Increased group IV cytosolic phospholipase
A2 activity in lungs of sheep
after smoke inhalation injury
Taeko
Fukuda,
Dae Kyong
Kim,
Mi-Reyoung
Chin,
Charles A.
Hales, and
Joseph V.
Bonventre
Medical Services, Massachusetts General Hospital, Charlestown 02129;
and Department of Medicine, Harvard Medical School, Boston,
Massachusetts 02115
 |
ABSTRACT |
Increased phospholipase
A2
(PLA2) activity was measured in
cytosolic fractions of lungs from sheep exposed to smoke from burning cotton or to synthetic smoke consisting of carbon and acrolein, a
cotton smoke toxin. Three peaks of
PLA2 activity were identified by
heparin-Sepharose chromatography. The heparin-nonbinding
PLA2 activity was twofold higher
in the extracts from lungs exposed to smoke than in normal lungs. This
activity was identified as the group IV 85-kDa cytosolic
PLA2
(cPLA2). The activities of the
forms of PLA2 that bound to
heparin did not change after smoke exposure. Those activities showed a
pH optimum of 9.0, required a millimolar
Ca2+ concentration for full
activity, and were inhibited by 5 mM dithiothreitol. One activity
eluted at an NaCl concentration typical for group Ib and V
PLA2 and had the expected
substrate specificity. The other form of lung
PLA2 that bound heparin was a
group II PLA2. Lung
myeloperoxidase activity increased progressively with increased exposure to smoke. cPLA2 was
identified in sheep neutrophils. With 30 breaths of smoke exposure,
there was an increase in cPLA2 activity without a difference in immunoreactivity on Western blot, indicating that the increased activity was not due to increased amounts
of protein. In conclusion, smoke induces increases in resident lung
cell cPLA2 activity that is likely
responsible for eicosanoid production, leading to lung inflammation and bronchoconstriction.
eicosanoids; polymorphonuclear leukocytes; acrolein; cotton
smoke
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INTRODUCTION |
LUNG INJURY FROM SMOKE INHALATION is a major cause of
mortality (34). Smoke inhalation initiates an inflammatory cascade, with leukocyte infiltration, cytokine release, and generation of
mediators such as reactive oxygen species, proteases, and products of
arachidonic acid (AA) metabolism (27), with a subsequent increase in
vascular permeability (28).
Heat, except when inhaled as steam, is not the primary agent causing
lung parenchymal injury. Rather, chemical toxins carried in either the
gas or particle phase confer most of the damage (19). Acrolein, a
potent aldehyde that is a major component of smoke from burning wood,
cotton, cigarettes, and smog, has a severe irritant effect on the
airways (25) and is a known cause of high-permeability pulmonary edema
(10).
Phospholipases A2 (EC. 3.1.1.4)
play key roles in the production of intracellular and extracellular
chemical mediators of inflammation, such as eicosanoids. Phospholipases
A2 hydrolyze fatty acids bound at
the sn-2 position of
glycerophospholipids (37, 38). A major product of phospholipase
A2
(PLA2) action on membrane lipids
is AA, the eicosanoid metabolites of which include prostaglandins and
leukotrienes that modulate membrane channel activity and signal
transduction, are vasoactive and chemotactic, and are implicated in
many pathophysiological mechanisms of inflammation and tissue injury
(3, 4). Eicosanoids have been identified in bronchoalveolar lavage
fluid (BALF) and lung lymph after inhalation of smoke from burning
cotton or synthetic smoke consisting of acrolein adherent to cotton
particles (12, 28). Blockade of the cyclooxygenase and lipoxygenase
cascades with BW-755C substantially ameliorates the appearance of
pulmonary edema after cotton smoke or acrolein smoke inhalation,
demonstrating a causative role for eicosanoids in smoke-induced injury
(11).
The superfamily of mammalian phospholipases
A2 is composed of a number of
members that vary in size, tissue distribution, Ca2+ dependency, substrate
preference, and sensitivity to dithiothreitol (DTT) (7). Four related
genes encode four Ca2+-dependent
secretory phospholipases A2
(groups I, IIa, IIc, and V), with approximate molecular
masses of 14 kDa and requirements for
10
4 to
10
3 M
Ca2+ concentration for effective
hydrolysis of substrates. Group I (29) and II phospholipases
A2 (26) have been found in the lung. Inflammatory cytokines, such as tumor necrosis factor,
interleukin-1, and interleukin-6 induce transcription of the group II
PLA2 gene, resulting in increased
eicosanoid generation in a variety of target cells (31).
The intracellular high-molecular-mass (85-kDa) group IV
PLA2 [cytosolic
PLA2
(cPLA2)] has been
implicated in Ca2+- and
receptor-mediated liberation of arachidonic acid (AA) (4, 9, 15,
31). cPLA2 has been
purified from various tissues including bovine lung, bovine kidney, pig
spleen (13), human monocytic U937 cells (6, 32), rabbit platelets (16),
and the rat macrophage cell line RAW 264.7 (22).
cPLA2 is important for
methacholine-induced bronchial reactivity (35). cPLA2
activity is also activated by reactive oxygen species (30), which are important mediators of smoke-induced lung injury.
To evaluate whether increased levels of
PLA2 activity might contribute to
eicosanoid production and inflammation after short exposures to smoke,
we identified and characterized
PLA2 activity in sheep lungs after
exposure to smoke from burning cotton or to a synthetic smoke
containing only carbon and acrolein, which is likely the major toxin in
cotton smoke. In the acrolein-carbon- or cotton smoke-exposed lungs,
there was an increase in PLA2
activity that was identified biochemically and immunochemically as
cPLA2. With longer periods of
smoke exposure, myeloperoxidase (MPO) activity in the lung was
increased, which suggests that neutrophil infiltration may be
responsible for some but not all of the increased lung cPLA2 activity. This increased
cPLA2 activity, with its substrate specificity for AA-containing phospholipids, may account for the generation of inflammatory mediators that lead to pulmonary edema and
respiratory distress.
 |
METHODS |
Materials.
1-Acyl-2-[1-14C]arachidonyl-sn-glycero-3-phosphoethanolamine
(2-[1-14C]AA-GPE; 55.1 mCi/mmol) and
1-stearoyl-2-[1-14C]arachidonyl-sn-glycero-3-phosphocholine
(2-[1-14C]AA-GPC; 56.7 mCi/mmol) were purchased from Amersham. Heparin-Sepharose HiTrap, Mono
Q, and Superose 12 columns were obtained from Pharmacia LKB
Biotechnology. All other chemicals were of the highest purity available
from commercial sources.
Preparation of animals and harvesting of
lungs. Domesticated sheep weighing from 25 to 27 kg
were anesthetized with intravenous pentobarbital sodium (25 mg/kg
induction; 150- to 200-mg maintenance dose given intermittently to
maintain deep anesthesia), intubated with a cuffed endotracheal tube
(10 mm ID, 33 cm long), and mechanically ventilated with 35% oxygen
with a volume ventilator (Harvard Apparatus, Millis, MA) set initially
at a tidal volume of 15 ml/kg at 15 breaths/min and 2 Torr positive
end-expiratory pressure. The respiratory rate was adjusted to achieve
an arterial PCO2 of 33-42 Torr.
Blood gases and pH were measured at 38°C with an Instrumentation
Laboratory 1306 blood gas analyzer (Watertown, MA). An oral tube was
passed into the stomach to evacuate the contents. A catheter was
inserted into a femoral vein to permit infusion of lactated Ringer
solution at a rate sufficient to maintain a pulmonary capillary wedge
pressure of 5 mmHg. A right thoracotomy was performed, and a lymph
fistula was established in the caudal mediastinal lymph node with the
use of a modification of the technique of Staub et al. (33). The distal
node coming from the abdomen was ligated with a double suture to
decrease contamination. As the thorax was closed, a tube was placed and
connected to a sealed collection system (Pleur-Evac, Deknatel, Floral
Park, NY) with
20 cmH2O applied. A Swan-Ganz pulmonary arterial catheter (model 93A-13H-7F, American Edwards Laboratories, Santa Ana, CA) was inserted via an
internal jugular vein and positioned in the pulmonary artery by
continuously following the wave forms on the monitor. Pulmonary arterial pressure, intermittent pulmonary capillary wedge pressure, and
tracheal pressure were monitored throughout the experiments with
transducers (model P23 XL, Spectromed, Oxnard, CA) mounted at the
midthoracic level. Data were continuously recorded on a Gould
(Cleveland, OH) model 3400 chart recorder. Cardiac output was determined in duplicate by using thermal dilution and a Cardiac OutPUT computer (COM-1, American Edwards Laboratories).
Lung lymph was continuously collected from below the level of the lymph
fistula and measured every 30 min. The samples were placed immediately
on ice at the time of collection and were later centrifuged for 10 min
at 2,300 rpm at 4°C. Protein content was determined on the
supernatant with a protometer (National Instrument, Baltimore, MD).
Sytemic and pulmonary arterial pressures and cardiac output were monitored.
Exposure to smoke. The animals were
allowed to stabilize on the anesthetic for 1-1.5 h, during which
time baseline measurement of the hemodynamic parameters and lymph flow
were measured every 30 min. Smoke generation and administration were
done in one of two ways, involving either a smoke generator on which
Hales et al. (10) have previously published or real smoke
from burning cotton (11).
The synthetic smoke system allowed the delivery of smoke containing
only one toxin at a time. Each breath of room air from the ventilator
was passed through the smoke generator, which divided flow into two
streams according to the settings of the two needle valves. The
resultant flows were measured on flow meters (Lab Crest, Waltham, MA).
One stream was fed into an ultrasonic nebulizer (model US-1,
Puritan-Bennett, Los Angeles, CA) that was filled with distilled water
and the toxin acrolein. The other stream was fed through a jet into a
cup filled with carbon particles (D-Dacro G-60, Fisher Scientific, Fair
Lawn, NJ) with a mean geometric diameter of 3.9 ± 2 (SD)
µm as determined by a particle impactor (Anderson
Sampler, Atlanta, GA). The carbon particles were then elutriated into
the exit tube. The airflows from the nebulizer and carbon chimney were
mixed and fed through a thermostat-controlled heating system (Hot-Watt
6 Fo10, Danvers, MA, and a Fenwall controller 551, Ashland,
MA) into a condensing section, allowing hot gases and
carbon to cool to body temperature. The vaporization-condensation process produces an aerosol that has characteristics independent of the
ultrasonic nebulizer. In addition, the carbon provides nucleation
sites, thus being incorporated into the droplets. The smoke was then
provided to the animal through an endotracheal tube. Immediately after
exposure to smoke in room air, the inspired fraction of
O2 was increased to 0.35.
The toxin used in the present study was acrolein (100 µl, density
0.841 g/ml; Kodak, Rochester, NY) that was mixed with 100 ml of
distilled water and placed in the nebulizer that was set to deliver 5 ml of distilled water and toxin over the 10-min exposure. Carbon
particles were weighed before being placed in the chimney. The
temperature of the heater was set at 120°C, and the condensing section was cooled by airflow to lower the effluent gas temperature to
40°C. Liquid remaining in the nebulizer and carbon remaining in the
chimney at the end of exposure were weighed. The concentrations of
inhaled acrolein in the gas mixture at the first, middle, and last
minutes of acrolein smoke exposure were 50-100 parts/million (10).
Real smoke was generated with a modified Bee smoker (The Bee Keeper,
Woburn, MA) as originally described by Walker et al. (39) and
subsequently modified by Kimaura et al. (17). Ten pure cotton pledgits
(14 g) were packed in the chamber and ignited instantly with a
blowtorch. The smoker was attached to the sheep via the endotracheal
tube while 16 breaths of smoke were delivered. The sheep was then
returned to the ventilator while the smoker was recharged and refired.
A total of 15-130 breaths was delivered to each sheep. The tidal
volume was 640 ± 35 ml as determined by measuring exhaled minute
ventilation from the sheep and dividing by the respiratory rate. We
have grouped together sheep receiving 40-50 breaths of cotton
smoke and those receiving 120-130 breaths of cotton smoke,
referring to them as 40- or 120-breath groups, respectively. In all
cases, the sheep exposed to acrolein-carbon received 120 breaths.
All hemodynamic measurements were continued for 4 h after smoke
exposure. At the end of this 4-h period, the sheep were killed with
intravenous thiopental and potassium chloride. The lungs were removed
and kept at
80°C. For gravimetric analysis, the lungs were
trimmed of extra parenchymal airway and blood vessels, blotted dry,
weighed, and then placed in an oven at 80°C for 48 h. The lung was
weighed and dried for another 24 h at 80°C. If the weight fell
between the two readings, the lung was dried for another 24 h.
PLA2 assay.
PLA2 activity was assayed with
ethanol-solubilized
2-[1-14C]AA-GPE (55.1 mCi/mol; Amersham). The standard
PLA2 assay buffer (100 µl)
contained 100 mM Tris · HCl, 5 mM
CaCl2, and 0.5 nmol of radioactive
phospholipids (~65,000 counts/min) at pH 9.0. The reaction was
carried out at 37°C for 30 min and was stopped by adding 0.56 ml of
Dole's reagent (78% propan-2-ol, 20%
n-heptane, and 2% of 2 N
H2SO4
in water) (8). Water (0.11 ml) was added, and the sample was vortex
mixed and centrifuged at 10,000 g for 2 min, then 0.15 ml of the upper phase was transferred to a new Eppendorf tube to which 50 mg of silica gel and 0.8 ml of
n-heptane were added, vortex mixed,
and centrifuged again at 10,000 g for 2 min. Finally, 0.8 ml of supernatant, which contained released fatty
acid, was counted for radioactivity in a beta liquid scintillation counter. Protein concentration was determined with the Bradford protein
assay (Bio-Rad Laboratories, Melville, NY), with bovine serum albumin
(BSA) as the standard.
Heparin-Sepharose HiTrap column
chromatography. Sheep lungs,
frozen at
80°C, were thawed at 37°C, minced, and
homogenized in six volumes (vol/wt) of homogenizing buffer (50 mM
Tris · HCl, pH 7.4, 1 mM EDTA, and 0.15 M NaCl) with
a polytron (Brinkmann homogenizer). The homogenates were centrifuged at
1,000 g for 20 min. All subsequent
manipulations of lung tissue for characterization were performed at
4°C in a cold room or on ice unless otherwise indicated. The
supernatants were centrifuged at 100,000 g for 60 min at 4°C. The resulting
supernatants were filtered with MILEX-HA (0.45-µm-pore size;
Millipore, Bedford, MA), diluted in the same volume of
buffer A (50 mM
Tris · HCl, pH 7.4, and 1 mM EDTA), and applied at a
flow rate of 2 ml/min to a heparin-Sepharose column (HiTrap 5 ml,
Pharmacia LKB Biotechnology) preequilibrated with
buffer A. The column was washed with
40 ml of buffer A. Bound fractions
were eluted at a flow rate of 2 ml/min, with a 40-ml linear gradient of
0-2.0 M NaCl. Fractions (2 ml) were collected and assayed for
PLA2 activity.
Ion-exchange chromatography on Mono Q fast-performance
liquid column. The pooled heparin-nonbinding fractions
of sheep lung were loaded onto a Mono Q fast-performance liquid
chromatography (FPLC) column (0.5 × 5 cm; Pharmacia LKB
Biotechnology) preequilibrated with buffer
A. Proteins were eluted at a flow rate of 1.0 ml, with
a 20-ml linear gradient of 0-1.0 M NaCl. Fractions (1.0 ml) were
collected and assayed for PLA2 activity.
Gel-filtration chromatography on Superose 12 FPLC
column. The active pool
(fractions
9-11; 3 ml) from
the Mono Q column was concentrated to ~200 µl with a Centricon 10 (Amicon, Danvers, MA). The concentrated sample was injected onto a
Superose 12 FPLC column preequilibrated with 50 mM
Tris · HCl, pH 7.4, 1 mM EDTA, and 0.5 M NaCl.
Proteins were eluted at a flow rate of 0.5 ml/min. Fractions (1.0 ml)
were collected and assayed for
PLA2 activity. For molecular-mass
determination, a mixture of molecular-mass markers, RNase A (13.7 kDa),
chymotrypsinogen A (25 kDa), ovalbumin (43 kDa), BSA (67 kDa), and blue
dextran 2000 (2,000 kDa), was injected onto the column under the same conditions.
Immunoprecipitation of PLA2 activity.
Packed protein A-Sepharose beads saturated with rabbit anti-porcine
spleen 100-kDa PLA2 antiserum or
rabbit normal serum were prepared as previously described (13). Rabbit
antiserum made against porcine spleen 100-kDa
cPLA2 or rabbit preimmune serum was mixed with packed protein A-Sepharose beads preequilibrated with 20 mM Tris · HCl buffer (pH 7.5) containing 5 mg/ml of
BSA (2:1 vol/vol) and incubated for 24 h at 4°C with constant
shaking. The beads were washed six times with 20 mM
Tris · HCl (pH 7.5) containing 1 mM EDTA and 5 mg/ml
of BSA. Fifty microliters of protein A-Sepharose beads were incubated
for 4, 8, and 16 h at 4°C with 450 µl of the active pool of
PLA2 activity collected from Mono
Q FPLC with constant shaking. The beads were then pelleted by
centrifuging at 10,000 g for 5 min,
and the supernatants were assayed for
PLA2 activity.
Immunoblotting. Proteins were
separated by SDS-PAGE (10% gels) (18) and electrophoretically
transferred to nitrocellulose membranes (Schleicher & Schuell) in 25 mM
Tris · HCl (pH 8.3)-190 mM glycine-20% methanol.
Nonspecific binding of antisera to nitrocellulose was prevented by
preincubation of the nitrocellulose in 3% BSA in Tris-buffered saline
(24.8 mM Tris · HCl, pH 8.0, 2.7 mM KCl, and 137 mM
NaCl) for 2 h at room temperature. The blocked nitrocellulose membrane
was incubated with 1:5,000 diluted anti-cPLA2 antiserum overnight at room temperature with constant shaking. Unbound antibodies were removed with three washes of Tris-buffered saline containing 0.1%
Tween 20, and the sites of antibody binding were developed with an
enhanced chemiluminescence system (Amersham).
MPO activity. MPO activity, used as an
indicator of leukocyte, primarily neutrophil, infiltration, was
measured in lung tissue after 2 h of smoke exposure or on
non-smoke-exposed lung tissue. An aliquot (0.4 ml) of the
40,000-g supernatants of lung
homogenates, prepared as described by Bradley et al. (5), was added to
2.6 ml of reaction mixture containing 50 mM potassium phosphate buffer (pH 6.0), 0.2 mg o-dianisidine
dihydrochloride/ml, and 0.0006% H2O2.
Absorbance was measured at 460 nm. Assay linearity was confirmed. MPO
activity, normalized to protein content of the supernatant, is
expressed as the percentage of the levels in normal lung.
Statistics. Data are expressed as
means ± SE. Differences among mean values of
PLA2 specific activities and among
groups were evaluated by analysis of variance. Student's
t-test was used for comparisons
between two groups, and differences were considered significant if
P was <0.05.
 |
RESULTS |
Hemodynamic and functional effects of smoke
inhalation. Pulmonary arterial pressure did not rise
significantly after 15 breaths of cotton smoke (Table
1), although by 4 h after
smoke, lung lymphatic clearance of protein had risen. This suggested an
increase in lung microvascular permeability because the lymph-to-plasma protein ratio was constant (Table 2).
However, the wet-to-dry weight ratio did not rise (Fig.
1), presumably because the lymph drainage
was adequate to prevent pulmonary edema. In two sheep that received 30 breaths of smoke and five that received 40-50 breaths, there was a
modest rise in pulmonary arterial pressure of 3-4 mmHg, which was
partly, but not entirely, due to a rise in cardiac output because
pulmonary vascular resistance rose (P < 0.05; Table 1). Pulmonary vascular resistance was calculated as
(mean pulmonary artery pressure
mean pulmonary capillary wedge
pressure)/cardiac output. Lung lymph flow and lymph protein clearance
rose significantly (Table 2) but the lymph-to-plasma protein ratio
fell. Thus lung microvascular fluid movement into the extravascular
space was likely due to a combination of pressure rise and an increase
in microvascular permeability. Again, the increase in lymph flow
prevented a significant increase in pulmonary edema as the lung
wet-to-dry weight ratio did not rise significantly in this small group,
although there was a small trend up in this ratio with an increased
number of breaths of smoke (Fig. 1). Both 120-130 breaths of
cotton smoke and synthetic smoke with acrolein-carbon caused mild
pulmonary edema (Fig. 1) as well as an increase in lymph flow and lymph
protein clearance (Table 2). Pulmonary arterial pressure rose with
acrolein-carbon exposure and probably contributed to increased lymph
flow, although the lymph-to-plasma protein ratio did not fall, showing
that there was also a change in microvascular permeability. High-dose
smoke, unlike lower doses, did not increase pulmonary arterial pressure
or pulmonary vascular resistance (Table 1). Thus the increased lymph
flow and wet-to-dry weight ratio of the lung were likely due to
increases in microvascular permeability.

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Fig. 1.
Blood corrected wet-to-dry (Wet/Dry) weight ratios in smoke-exposed
lungs. No. of animals/group are the same as in Table 1.
* P < 0.05 compared with
control group.
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PLA2 activity of 100,000-g supernatants
of sheep lungs.
PLA2 activities of the
100,000-g supernatants from sheep
lungs after 10 min of exposure to acrolein-carbon smoke or burning cotton smoke or no treatment were assayed with
2-[1-14C]AA-GPE as the
substrate (Fig. 2).
PLA2 activities in both types of
smoke-exposed lungs were significantly higher than those in normal
lungs. There was no significant difference between the PLA2 activities of lungs exposed
to acrolein-carbon or burning cotton smoke. As few as 15 breaths of
cotton smoke resulted in a significant increase in lung
PLA2 activity (see
Correlation between PLA2 activity and MPO
activity).


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Fig. 2.
Comparison of phospholipase A2
(PLA2) activity of
100,000-g supernatant from normal
sheep lungs or lungs exposed to 120 breaths of smoke derived from
acrolein or burning cotton. A:
PLA2 activity after exposure to
acrolein or cotton smoke. Each aliquot of
100,000-g supernatant was assayed for
PLA2 activity with
1-stearyl-2-[1-14C]arachidonyl-sn-glycero-3-phosphoethanolamine
(2-[1-14C]AA-GPE) for
30 min at 37°C. Results are means ± SE;
n = 9 animals/group.
B: assay measurement of
PLA2 activity with varying amounts
of 100,000-g supernatant protein added
to assay. cpm, Counts/min.
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Characterization of sheep lung PLA2
activities.
The PLA2 activity of sheep lungs
was resolved into three peaks by heparin-Sepharose chromatography (Fig.
3). Two peaks of PLA2 activities were present in
the heparin-binding fractions and one in the heparin-nonbinding
fractions. In the fractions that bound to heparin, the
PLA2 activities in the two peaks
eluted from the column were equivalent in normal and smoke-exposed
lungs. In contrast, the activity in the heparin-nonbinding fractions from the smoke-exposed lungs was higher by more than threefold than
that in the fractions from normal lungs.

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Fig. 3.
Heparin-Sepharose column chromatography of normal and acrolein- and
burning cotton smoke-exposed sheep lungs. Each aliquot of filtered
100,000-g supernatant was diluted by
the same volume of 50 mM Tris · HCl (pH 7.4) and 1 mM
EDTA and applied to a heparin-Sepharose HiTrap (5-ml) column
preequilibrated with the same buffer. Bound fractions were eluted at a
flow rate of 2 ml/min, with a 40-ml linear gradient of 0-2.0 M
NaCl. Fractions (2 ml) were collected, and a 40-µl aliquot from each
fraction was assayed for PLA2
activity with
2-[1-14C]AA-GPE.
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The heparin-binding activity was further characterized and compared
with group Ib and IIa PLA2
activities derived from bovine pancreas and rat platelets,
respectively. Supernatants (100,000 g) of rat platelets
alone or a mixture of purified pancreatic PLA2 with BSA (Sigma, St. Louis,
MO) and 100,000-g supernatants of rat
platelets were applied to a heparin-5PW FPLC column (0.75 × 7.5 cm; TosoHaas, Montgomeryville, PA). The first peak of heparin-binding lung PLA2 activity eluted in the
same fractions as those of porcine pancreatic group I
PLA2. The second peak of
heparin-binding PLA2 activity
eluted at the same NaCl concentration as that of the rat platelet group
II (data not shown). In addition, both heparin-binding forms of
PLA2 activity hydrolyzed
2-[1-14C]AA-GPE
preferentially to
2-[1-14C]AA-GPC as the
substrate. As shown in Table 3, the
activity of the first peak of heparin-binding activity in both normal
and smoke-treated lungs against
2-[1-14C]AA-GPE was
<1.5 times that with
2-[1-14C]AA-GPC as the
substrate, which is consistent with what is seen with group I
PLA2 where this ratio is generally
<2 (16). Group V secretory PLA2 has phospholipid
head-group specificity and heparin binding characteristics that are
more similar to group Ib than to group IIa secretory PLA2
(12a). Thus the first peak may include both group Ib and V secretory
PLA2. In contrast, the activities of peak
2 against
2-[1-14C]AA-GPE were
five times greater than those seen with
2-[1-14C]AA-GPC as
the substrate, a property of group II
PLA2. Both heparin-binding PLA2 activities of lungs
were almost completely inhibited by preincubation with 5 mM DTT
at 37°C for 30 min (data not shown). All of these properties are
consistent with identification of the two heparin-bound activities as
group I and group II phospholipases
A2.
Characterization of the PLA2 activity
detected in the heparin-nonbinding fractions of sheep lungs.
To characterize the PLA2 activity
in the heparin-nonbinding fractions, the active pools from normal and
smoke-exposed sheep lungs were loaded onto a Mono Q FPLC column
preequilibrated with 50 mM Tris · HCl, pH 7.4, and 1 mM EDTA. The PLA2 activities were eluted as a major peak in fractions
9-11 with
increasing salt concentrations (Fig. 4).
The specific activity of the heparin-nonbinding fractions from the
smoke-exposed sheep lungs was higher than that of corresponding
fractions from the normal lungs. To examine whether or not this
activity is cPLA2, as a positive
control, we partially purified
cPLA2 from porcine spleen (13) and
human monocytic U937 cells. These enzymes were eluted in the same
fractions from the Mono Q column (data not shown).

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Fig. 4.
Ion-exchange chromatography by Mono Q fast-performance liquid
chromatography (FPLC) of heparin-nonbinding
PLA2 activity of sheep lung.
Heparin-nonbinding fractions, obtained in experiments described in Fig.
2, were directly loaded to a Mono Q column that was preequilibrated
with 50 mM Tris · HCl (pH 7.4) and 1 mM EDTA.
Proteins were eluted at a flow rate of 1.0 ml/min, with a 20-ml linear
gradient of 0-1.0 M NaCl. Fractions (1.0 ml) were collected, and
40 µl from each fraction was assayed for
PLA2 activity for 30 min at
37°C with
2-[1-14C]AA-GPE as
substrate.
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The heparin-nonbinding activity was further characterized by examining
the biochemical properties, including measurement of apparent molecular
mass by gel-filtration chromatography, substrate specificity,
Ca2+ requirement, pH dependency,
and DTT sensitivity as well as the immunochemical properties such as
immunoprecipitation and immunoblotting. To examine the apparent
molecular mass, the active pool (3 ml) was concentrated to 200 µl
with a Centricon 10 (Amicon) and injected onto Superose 12 gel-filtration FPLC preequilibrated with 50 mM Tris · HCl, pH 7.4, 1 mM EDTA, and 0.5 M NaCl and
eluted with the same buffer. The molecular mass was estimated to be
70-80 kDa by comparison with molecular-mass standards for
gel-filtration chromatography (Pharmacia LKB Biotechnology) (Fig.
5). In a parallel experiment, the partially
purified porcine spleen cPLA2,
which has a true molecular mass >80 kDa (13), was found to elute in the same fractions (data not shown).

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Fig. 5.
Gel-filtration chromatography of the Mono Q-binding
PLA2 activity with Superose 12 FPLC. Pooled sample (3 ml) eluted by Mono Q column (Fig. 4) was
concentrated to ~200 µl with a Centricon 10. Concentrated sample
was introduced onto a Superose 12 FPLC column, preequilibrated with 50 mM Tris · HCl (pH 7.4), 1 mM EDTA, and 0.5 M NaCl.
Sixty microliters of each 1-ml fraction were assayed for
PLA2 activity with
1-[1-14C]AA-GPE as
substrate. Major peak of activity eluted with estimated molecular mass
of 70-80 kDa. A mixture of molecular-mass standards, including
RNase A (13.7 kDa), chymotrypsinogen A (25 kDa), ovalbumin (43 kDa),
bovine serum albumin (67 kDa), and blue dextran 2000 (2,000 kDa), was
injected (arrows) onto column under the same conditions as
PLA2 sample.
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The pH dependency of this lung
PLA2 activity revealed an optimal
activity at pH 9.0 at 5 mM Ca2+
with both
2-[1-14C]AA-GPE and
2-[1-14C]AA-GPC as
substrates. Fractions showed full
PLA2 activity at 10
5 M
Ca2+ (data not shown).
Immunoprecipitation of PLA2 activity with
anti-cPLA2 antiserum.
The effect of anti-PLA2 antiserum
on PLA2 activity of Mono Q active
fractions was evaluated by measuring
2-[1-14C]AA-GPE- and
2-[1-14C]AA-GPC-hydrolyzing
activities after immunoprecipitation with anti-cPLA2 antibody. As shown in
Fig 6, incubation of 450 µl of a Mono Q
active fraction with 50 µl of packed protein A-Sepharose beads
saturated with rabbit anti-porcine spleen 85-kDa
PLA2 antiserum resulted in a loss
of PLA2 activity in the
supernatant. As expected incubation of partially purified porcine
spleen cPLA2 with 50 µl of
packed protein A-Sepharose beads saturated with the
anti-cPLA2 antiserum resulted in a
loss of activity in the supernatants in a time-dependent manner, with
an ~90% reduction in supernatant activity after 4 h of incubation.
Incubation of preimmune serum-saturated beads had no effect on
PLA2 activity.

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Fig. 6.
Immunoprecipitation of heparin-nonbinding
PLA2 activity with anti-cytosolic
PLA2
(cPLA2) antiserum. Packed
protein A-Sepharose beads preequilibrated with 20 mM
Tris · HCl (pH 7.5) buffer containing 5 mg/ml of BSA
were mixed with rabbit antiserum or preimmune serum (2:1 vol/vol) for
24 h at 4°C with constant shaking. Beads were washed 6 times with
1.0 ml of 20 mM Tris · HCl (pH 7.5) containing 1 mM
EDTA and 5 mg/ml of BSA. Fifty microliters of washed beads were
incubated with 450 µl of Mono Q FPLC active fractions from sheep
lungs or partially purified cPLA2
from porcine spleen for 4, 8, or 16 h at 4°C with constant shaking.
To estimate rabbit antiserum-specific binding of 100-kDa
PLA2, samples were centrifuged,
and supernatants were assayed for
PLA2 activity with
1-[1-14C]AA-GPE as
substrate.
|
|
Western blot analysis of PLA2 derived
from sheep lungs by using anti-cPLA2
antiserum.
Western blot analysis was performed on lysates from normal and
smoke-exposed sheep lungs removed from the animals 4 h after a
30-breath exposure to cotton smoke. As shown in Fig.
7,
anti-cPLA2 antiserum reacted
strongly with 100-kDa bands from normal and smoke-exposed sheep lungs
as well as with partially purified pig spleen
cPLA2 used as a standard. It is
usual for cPLA2 to migrate at a
higher molecular mass than that predicted by its amino acid sequence.
Although cPLA2 activity was higher
in the smoke-exposed lungs, there was no increase in
cPLA2 protein, indicating that the
increased activity was not related to increased synthesis of protein or
cells containing cPLA2 that
infiltrated the lungs.

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Fig. 7.
Western blot analysis of protein from sheep lungs. Two samples (25 and
50 µg) were prepared from active fractions of Mono Q column and
loaded onto SDS-PAGE as described in
METHODS. Arrow,
cPLA2 protein that migrates at 100 kDa. Porcine spleen cPLA2
(purified cPLA2) was used as a
standard.
|
|
Correlation between PLA2 activity and MPO
activity.
MPO activity was measured in lung homogenates as an index of neutrophil
infiltration. There were significant differences in MPO activity
between smoke-exposed and normal sheep lungs. Although there were
increases in both PLA2 and MPO
activities after acrolein-carbon and smoke exposure, shorter exposures
to smoke (15 breaths) were associated with a significant increase in
PLA2 without a significant increase in MPO activity above normal levels (Fig.
8). Thus the increase in
PLA2 activity cannot be entirely
accounted for by infiltration of MPO-positive cells into the tissue. It
could derive partially from parenchymal lung cells and from endogenous
normally present intravascular neutrophils.


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Fig. 8.
Myeloperoxidase (MPO; A) and
PLA2
(B) activities in sheep lung
extracts after exposure to acrolein or varying number of breaths of
cotton smoke. Each aliquot of
100,000-g supernatants from normal and
smoke-exposed lungs was assayed for MPO and
PLA2 activities by methods
described in text. Values are means ± SE;
n = 9 animals/group.
* P < 0.01 compared with
nontreated group. ** P < 0.001 compared with nontreated group.
|
|
Sheep neutrophils contain cPLA2.
Supernatants (100,000 g) from sheep
neutrophils were applied to a Mono Q column, and bound proteins were
eluted with a 20-ml linear gradient of 0-1.0 M NaCl (Fig.
9). Extracts (100,000 g) from U937 cells were also loaded
on the same column. The main PLA2
activity in each sample measured with
2-[1-14C]AA-GPE as the
substrate eluted with the same profiles as those predicted for
cPLA2. Thus sheep neutrophils
contain cPLA2.

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Fig. 9.
Mono Q chromatography of 100,000-g
supernatants of sheep neutrophils and U937 cells. Activity was eluted
with a 20-ml linear gradient of 0-1.0 M NaCl. U937 cell
supernatant PLA2 activity is
cPLA2. This activity elutes with
the same characteristics as activity of sheep neutrophils.
|
|
 |
DISCUSSION |
This study demonstrates that there is an increase in
cPLA2 activity in sheep lungs
after exposure to synthetic smoke of acrolein-carbon or cotton smoke.
In contrast, at a time when cPLA2
activity is increased with smoke inhalation, there is no apparent
change in the small-molecular-mass secretory
PLA2 activities that bind to heparin. We have previously identified
cPLA2 in normal lungs (13), reported increased levels of PLA2
activity in patients with acute respiratory distress syndrome, and
suggested that increased PLA2 activity may contribute to the pathophysiology of this syndrome (14).
Small-molecular-mass forms of PLA2
have been purified by others from soluble fractions of guinea pig lungs
(2) and rat lungs (23).
Acute lung damage can be caused by smoke inhalation alone without
surface burns (40). Chemical toxins in smoke, not heat, produce a
delayed-onset noncardiogenic pulmonary edema. In our experiments, we
used two models of smoke inhalation: exposure to smoke derived from
burning cotton and exposure to acrolein-carbon for 10 min.
Acrolein-carbon exposure results in an accumulation of extravascular
lung water in a dose-dependent fashion (10). Acrolein-carbon inhalation
resulted in increases in cPLA2
activity equivalent to those observed with exposure to burning cotton. This increase in phospholipase activity may explain the rapid increases
in bronchoconstrictor eicosanoids,
PGF2
, thromboxane B2, and leukotriene
C4 that are seen immediately after
acrolein exposure, before neutrophil infiltration into the lung (20, 21). Enhanced cPLA2 activity will
generate increased amounts of AA that can then be converted by
cyclooxygenases and lipoxygenases to eicosanoids.
The BALF of smokers contains an increased number of cells, with higher
percentages of macrophages and a lower percentage of lymphocytes than
normal lung BALF (24). Macrophages can be a source of
cPLA2. In the present study, the
percentage of alveolar macrophages in the BALF was unchanged (90% of
total cells; data not shown); however, lung tissue levels of MPO
activity were increased after exposure to cotton or acrolein-carbon
smoke. This increased MPO content is likely due to infiltrating
polymorphonuclear neutrophils (PMNs). PMNs infiltrate the lung in
response to an inflammatory stimulus. PMNs can contribute to the
generation of reactive oxygen species, proteolytic enzymes, AA
metabolites, and platelet-activating factor, which contribute to the
increases in pulmonary microvascular permeability and pulmonary edema
seen after smoke inhalation. Neutrophil depletion has been reported to
prevent pulmonary edema associated with smoke inhalation (1). In this
study, we found that sheep neutrophils contain
cPLA2. With short periods of smoke exposure, cPLA2 activity increases
more than MPO activity, and there is no increased amount of
cPLA2 protein found on Western blot. Thus the increase in
cPLA2 activity reflects increased
endogenous cPLA2 activity in
resident lung cells, a conclusion also consistent with the immediate
increase in eicosanoids measured after acrolein exposure by others (20,
21). The increase in cPLA2
activity is likely related to phosphorylation of the protein, perhaps
due to activation of one of the mitogen-activated protein kinase
signaling pathways (31). In addition to the bronchoconstrictive,
vasoconstrictive, and chemotactic effects of eicosanoids, the
metabolism of AA by cyclooxygenases and lipoxygenases generates
reactive oxygen species that can also contribute to the lung injury
(36). Increased cellular cPLA2
activity also enhances the susceptibility of the cell to
oxidant-induced injury (30).
In conclusion, our findings reveal an increase in
cPLA2 activity in the lungs of
sheep exposed to cotton smoke or synthetic smoke containing
acrolein-carbon, an important component of cotton smoke. This
PLA2 activity may serve as an
important mediator of lung injury.
cPLA2 likely contributes to high
vascular permeability, bronchoconstriction, vasoconstriction, cellular
injury, and inflammation by direct effects on membranes, by indirect
effects mediated by free fatty acids and lysophospholipids released by
cPLA2, and as a result of the
eicosanoids and reactive oxygen species produced as a result of the
metabolism of released AA.
 |
ACKNOWLEDGEMENTS |
This work was supported by National Institute of Diabetes and
Digestive and and Kidney Diseases Grants DK-39773 and DK 38452; National Institute of Neurological Disorders and Stroke Grant NS-10828;
National Heart, Lung, and Blood Institute Grant HL-36829; and Shriners
Burn Institute Grant 15872.
 |
FOOTNOTES |
D. K. Kim was supported by a Paul Dudley White Fellowship from the
Massachusetts Chapter of the American Heart Association.
Address for reprint requests and other correspondence: J. V. Bonventre,
Suite 4002, Massachusetts General Hospital-East, 149 13th St.,
Charlestown, MA 02129 (E-mail:
joseph_bonventre{at}hms.harvard.edu).
Received 6 May 1997; accepted in final form 22 April 1999.
 |
REFERENCES |
1.
Basadre, J.,
K. Sugi,
D. Traber,
R. Traber,
G. Niehause,
and
D. Herndon.
The effect of leukocyte depletion on smoke inhalation injury in sheep.
Surgery
104:
208-215,
1988[Medline].
2.
Bennett, C. F.,
A. McCarte,
and
S. T. Crooke.
Purification and characterization of a soluble phospholipase A2 from guinea pig lung.
Biochim. Biophys. Acta
1047:
271-283,
1990[Medline].
3.
Bonventre, J. V.
Phospholipase A2 and signal transduction.
J. Am. Soc. Nephrol.
3:
128-150,
1992[Abstract].
4.
Bonventre, J. V.,
Z. Huang,
M. R. Taheri,
E. O'Leary,
E. Li,
M. A. Moskowitz,
and
A. Sapirstein.
Reduced fertility and postischaemic brain injury in mice deficient in cytosolic phospholipase A2.
Nature.
390:
622-625,
1997[Medline].
5.
Bradley, P.,
D. Priebat,
R. Christensen,
and
G. Rothstein.
Measurement of cutaneus inflammation: estimation of neutrophil content with an enzyme marker.
J. Invest. Dermatol.
78:
206-209,
1982[Abstract].
6.
Clark, J. D.,
L.-L. Lin,
R. W. Kriz,
C. S. Ramesha,
L. A. Sultzman,
A. Y. Lin,
N. Milona,
and
J. L. Knopf.
A novel arachidonic acid-selective cytosolic PLA2 contains a Ca++ dependent translocation domain with homology to PKC and GAP.
Cell
65:
1043-1051,
1991[Medline].
7.
Dennis, E. A.
The growing phospholipase A2 superfamily of signal transduction enzymes.
Trends Biochem. Sci.
22:
1-2,
1997[Medline].
8.
Dole, V. P.,
and
H. Meimertz.
Microdetermination of long-chain fatty acids in plasma and tissues.
J. Biol. Chem.
235:
2595-2599,
1960[Medline].
9.
Gronich, J. H.,
J. V. Bonventre,
and
R. A. Nemenoff.
Purification of a high-molecular-mass phospholipase A2 from rat kidney activated at physiological calcium concentrations.
Biochem. J.
271:
37-43,
1990[Medline].
10.
Hales, C.,
P. Barkin,
W. Jung,
E. Trautman,
D. Lamborghini,
N. Herrig,
and
J. Burke.
Synthetic smoke with acrolein but not HCl produces pulmonary edema.
J. Appl. Physiol.
64:
1121-1133,
1988[Abstract/Free Full Text].
11.
Hales, C. A.,
S. Musto,
W. G. Hutchison,
and
E. Mahoney.
BW-755C diminishes smoke-induced pulmonary edema.
J. Appl. Physiol.
78:
64-69,
1995[Abstract/Free Full Text].
12.
Hales, C. A.,
S. Musto,
S. Janssens,
W. Jung,
D. Quinn,
and
M. Witten.
Smoke aldehyde component influences pulmonary edema.
J. Appl. Physiol.
72:
555-561,
1992[Abstract/Free Full Text].
12a.
Han, S.-K.,
E. T. Yoon,
and
W. Cho.
Bacterial expression and characterization of human secretory class V phospholipase A2.
Biochem. J.
331:
353-357,
1998[Medline].
13.
Kim, D. K.,
and
J. V. Bonventre.
Purification of a 100 kDa phospholipase A2 from spleen, lung and kidney: antiserum raised to pig spleen phospholipase A2 recognizes a similar form in bovine lung, kidney and platelets, and immunoprecipitates phospholipase A2 activity.
Biochem. J.
294:
261-270,
1993[Medline].
14.
Kim, D. K.,
T. Fukuda,
B. T. Thompson,
B. Cockrill,
C. Hales,
and
J. V. Bonventre.
Bronchoalveolar lavage fluid phospholipase A2 activity is increased in human adult respiratory distress syndrome.
Am. J. Physiol.
269 (Lung Cell. Mol. Physiol. 13):
L109-L118,
1995[Abstract/Free Full Text].
15.
Kim, D. K.,
I. Kudo,
and
K. Inoue.
Detection in human platelets of phospholipase A2 activity which preferentially hydrolyzes an arachidonoyl residue.
J. Biochem. (Tokyo)
104:
492-494,
1988[Abstract].
16.
Kim, D. K.,
I. Kudo,
and
K. Inoue.
Purification and characterization of rabbit platelet cytosolic phospholipase A2.
Biochim. Biophys. Acta
1083:
80-88,
1991[Medline].
17.
Kimaura, R.,
L. Traber,
D. Herndon,
G. Niehaus,
J. Flynn,
and
D. L. Traber.
Ibuprofen reduces the lung lymph flow changes associated with inhalation injury.
Circ. Shock
24:
183-191,
1988[Medline].
18.
Laemmli, U. K.
Cleavage of structure proteins during assembly of the head of bacteriophage T4.
Nature
227:
680-685,
1970[Medline].
19.
Lalonde, C.,
R. Demling,
J. Brain,
and
J. Blanchard.
Smoke inhalation injury in sheep is caused by the particulate phase, not the gas phase.
J. Appl. Physiol.
77:
15-22,
1994[Abstract/Free Full Text].
20.
Leikauf, G. D.,
C. A. Doupnik,
L. M. Leming,
and
H. E. Wey.
Sulfidopeptide leukotrienes mediate acrolein-induced bronchial hyperresponsiveness.
J. Appl. Physiol.
66:
1838-1845,
1989[Abstract/Free Full Text].
21.
Leikauf, G. D.,
L. Leming,
J. O'Donnell,
and
C. Doupnik.
Bronchial responsiveness and inflammation in guinea pig exposed to acrolein.
J. Appl. Physiol.
66:
171-178,
1989[Abstract/Free Full Text].
22.
Leslie, C. C.,
D. R. Voelker,
J. Y. Cannon,
M. M. Wall,
and
P. T. Zelarney.
Properties and purification of an arachidonoyl-hydrolyzing phospholipase A2 from a macrophage cell line, RAW 264.7.
Biochim. Biophys. Acta
963:
476-492,
1988[Medline].
23.
Lindahl, M.,
H. von Schenck,
and
C. Tagesson.
Isolation and characterization of phospholipase A2 from rat lung with affinity chromatography and two-dimensional gel electrophoresis.
Biochim. Biophys. Acta
1005:
282-288,
1989[Medline].
24.
Merchant, R.,
D. Schwartz,
R. Helmers,
C. Dayton,
and
G. Hunninghake.
Bronchoalveolar lavage cellularity. The distribution in normal volunteers.
Am. Rev. Respir. Dis.
146:
448-453,
1992[Medline].
25.
Morikawa, T.
Acrolein formaldehyde and volatile fatty acids from smoldering combustion.
J. Combust. Toxicol.
3:
135-150,
1976.
26.
Murakami, M.,
T. Kobayashi,
M. Umeda,
I. Kudo,
and
K. Inoue.
Monoclonal antibodies against rat platelet phospholipase A2.
J. Biochem. (Tokyo)
104:
884-888,
1988[Abstract].
27.
Pruzanski, W.,
and
P. Vadas.
Phospholipase A2
a mediator between proximal and distal effects of inflammation.
Immunol. Today
12:
143-146,
1991[Medline].
28.
Quinn, D. A.,
D. Robinson,
W. Jung,
and
C. A. Hales.
Role of sulfidopeptide leukotrienes in synthetic smoke inhalation injury in sheep.
J. Appl. Physiol.
68:
1962-1969,
1990[Abstract/Free Full Text].
29.
Sakata, T.,
E. Nakamura,
Y. Tsuruta,
M. Takami,
H. Teraoka,
H. Tojo,
T. Ono,
and
M. Okamoto.
Presence of pancreatic-type phospholipase A2 mRNA in rat gastric mucosa and lung.
Biochim. Biophys. Acta
1007:
124-126,
1989[Medline].
30.
Sapirstein, A.,
R. A. Spech,
R. Witzgall,
and
J. V. Bonventre.
Cytosolic phospholipase A2 (PLA2), but not secretory PLA2, potentiates hydrogen peroxide cytotoxicity in kidney epithelial cells.
J. Biol. Chem.
271:
21505-21513,
1996[Abstract/Free Full Text].
31.
Serhan, C. N.,
J. Z. Haeggström,
and
C. C. Leslie.
Lipid mediator networks in cell signaling: update and impact of cytokines.
FASEB J.
10:
1147-1158,
1996[Abstract/Free Full Text].
32.
Sharp, J. D.,
D. L. White,
X. G. Chiou,
T. Goodson,
G. C. Gamboa,
D. McClure,
S. Burgett,
J. Hoskins,
P. L. Skatrud,
J. R. Sportsman,
G. W. Becker,
L. H. Kang,
E. F. Roberts,
and
R. M. Kramer.
Molecular cloning and expression of human Ca2+-sensitive cytosolic phospholipase A2.
J. Biol. Chem.
266:
14850-14853,
1991[Abstract/Free Full Text].
33.
Staub, N. C.,
K. L. Bland,
K. L. Brigham,
R. Demling,
A. J. Erdman,
and
W. C. Wolverton.
Preparation of chronic lymph fistulas in sheep.
J. Surg. Res.
19:
315-320,
1975[Medline].
34.
Thompson, P. B.,
D. N. Herndon,
D. L. Traber,
and
S. Abston.
Effect on mortality of inhalation injury.
J. Trauma
26:
163-165,
1986[Medline].
35.
Uozumi, N.,
K. Kume,
T. Nagase,
N. Nakatani,
S. Ishii,
F. Tashiro,
Y. Komagata,
K. Maki,
K. Ikuta,
Y. Ouchi,
J.-I. Miyazaki,
and
T. Shimizu.
Role of cytosolic phospholipase A2 in allergic response and parturition.
Nature
390:
618-622,
1997[Medline].
36.
Van Antwerpen, L.,
A. J. Theron,
M. S. Myer,
G. A. Richards,
L. Wolmarans,
U. Booysen,
C. A. van der Merwe,
G. K. Sluis-Cremer,
and
R. Anderson.
Cigarette smoke-mediated oxidant stress, phagocytes, vitamin C, vitamin E, and tissue injury.
Ann. NY Acad. Sci.
686:
53-65,
1993[Medline].
37.
Van den Bosch, H.
Intracellular phospholipases A.
Biochim. Biophys. Acta
604:
191-246,
1980[Medline].
38.
Waite, M.
Approaches to the study of mammalian cellular phospholipases.
J. Lipid Res.
26:
1379-1388,
1985[Abstract].
39.
Walker, H. L.,
C. G. J. McLeod,
and
W. F. McManus.
Experimental inhalation injury in the goat.
J. Trauma
21:
962-964,
1981[Medline].
40.
Whitener, D.,
L. Whitener,
K. Robertson,
C. Baxter,
and
A. Pierce.
Pulmonary function measurement in patients with thermal injury and smoke inhalation.
Am. Rev. Respir. Dis.
122:
731-739,
1980[Medline].
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