difference in the two regression intercepts was not
significant.
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Fig. 2.
Recovery of intratracheally instilled 1 and 2% surfactant-associated
protein C (SP-C) fluorescently labeled with
succinimidyl-6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino hexanoate (NBD)
and its fluorescently labeled dimeric form (2% NBD-[SP-C]2) from
mouse lungs at various time points after instillation. Total
fluorescence instilled was considered to be 100%. Vertical bars, SD of
means of >5 animals. Linear regression curves and equations were
obtained with computer software (CA-Cricket Graph III; Computer
Associates International). For clarification, symbols of NBD-SP-C (1%)
are shown by moving them slightly to the right.
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As shown in Fig. 3, both proteins showed
elution profiles different from the original proteins in liposomes. In
NBD-SP-C, a small shoulder appeared before the main original SP-C peak, which was eluted at the fractions corresponding to those of
NBD-[SP-C]2. In
NBD-[SP-C]2, a
relatively large second peak was found at the elution position
corresponding to SP-C. The SDS-PAGE profile of the protein fractions
obtained by the column is shown in Fig. 4.
In NBD-[SP-C]2, two
bands were found, one corresponding to original protein and the other
migrating at the position of NBD-SP-C (lane
3). In SP-C, when total proteins recovered were
examined, no obvious band corresponding to a dimeric form was found
(lane 4). But when only the
fractions corresponding to
[SP-C]2 were examined,
a very faint band migrating at the position of a dimer was found
(lane 5), and this band disappeared
after reduction with 2-mercaptoethanol (lane
6). These results show that a significant amount of
NBD-[SP-C]2 was
converted to the monomeric form; however, the conversion of SP-C to the
dimeric form, although it did occur, was not remarkable.
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Fig. 3.
Representative elution profile of recovered protein (solid line) from
mouse lung and original fluorescence-labeled proteins (dashed line)
from Sephadex LH60 column. A:
NBD-SP-C; B:
NBD-[SP-C]2. For
quantitation, area of converted protein was calculated by subtracting
area occupied by original protein from total area of recovered protein
after adjusting peak tops of original proteins between the 2 chromatograms.
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Fig. 4.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
profile under ultraviolet light of NBD-SP-C and
NBD-[SP-C]2 recovered
from mouse lung homogenate at 24 h after instillation. Lane 1, original
NBD-[SP-C]2 (1 µg); lane 2,
original NBD-SP-C (1 µg); lane
3, total proteins recovered from mice instilled with NBD-[SP-C]2 liposome;
lane 4, total proteins recovered from
mice instilled with NBD-SP-C liposome; lanes
5 and 6, proteins
eluted at position of
[SP-C]2 recovered from
mice instilled with NBD-SP-C-liposome. Lanes
1-5, unreduced with 2-mercaptoethanol;
lane 6, reduced with 2-mercaptoethanol. Lanes 1-4 and
lanes 5 and
6 are from 2 different experiments.
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We then examined whether the conversion of
NBD-[SP-C]2 to
NBD-SP-C was mediated through GSH. GSH content in the lung after BSO
and DA treatment is shown in Fig. 5.
Intra-alveolar GSH concentration decreased to about one-fourth of
control levels after 4 h of BSO treatment and 2 h of DA treatment at
the time when liposomes were instilled. However, content of GSH in the
lung tissue decreased gradually after 2 h of instillation and attained
one-half of the control value at 4 and 8 h after instillation. GSSG
content increased from 2 h after instillation and attained 20% of
total GSH at 8 h after instillation. The conversion of
NBD-[SP-C]2 to
NBD-SP-C in control and BSO- and DA-treated groups over a short period is shown in Fig. 6. In the control group,
SP-C increased steadily from 2 h after instillation of liposomes. The
mean conversion rate from 2 to 8 h was calculated to be 0.22 µg · h
1 · mouse1
(y = 0.217x-0.166). In BSO- and DA-treated
groups, the amount of SP-C formed was significantly lower at 4 h after
instillation in the group treated with a low dose of DA
(P < 0.05) and was significantly
lower at all time points examined in the group treated with a high dose
of DA (P < 0.05).
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Fig. 5.
A: total GSH concentration in
first lung lavage fluid. B: reduced
and oxidized GSH (GSH and GSSG, respectively) content in lung tissues
before and after treatment with
L-buthionine-(S,R)-sulfoximine (BSO) and high dose of diamide (DA). Arrows, times of injection of
drugs; arrowhead, time of intratracheal instillation of liposomes containing NBD-[SP-C]2
shown in Fig. 6. Vertical bars, SD of means of 4 animals. SD bar is
within symbol at 2 h of tissue GSH.
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Fig. 6.
Total recovery of intratracheally instilled
NBD-[SP-C]2
(A) and monomeric SP-C formed at
various times after instillation of liposomes
(B) in control mice and in mice
treated with BSO and low or high dose of DA. Vertical bars, SD of means
of 4 animals. SD bar is within the symbol at 8 h of BSO + high DA
group.  Different from control;
P < 0.05.
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Figure 7 shows the distribution of
fluorescence between lavage fluid and lung tissue in controls and in
the group given GSH-treated liposomes at 0 and 24 h after instillation.
At time 0, in liposomes containing
NBD-[SP-C]2 pretreated
with GSH, 23.1 ± 1.9% were found as NBD-SP-C and this increased to
30.4 ± 1.9% at 24 h. These values were significantly higher than
those of control
NBD-[SP-C]2
(P < 0.001, see Table
1). The GSH-treated
NBD-[SP-C]2 was
removed from lavage fluid faster than untreated
NBD-[SP-C]2
(P < 0.05) at 24 h after
instillation. In comparison with NBD-SP-C, a larger fraction of
NBD-[SP-C]2 was
recovered in lung tissue than in lung lavage. Although a higher
percentage of NBD-SP-C was recovered in lavage fluid in liposomes
containing 1% SP-C than in those with 2% SP-C (96.0 ± 0.7 vs.
89.0 ± 3.3%) at time 0, no
differences were seen in the distribution between lavage fluid and
tissue at 24 h after instillation (data not shown).
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Fig. 7.
Distribution of fluorescence recovered in lung lavage fluid
(A) and in lung tissue
(B) at 0 and 24 h after
intratracheal instillation of liposomes containing 2%
NBD-[SP-C]2,
GSH-treated liposomes containing 2%
NBD-[SP-C]2
(GSH-treated-NBD-[SP-C]2),
and liposomes containing 2% SP-C (NBD-SP-C). Total fluorescence
(lavage fluid + lung tissue) at time 0 in each group was considered to be 100%. Vertical bars, SD of means of
at least 5 animals. Different from
NBD-[SP-C]2 group;
 different from GSH-treated
NBD-[SP-C]2 group
(P < 0.05).
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Table 1.
Conversion of fluorescence-labeled dimeric form of
surfactant-associated protein C (NBD-[SP-C]2) to
NBD-SP-C after intratracheal instillation of liposomes
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In vitro conversion of SP-C from a dimeric to a monomeric form by GSH
was examined. Because total protein concentration did not change during
incubation, the results were expressed as percentages of SP-C formed.
The reaction was temperature-, time-, and concentration-dependent as
shown in Fig. 8,
A and
B. Maximum change was attained at 2 h
of incubation. At the highest concentration of GSH (20 mM), ~40% of
NBD-[SP-C]2 was
converted to SP-C. When the unchanged NBD-[SP-C]2 was
isolated and incubated again with GSH in reconstituted liposomes, a
further 15% were converted to SP-C monomer (data not shown). Direct
addition of GSH-containing buffer to dried lipids and proteins did not
increase the conversion efficiency. These results suggested that the
disulfide linkage of more than one-half of the proteins occurs in a
hydrophobic environment that inhibits contact between GSH and proteins.
Decrease in GSH concentration from 1 to 0.1 mM decreased the conversion
only to about one-half (Fig. 8B). DA
inhibited the conversion more effectively in vitro than
in vivo, and inhibition was almost complete at a DA
concentration of one-half of the GSH concentration (0.2 mM DA vs. 0.4 mM GSH) (Fig. 8C).
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Fig. 8.
In vitro conversion by GSH of fluorescence labeled SP-C from
NBD-[SP-C]2 to
monomeric form (NBD-SP-C). Liposomes containing 500 µg phospholipids and 10 µg
NBD-[SP-C]2 were
incubated with GSH in 0.15 M KCl buffered with 0.1 M
tris(hydroxymethyl)aminomethane · HCl (pH 8.1)
containing 1 mM EDTA. After incubation, mixtures were extracted with
chloroform-methanol, and NBD-SP-C and
NBD-[SP-C]2 were
separated on Sephadex LH60 column. Conversion is expressed as
%NBD-SP-C formed. A: time and
temperature dependency, where liposomes were incubated with 0.4 and 5 mM GSH at 4 and 37°C; B:
concentration dependency, where liposomes were incubated with GSH at
37°C for 4 h; and C: inhibition of
conversion by DA, where liposomes were incubated with 0.4 mM GSH at
37°C for 4 h. Value without DA is considered as 100%; vertical
bars indicate SD of mean of 3 experiments.
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Figure 9 shows the SDS-PAGE profile of
unlabeled proteins treated with GSH in vitro. The bands corresponding
to [SP-C]2 became faint compared with those of SP-C. Similar changes were observed in
SP-B, and new bands appeared that migrated faster than SP-B. Relative
abundance of [SP-C]2 to SP-C on
SDS-PAGE was determined by densitometry in liposomes treated with GSH
(Fig. 10). GSH treatment significantly
lowered the ratio from 0.81 to 0.58 (P < 0.05).
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Fig. 9.
SDS-PAGE profile of unlabeled hydrophobic SP after treatment with GSH.
Liposomes prepared from total lipid extract of lung lavage fluid of
patients with alveolar proteinosis were incubated with 10 mM of GSH at
37°C for 2 h. Proteins were fractionated with a Sephadex LH60
column, and fractions containing proteins were examined by using 12.5%
gel without reduction. A: control incubated without GSH; B: GSH treated.
Closed arrows, bands of dimeric SP-C; open arrow, appearance of new
bands. Gel stained with Coomassie brilliant blue.
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Fig. 10.
Densitometric quantification of alteration of unlabeled
[SP-C]2 and SP-C
contents in liposomes after incubation with GSH. Incubation conditions
for control and GSH-treated liposomes were same as those shown in Fig.
9. Coomassie brilliant blue-stained gels were analyzed by densitometry,
and ratio of [SP-C]2
to SP-C was calculated. Each bar represents mean ± SD of 3 experiments. Statistically significant difference at
P < 0.05.
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DISCUSSION |
The PL content in the first of the sequential lung
washes was about one-half of the total intra-alveolar PL. The rapid
increase in GSH after the second wash was possibly caused by leakage
from cells without cell disintegration, because the increase in
GSH preceded the increase in LDH. These results suggest
that the content of GSH in the first wash (0.6 µg/lungs) represents one-half of the actual intra-alveolar content of GSH (1.2 µg). Intra-alveolar concentration of GSH is,
therefore, calculated to be 0.4-0.8 mM, assuming that the original
alveolar lining fluid is 5-10 µl (total area,
1,000 cm2; mean depth of lining
layer, 0.05-0.1 µm) (29).
[SP-C]2 has been
detected in lungs in several studies (1, 5, 18, 26, 27).
Interconversion between the monomer and dimer has been suspected (5)
and was demonstrated in the present experiment. Furthermore, we have
shown that GSH participates in this conversion.
Supposing the conversion of NBD-SP-C to
NBD-[SP-C]2 to be
negligible, the half-life of NBD-SP-C was shown to be almost the same
in liposomes containing different contents of SP-C: 17.7 h for
liposomes containing 1% SP-C and 18.8 h for those with 2% SP-C. This
suggests that the clearance pathways of SP-C are not saturable under
the present experimental conditions. Pettenazzo and colleagues (15)
reported that the clearance pathways of DPPC in the rabbit were
relatively nonsaturable despite exogenous administration of large
amounts of lipids. On the other hand, the half-life of
NBD-[SP-C]2 as
calculated from the curve obtained in Fig. 2 was 30.1 h. However, some
NBD-[SP-C]2 was
removed as NBD-SP-C, which has a shorter half-life. To correct for
this, we obtained an equation composed of two parts with different
removal rates (assuming 20 µg as the starting amount of
NB-[SP-C]2); that is,
where
0.22 denotes the rate of
NBD-[SP-C]2 converted
to NBD-SP-C per hour (Fig. 6), 17.7 is the half-life of NBD-SP-C (1%) described above, and
t1/2C2
represents the half-life of
NBD-[SP-C]2. We
obtained a half-life of 37.5 h. The percentage of
NBD-SP-C in the total recovered proteins was calculated as
0.22t × 2t/17.7
divided by C2t
and was 17.9 and 24.3% at 24 and 48 h after instillation, respectively. These calculated values were in accordance with the
observed values as summarized in Table 1.
These results suggest that the dimeric form of NBD-SP-C has a
biological half-life about twice as long as the monomeric form. Interconversion of
NBD-[SP-C]2 to
NBD-SP-C accelerates the apparent removal of the former. The half-lives
obtained here for these proteins are, however, very long compared with
those obtained for SP-A, SP-B, and DPPC in rabbit lungs (24, 25).
Extrapolation of the present results directly to the metabolism of
these proteins in humans may be limited, because the amounts of lipids
and proteins we used were 10- to 20-fold larger than the endogenous
pools of lipids and proteins in the mouse lung, and human proteins
might behave differently in the mouse lungs. Therefore, the actual
clearance times of these proteins in humans should be investigated
further.
BSO and DA, potent inhibitors of GSH, are widely used to inhibit GSH
action (11, 23). In vivo, we could lower the GSH level
in lavage fluid to about one-fourth of the control value with BSO and
with a high dose of DA. The decrease in GSH content correlated with
decreased formation of NBD-SP-C; thus GSH may be one of the substances
in the lung which reduces the dimer. Direct chemical oxidation of
biliverdin reductase by DA was shown to induce dimerization of the
enzyme (23), but treatment of NBD-SP-C with DA in vitro
did not produce
NBD-[SP-C]2. The
decreased conversion of the dimeric form of NBD-SP-C is, therefore,
considered to be caused mainly by the decrease in GSH.
In vitro, we showed that GSH sufficiently converted
NBD-[SP-C]2 to
NBD-SP-C. This reaction was not dependent on the presence of enzymes
and proceeded even at 4°C at a high concentration of GSH. The
concentration range (0.2-1 mM), which corresponded to that of GSH
in mouse alveoli, effectively induced the conversion. The efficiency of
conversion decreased to only one-half despite the decrease in the
concentration of GSH from 1 to 0.1 mM. This explains why the reaction
proceeded in animals, extensively treated with BSO and DA, in which GSH
concentration was reduced to one-fourth of the control value.
The reason for the presence of a large amount of
[SP-C]2 in PAP is not
clear. More than one-half of the cysteinyl residues of SP-C in PAP are
depalmitoylated (19), and, if the reducing activity in the lung is not
sufficient, dimers may be formed. However, as shown here, the
conversion of a monomer to a dimer is negligible in normal lung. GSH
treatment of liposomes slightly accelerated the removal of
[SP-C]2 at 24 h after
instillation, and GSH may participate in the metabolism of these
hydrophobic proteins. However, we failed to reduce GSH levels
continuously for a prolonged period, as there was significant rebound
of these levels. Therefore it is a matter for further investigation
whether the inhibition of reducing activity in the lung is accompanied by an overaccumulation and a delay in the removal of the dimeric form
of SP-C. Recently, it was reported that mice deficient in granulocyte/macrophage colony-stimulating factor exhibited
overaccumulation of surfactant lipids and proteins, similar to the
conditions in human PAP and the abnormalities in type II cell
and/or macrophage function that are suspected to
cause PAP (13, 14). Precise comparison of
the status of the hydrophobic SP is required between the mouse model
and human PAP, and such comparison may give further insight into the
pathogenesis of these conditions.
Address for reprint requests: Y. Suzuki, Dept. of Molecular
Pathology, Chest Disease Research Institute, Kyoto Univ., Sakyo-ku,
Kyoto 606, Japan.
| 1.
|
Baatz, J. E.,
K. L. Smyth,
J. A. Whitsett,
C. Baxter,
and
D. R. Absolom.
Structure and function of a dimeric form of surfactant protein SP-C: a Fourier transform infrared and surfactometry study.
Chem. Phys. Lipids
63:
91-104,
1992[Medline].
|
| 2.
|
Bartlett, G. R.
Phosphorus assay in column chromatography.
J. Biol. Chem.
234:
466-468,
1959[Free Full Text].
|
| 3.
|
Bates, S. R.,
M. F. Beers,
and
A. B. Fisher.
Binding and uptake of surfactant protein B by alveolar type II cells.
Am. J. Physiol.
263 (Lung Cell. Mol. Physiol. 7):
L333-L341,
1992[Abstract/Free Full Text].
|
| 4.
|
Bates, S. R.,
and
A. B. Fisher.
Degradation of surfactant protein B by alveolar type II cells.
Am. J. Physiol.
265 (Lung Cell. Mol. Physiol. 9):
L448-L455,
1993[Abstract/Free Full Text].
|
| 5.
|
Creuwels, L. A. J. M.,
R. A. Demel,
L. M. G. van Golde,
and
H. P. Haagsman.
Characterization of a dimeric canine form of surfactant protein C (SP-C).
Biochim. Biophys. Acta
1254:
326-332,
1995[Medline].
|
| 6.
|
Curstedt, T.,
H. Jornvall,
B. Robertson,
T. Bergman,
and
P. Berggren.
Two hydrophobic low-molecular-mass protein fractions of pulmonary surfactant. Characterization and biological activity.
Eur. J. Biochem.
168:
255-262,
1987[Medline].
|
| 7.
|
Dobbs, L. G.,
J. R. Wright,
S. Hawgood,
R. Gonzalez,
K. Venstrom,
and
J. Nellenbogen.
Pulmonary surfactant and its components inhibit secretion of phosphatidylcholine from cultured rat alveolar type II cells.
Proc. Natl. Acad. Sci. USA
84:
1010-1014,
1987[Abstract/Free Full Text].
|
| 8.
|
Folch, J.,
M. Lees,
and
G. H. Sloane Stanley.
A simple method for the isolation and purification of total lipids from animal tissues.
J. Biol. Chem.
226:
497-509,
1957[Free Full Text].
|
| 9.
|
Gilmore, L. B.,
F. A. Talley,
and
E. R. Hook.
Classification and morphometric quantification of insoluble materials from the lungs of patients with alveolar proteinosis.
Am. J. Pathol.
133:
252-264,
1988[Abstract].
|
| 10.
|
Goto, Y.,
and
K. Hamaguchi.
Formation of the interchain disulfide bond in the constant fragment of the immunoglobulin light chain.
J. Mol. Biol.
146:
321-340,
1981[Medline].
|
| 11.
|
Griffith, O. W.
Mechanism of action, metabolism, and toxicity of buthionine sulfoximine and its higher homologs, potent inhibitors of glutathione synthesis.
J. Biol. Chem.
257:
13704-13712,
1982[Free Full Text].
|
| 12.
|
Horowitz, A. D.,
J. E. Baatz,
and
J. A. Whitsett.
Lipid effects on aggregation of pulmonary surfactant protein SP-C studied by fluorescence energy transfer.
Biochemistry
32:
9513-9523,
1993[Medline].
|
| 13.
|
Huffman, J. A.,
W. M. Hull,
G. Dranoff,
R. C. Mulligan,
and
J. A. Whitsett.
Pulmonary epithelial cell expression of GM-CSF corrects the alveolar proteinosis in GM-CSF-deficient mice.
J. Clin. Invest.
97:
649-655,
1996[Medline].
|
| 14.
|
Ikegami, M.,
T. Ueda,
W. Hull,
J. A. Whitsett,
R. C. Mulligan,
G. Dranoff,
and
A. H. Jobe.
Surfactant metabolism in transgenic mice after granulocyte macrophage-colony stimulating factor ablation.
Am. J. Physiol.
270 (Lung Cell. Mol. Physiol. 14):
L650-L658,
1996[Abstract/Free Full Text].
|
| 15.
|
Pettenazzo, A.,
A. H. Jobe,
M. Ikegami,
E. Rider,
S. R. Seider,
and
T. Yamada.
Cumulative effects of repeated surfactant treatments in the rabbit.
Exp. Lung Res.
16:
131-143,
1990[Medline].
|
| 16.
|
Pinto, R. A.,
J. R. Wright,
D. Lesikar,
B. J. Benson,
and
J. A. Clements.
Uptake of pulmonary surfactant protein C into adult rat lung lamellar bodies.
J. Appl. Physiol.
74:
1005-1011,
1993[Abstract/Free Full Text].
|
| 17.
|
Suzuki, Y.
Effect of protein, cholesterol, and phosphatidylglycerol on the surface activity of the lipid protein complex reconstituted from pig pulmonary surfactant.
J. Lipid Res.
23:
62-69,
1982[Abstract].
|
| 18.
|
Suzuki, Y.,
H. Q. Shen,
A. Sato,
and
Y. Fujita.
Characterization of surfactant apoproteins in pulmonary alveolar proteinosis.
J. Jpn. Med. Soc. Biol. Interface
24:
93-98,
1993.
|
| 19.
|
Suzuki, Y.,
H. Q. Shen,
A. Sato,
and
S. Nagai.
Analysis of fused-membrane structures in bronchoalveolar lavage fluid from patients with alveolar proteinosis.
Am. J. Respir. Cell Mol. Biol.
12:
238-249,
1995[Abstract].
|
| 20.
|
Tenner, A. J.,
S. L. Robinson,
J. Borchelt,
and
J. R. Wright.
Human pulmonary surfactant protein (SP-A), a protein structurally homologous to C1q, can enhance FcR- and CR-1 mediated phagocytosis.
J. Biol. Chem.
264:
13923-13928,
1989[Abstract/Free Full Text].
|
| 21.
|
Thompson, J. F.,
and
G. R. Morrison.
Determination of organic nitrogen. Control of variables in the use of Nessler's reagent.
Anal. Chem.
23:
1153-1157,
1951.
|
| 22.
|
Tietze, F.
Enzymatic method for quantitative determination of nanogram amounts of total and oxidized glutathione: application to mammalian blood and other tissues.
Anal. Biochem.
27:
502-522,
1969[Medline].
|
| 23.
|
Tomaro, M. L.,
J. Frydman,
and
R. B. Frydman.
The in vivo and in vitro oxidation of molecular form 1 of biliverdin reductase to molecular form 3 by diamide.
FEBS Lett.
263:
38-42,
1990[Medline].
|
| 24.
|
Ueda, T.,
M. Ikegami,
M. Henry,
and
A. H. Jobe.
Clearance of surfactant protein B from rabbit lungs.
Am. J. Physiol.
268 (Lung Cell. Mol. Physiol. 12):
L636-L641,
1995[Abstract/Free Full Text].
|
| 25.
|
Ueda, T.,
M. Ikegami,
and
A. H. Jobe.
Clearance of surfactant protein A from rabbit lungs.
Am. J. Respir. Cell Mol. Biol.
12:
89-94,
1995[Abstract].
|
| 26.
|
Voss, T.,
K. P. Schafer,
P. F. Nielsen,
A. Schafer,
C. Maier,
E. Hannappel,
J. Maassen,
B. Landis,
K. Klemm,
and
M. Przybylski.
Primary structure difference of human surfactant-associated proteins isolated from normal and proteinosis lung.
Biochim. Biophys. Acta
1138:
261-267,
1992[Medline].
|
| 27.
|
Warr, R. G.,
S. Hawgood,
D. I. Buckley,
T. M. Crisp,
J. Schilling,
B. J. Benson,
P. L. Ballard,
J. A. Clements,
and
R. T. White.
Low molecular weight human surfactant protein (SP5): isolation, characterization, and cDNA and amino acid sequences.
Proc. Natl. Acad. Sci. USA
84:
7915-7919,
1987[Abstract/Free Full Text].
|
| 28.
|
Weber, K.,
and
M. Osborn.
The reliability of molecular weight determination by dodecyl sulfate-polyacrylamide gel electrophoresis.
J. Biol. Chem.
244:
4406-4412,
1969[Abstract/Free Full Text].
|
| 29.
|
Weibel, E. R.
Morphological basis of alveolar-capillary gas exchange.
Physiol. Rev.
53:
419-495,
1973[Free Full Text].
|
| 30.
|
Wright, J. R.,
and
D. C. Youmans.
Degradation of surfactant lipids and surfactant protein A by alveolar macrophages in vitro.
Am. J. Physiol.
268 (Lung Cell. Mol. Physiol. 12):
L772-L780,
1995[Abstract/Free Full Text].
|
| 31.
|
Yu, S. H.,
and
F. Possmayer.
Role of bovine pulmonary surfactant-associated proteins in the surface-active property of phospholipid mixtures.
Biochim. Biophys. Acta
1046:
233-241,
1990[Medline].
|
Top
Abstract
Introduction
Clearance of hydrophobic surfactant-associated protein C (SP-C)
and its dimeric form
([SP-C]2) was
investigated. SP-C and
[SP-C]2 obtained from
proteinosis patients were fluorescently labeled and were instilled into
mouse lungs as lipid-protein complexes.
[SP-C]2 was removed
more slowly than SP-C, with apparent half-lives of 30 and 18 h,
respectively. A significant amount of
[SP-C]2 was removed as
SP-C, and the conversion rate was 0.22 µg · h
