Edward A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, St. Louis, Missouri 63104-1079
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ABSTRACT |
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The purpose of
this study was to determine if the acute alveolar injury induced by
subcutaneous injections of
N-nitroso-N-methylurethane (NNMU) in rats is mediated by nitric oxide (NO ·). We show
that intraperitoneal injections of the NO · synthase (NOS)
inhibitor N-nitro-L-arginine
methyl ester (L-NAME) or
aminoguanidine significantly attenuate the NNMU-induced alveolar injury
as assessed by 1) normalization of
the alveolar-arterial O2
difference, 2) attenuation of the lowered phospholipid-to-protein ratio in the crude surfactant pellet
(CSP), 3) attenuation of the
elevated minimal surface tension of the CSP, and
4) attenuation of polymorphonuclear
neutrophilic infiltration into the alveolar space. Injections of
N
-nitro-D-arginine
methyl ester, the inactive stereoisoform of L-NAME, did not affect the acute
lung injury. Western blot analysis of whole lung homogenates
demonstrate an elevated expression of transcriptionally inducible,
Ca2+-independent NOS (iNOS) in
NNMU-injected rats compared with control saline-injected rats. NOS
inhibitors did not affect NNMU-induced iNOS expression. These
investigations demonstrate that the inhibition of NOS attenuates
NNMU-induced acute lung injury, suggesting a role for NO · in
the progression of acute respiratory distress syndrome.
reactive oxygen species; acute respiratory distress syndrome; lung injury; surfactant; N-nitroso-N-methylurethane
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INTRODUCTION |
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N-nitroso-n-methylurethane (NNMU)-induced acute lung injury is an in vivo animal model of acute respiratory distress syndrome (ARDS; see Refs. 18, 24, and 33). The NNMU injury exhibits ARDS-like characteristics such as a slow subacute development of injury, hypoxia, and alterations in surfactant composition and function (18).
Recently, nitric oxide (NO ·) has been shown to inhibit type
II cell metabolism and surfactant synthesis in vitro (16, 27), and
peroxynitrite, produced by the reaction of NO · and
superoxide (), has been shown to
inhibit ion transport in type II pneumocytes (21). Furthermore,
isolated, perfused lung studies have shown that lung injury induced by
paraquat, immune complexes, or ischemia appears to be mediated by
NO · (3, 29) and/or peroxynitrite (22). The
mechanisms by which NO · can participate in lung injury may
be through the inactivation of mitochondrial iron-sulfur-containing
enzymes (10), the induction of DNA damage (30), and the potentiation of
inflammatory responses by the activation of cyclooxygenase and the
increase in prostaglandin synthesis (7, 35). In addition, peroxynitrite
has been shown to diminish surface activity of calf lung surfactant
extract by damaging surfactant proteins and lipid peroxidation (15). In contrast, NO · has been reported to protect alveolar
epithelial cells from neutrophil-mediated cell death (36) and
-mediated toxicity (13), and
NO · inhalation [50 parts/million (ppm)] decreases interleukin (IL)-1-stimulated neutrophil influx to the alveolar space (12). Clinically, NO · inhalation (25 ppm)
reduces mean pulmonary capillary pressure in ARDS patients, thereby
preventing edema formation (34).
NO · synthase (NOS) catalyzes the five-electron oxidation of
L-arginine to produce citrulline
and NO ·. NOS isoforms are generally grouped into two
categories: the constitutively expressed,
Ca2+-dependent NOS (cNOS) and the
transcriptionally inducible,
Ca2+-independent NOS (iNOS). It
has been previously demonstrated that NO · production can be
inhibited with N-nitro-L-arginine
methyl ester (L-NAME), a
nonselective competitive inhibitor for cNOS and iNOS (28), or
aminoguanidine (AG), a selective competitive inhibitor for iNOS
(8).
In the present study, evidence is presented to support a role for the participation of NO · in the progression of NNMU-induced lung injury. We show that the NOS inhibitors L-NAME and AG attenuate NNMU-induced acute lung injury as assessed by 1) the alveolar-arterial O2 difference [(A-a)DO2], 2) the phospholipid-to-protein ratio (PL/Pr) in the crude surfactant pellet (CSP), 3) the minimal surface tension of the CSP, and 4) the percentage of polymorphonuclear neutrophils (PMNs) in the bronchoalveolar lavage fluid (BALF). In addition, we show elevated levels of the iNOS enzyme in whole lung homogenates from NNMU-injected rats with or without the administration of NOS inhibitors. Collectively, the results support a role for NO ·-mediated acute lung injury in this animal model.
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MATERIALS AND METHODS |
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Animals and materials. In all experiments, male pathogen-free Sprague-Dawley rats (250-350 g), obtained from Harlan Sprague Dawley (Indianapolis, IN), were used. Heparin-coated polyvinyl catheters were inserted into the left carotid artery of rats that were anesthetized with a mixture of ketamine and xylazine (55 and 7 mg/kg body wt, respectively) at least 1 wk before the injections. The external portion of the arterial catheter, which was used for the collection of blood samples, extended behind the base of the skull. The rats were individually housed in plastic cages on self-watering racks in semibarrier rooms and were fed normal rat chow ad libitum. All common chemical reagents were obtained from Sigma Chemical (St. Louis, MO) unless otherwise noted.
Injections.
The NNMU (Kings Laboratory, Greeneville, SC) injections were performed
as reported by Harris et al. (18). The injection scheme consisted of
two rounds of injections separated by 24 h. The first round of
injections, which included NNMU, an NOS inhibitor, and/or
saline, were performed on rats anesthetized by inhalation of halothane
(Halocarbon Laboratories, River Edge, NJ). NNMU injections were
subcutaneously administered at the dosage of 7.5 mg/kg body wt unless
otherwise noted. The NOS inhibitor
L-NAME was injected intraperitoneally at a dosage of 100-150 mg · kg body
wt1 · injection
1. In some
experiments, 150 mg/kg body wt of the inactive stereoisomer N
-nitro-D-arginine
methyl ester (D-NAME) were
injected intraperitoneally. The NOS inhibitor AG was administered
intraperitoneally at a dosage of 225-450 mg · kg body
wt
1 · injection
1. Saline
injections for control rats were of equal volume to that of the NOS
inhibitor injections. The second round of injections, 24 h later,
consisted of either the NOS inhibitor or normal saline given
intraperitoneally and were performed on unanesthetized rats. Selected
dosages were based on observations reported by DeLuca et al. (9) and
our preliminary trials of various dosages of NOS inhibitor
intraperitonal injections (data not shown).
(A-a)DO2 analysis. Arterial blood samples (0.7 ml) were collected directly into a heparin-flushed 1.00-ml syringe via an indwelling arterial catheter at 60-65 h after the first round of injections from spontaneously breathing, unanesthetized, manually restrained rats. Arterial blood gases (ABG) were measured using a Radiometer ABL 520 blood microsystem (Copenhagen, Denmark). PO2 and PCO2 from ABG determinations were used to calculate the alveolar-arterial gradient. The alveolar PO2 was calculated using the alveolar gas equation, and the arterial PO2 was obtained from ABG determinations. The respiratory quotient and the inspired O2 fraction were assumed to be constant at 0.8 and 0.21, respectively. Partial pressure values were corrected for animal body temperature and blood pH (23). (A-a)DO2 values >25 mmHg in spontaneously breathing rats were considered to demonstrate acute lung injury.
Cellular infiltrate analysis. After blood samples were drawn and ABGs were determined, the lungs were surgically removed, and ~40 ml of BALF were collected from each isolated rat lung. Briefly, this protocol involves the anesthetization of rats with 85 mg/kg body wt ip of pentobarbital sodium, insertion of a metal tracheostomy tube into the trachea, and exsanguination by severing the inferior vena cava. The lung was perfused through the pulmonary artery by inserting a cannula into the right ventricle of the heart with solution B (125 mM NaCl, 5 mM KCl, 2.5 mM Na2HPO4, 17 mM N-2hydroxyethylpiperazine-N'-2-ethanesulfonic acid, 1 mg/ml gentamicin, and 1 mg/ml dextrose, pH 7.4) to remove blood cells from the pulmonary vasculature. The intact lungs were then carefully removed and were lavaged five times with 8.0 ml of solution A (solution B containing 2.5 mM CaCl2 and 1.2 mM MgSO4). The lavage was centrifuged at 300 g for 15 min at 4°C, and the cell pellet was resuspended in 2.0 ml of solution A. An aliquot was centrifuged onto glass slides and was stained by a modified Wright's stain procedure (Diff-Quik; Baxter Scientific). The cell-free supernatant collected was used for protein and phospholipid determinations (see below).
Phospholipid and protein analysis. The BALF cell-free supernatant was centrifuged at 48,000 g for 1 h to obtain a CSP that was resuspended in 300 µl of solution A. An aliquot was taken, and the protein concentration was determined by a modified Lowry assay (see Refs. 25 and 31). Phospholipid concentration was determined by extracting the phospholipids from the CSP by chloroform-methanol lipid extraction as reported by Bligh and Dyer (5), with subsequent measurement of the Pi released by heat-acid digestion (6, 20).
Analysis of CSP surface activity. In these experiments, the 48,000-g CSP was resuspended in 300 µl of 154 mM NaCl containing 5 mM CaCl2. Phospholipid concentration was determined as noted above, and the pellet was diluted to a final phospholipid concentration of 1.5 µmol/ml (18). Surface tension at minimal bubble size was determined on a pulsating bubble surfactometer after 5 min at 37°C, 50% surface area compression, and 20 cycles/min (18).
Western blot analysis for iNOS.
In these experiments, rats were injected subcutaneously with 8.0 mg/kg
body wt of NNMU. At 60-65 h after NNMU treatment, intact lungs
were removed and were rinsed in saline. Connective tissues and large
airways were removed. The remaining lung parenchyma was placed in 4 ml
of homogenization buffer [25 mM tris(hydroxymethyl)aminomethane (Tris) · HCl, 2 mM EDTA, and 2 mM
4-(2-aminoethyl)benzenesulfonyl fluoride, pH 7.4] and was
homogenized for 15 s by Polytron at 4°C. The homogenate was
centrifuged at 1,500 revolutions/min at 4°C for 10 min, and 2 ml of
the supernatant were mixed with 2 ml of sample buffer [0.25 M
Tris · HCl, 20% -mercaptoethanol, and 4% sodium
dodecyl sulfate (SDS), pH 6.6] and were boiled for 4 min. Four
hundred microliters of sample dye (80% glycerol and 0.05% bromphenol
blue) were added, and the samples were stored at
70°C. An
aliquot of the supernatant was saved for Bradford protein determination
and was stored at
70°C. Homogenized lung protein (20 µg)
was separated by SDS gel electrophoresis and was transferred to a
nitrocellulose membrane under semidry conditions (Millipore
Milliblot Graphite Electroblotter Systems MBBDGE001). Detection of iNOS protein was performed by enhanced chemiluminescence (Amersham) using a rabbit anti-mouse iNOS antibody (Cayman Chemicals) at a dilution of 1:1,000 and a horseradish peroxidase-conjugated donkey
anti-rabbit antibody (Jackson Immunological Research) at a dilution of
1:7,000 (19). IL-1
-stimulated (1.0 U/ml for 18 h) RIN-m5F cells were
used as a positive control (see Fig. 5, lane
1 and Ref. 19).
Statistics. Statistical significance was calculated using one-way analysis of variance with a Bonferroni post hoc analysis. The data presented are means ± SE.
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RESULTS |
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NOS inhibitors attenuate NNMU-induced elevation in
(A-a)DO2.
Experiments were performed to determine if NO · mediates the
elevation in
(A-a)DO2
induced by NNMU injection. The NOS inhibitors L-NAME (100 or 150 mg · kg1 · injection
1)
or AG (225, 300, or 450 mg · kg
1 · injection
1)
were administered to NNMU-treated rats at the time of NNMU
administration and 24 h later. Sixty to sixty-five hours after NNMU
injection, arterial blood samples were collected, and blood gases were
determined. The administration of 100 or 150 mg · kg
1 · injection
1
of L-NAME significantly
(P < 0.001) attenuated the elevation of
(A-a)DO2
in rats treated with NNMU, a decrease of 43.3 or 84.2%, respectively
(Fig.
1A).
In addition, 150 mg/kg D-NAME,
the inactive D-isoform of
L-NAME, did not attenuate the
elevated NNMU-induced (A-a)DO2
(60.50 ± 10.60 mmHg; n = 4),
suggesting that the attenuation seen with
L-NAME is specific to the active
L-isoform of the NOS inhibitor and its inhibitory activity
on NO · production (Fig. 1A). Similar to the effects of
L-NAME, AG, a selective
inhibitor of iNOS (8), administered at dosages of 300 or 450 mg · kg body
wt
1 · injection
1
significantly (P < 0.05 and
P < 0.001, respectively) lowers the elevated
(A-a)DO2
in NNMU-treated rats compared with control rats, a decrease of 34.8 or
86.3%, respectively (Fig. 1B).
These results indicate that the administration of NOS inhibitors
attenuates NNMU-induced alterations in gas exchange.
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NOS inhibitors attenuate decreased PL/Pr in CSP associated with NNMU-induced acute lung injury. Harris et al. (18) have previously reported a lower PL/Pr in the CSP of NNMU-treated rats compared with normal control animals. The PL/Pr of the CSP is an indirect measure of the permeability of the alveolar epithelial barrier to vascular fluids and proteins. Therefore, a lowered PL/Pr (resulting from an increase in protein concentration) may indicate edema formation due in part to a damaged alveolar epithelial barrier. As shown in Fig. 2, control and NNMU-treated rats exhibited PL/Pr values of 3.05 ± 0.37 (n = 3) and 1.33 ± 0.17 (n = 3), respectively. Injections with 450 mg/kg AG or 150 mg/kg L-NAME attenuated the decrease, resulting in PL/Pr values of 2.39 ± 0.07 (n = 4) and 2.53 ± 0.15 (n = 4), respectively. Similar to their effects on (A-a)DO2, NOS inhibitors attenuated the reduction in the PL/Pr caused by NNMU administration.
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NOS inhibitors attenuate the elevated minimal surface tension of the CSP associated with NNMU-induced acute lung injury. It has been previously demonstrated that NO · can affect surfactant proteins and phospholipids, resulting in an impairment of the lipid aggregation and surface activity in vitro (15, 17). In addition, Harris et al. (18) demonstrated that a lowered PL/Pr of the CSP correlates with an impairment of surface activity (18). Therefore, it was predicted that the inhibition of NOS, resulting in the attenuation of the lowered PL/Pr associated with NNMU-induced lung injury (as noted above), would demonstrate an attenuation of surface activity impairment. As shown in Fig. 3, the surface tension at the minimal bubble size of the CSP derived from NNMU-treated rats was impaired compared with control animals [39.69 ± 1.27 (n = 4) and 7.52 ± 2.04 dyn/cm (n = 8), respectively]. As predicted, NNMU-treated rats that received either L-NAME or AG demonstrated a partial, but significant (P < 0.001), attenuation of the increased minimal surface tension [31.26 ± 0.83 (n = 6) or 26.41 ± 1.35 dyn/cm (n = 6), respectively]. This finding further suggests that NO · may participate in lung injury by damaging the pulmonary surfactant system and thereby lowering surface activity.
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Effect of NOS inhibitors and NNMU on lung inflammation. Experiments were conducted to determine if NO · mediates lung inflammation as measured by the cellular infiltrate composition in the BALF. NNMU-induced lung injury results in a significant increase in the total number of PMNs in the BALF (79.07 ± 2.25%, n = 11) compared with controls. A decrease in the elevated PMN infiltration was observed in NNMU-treated rats that were coinjected with either L-NAME (Fig. 4A) or AG (Fig. 4B). L-NAME at 100 and 150 mg/kg reduced the NNMU-induced PMN infiltration by 28.96 (n = 7) and 43.41% (n = 6), respectively. In addition, NNMU-treated rats that received 150 mg/kg D-NAME did not demonstrate a significant reduction in the NNMU-induced PMN increase (Fig. 4A). As shown in Fig. 4B, 300 and 450 mg/kg AG reduced the NNMU-induced PMN infiltration by 23.07 (n = 5) and 43.56% (n = 3), respectively. These findings indicate that NO · may participate in the mediation of NNMU-induced alveolar inflammation, as assessed by PMN infiltration, and that NOS inhibitors reduce the inflammation.
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NNMU-induced iNOS expression in whole lung homogenates.
The results presented above suggest that NOS inhibitors attenuate lung
injury in the NNMU animal model. Therefore, the expression of iNOS in
the whole lung homogenates of NNMU-treated rats with or without NOS
inhibitor administration was examined. At 60-65 h after NNMU
injection, whole intact lungs were removed and homogenized. As shown in
Fig. 5, control (saline-injected) rats did
not express detectable levels of iNOS
(lanes
2 and
3); however, iNOS expression was
detected from lung homogenates of NNMU-injected animals with an acute
lung injury (lanes
4 and
5). The administration of AG did not
affect the elevated iNOS expression in NNMU-treated rats (Fig. 5,
lanes
6 and
7). Similar results were observed in
rats receiving both NNMU and 150 mg/kg
L-NAME (data not shown). As a
positive control for iNOS expression (Fig. 5,
lane
1), we used IL-1-stimulated (1.0 U/ml for 18 h) RIN-m5F cells, a rat insulinoma cell line (19). The
presence of iNOS expression in lungs isolated from NNMU-treated rats
provides further evidence that NO ·, produced after the
expression of iNOS, participates in the progression of acute lung
injury.
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DISCUSSION |
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ARDS is a general term describing a pathological condition found in humans that displays the characteristics of increased pulmonary vascular permeability, lung inflammation with alveolar neutrophilic infiltration, and pulmonary edema. Alveolar epithelial damage compounds the injury and leads to a decreased lung compliance and an increased right-to-left blood shunting that, along with ventilation-perfusion mismatching, causes the severe hypoxemia found in ARDS (4). The NNMU animal model displays ARDS-like characteristics such as proteinosis, abnormal gas exchange, reduced lung compliance, and alterations in the surfactant phospholipid pools (18).
In this study, several lung injury characteristics were investigated to demonstrate the possible role(s) of iNOS expression and activity in the progression of the NNMU-induced lung injury. We show that NOS inhibitors significantly attenuate 1) the increase in the (A-a)DO2, 2) the decrease in PL/Pr of the CSP, 3) the decrease in surface activity of the CSP, and 4) the increase in PMN infiltration into the alveolar space associated with lung injury induced by NNMU injections in rats. In addition, NNMU-induced lung damage is associated with the expression of iNOS. NOS inhibitors AG and L-NAME, which attenuate NNMU-induced injury, do not inhibit the expression of iNOS in NNMU-treated rats. This latter result demonstrates that NNMU-induced iNOS expression is not affected by NOS inhibitors, whereas the ARDS-like characteristics of the NNMU model are attenuated by administration of NOS inhibitors. Collectively, these findings suggest that the generation of NO · may participate in the progression of acute lung injury.
Several previously published reports have led us to investigate the involvement of NO · in ARDS. The iNOS isoform has been detected in airway and alveolar epithelial cells, alveolar macrophages, and the pulmonary vasculature (1). Also, concerning lung inflammation in the asthmatic condition, NO · has been detected in exhaled air and BALF of patients with an asthmatic condition (2). Indirectly, the activation of cyclooxygenases by NO · may further augment the inflammatory condition by increased production of proinflammatory prostaglandins (7, 35). NO · can damage the proteins of the surfactant system in vitro (14, 16, 17, 26), which is suggestive of NO · participation in lung injury. Last, Vara et al. (37, 38) recently reported that proinflammatory cytokines (IL-1 and tumor necrosis factor) that are known to stimulate NO · production have demonstrated an inhibitory effect on surfactant synthesis in a manner that is mediated by NO · and prostaglandins. Therefore, within the pulmonary system there exists the potential to synthesize elevated levels of NO · that are detectable in the expired air of the inflamed lung. The activity of NO · can promote inflammation, inhibit the functioning of the pulmonary surfactant system, and inhibit surfactant synthesis.
Previous reports demonstrate that pulmonary surfactant phospholipids and proteins are damaged by NO · and peroxynitrite in vitro, resulting in a loss of surface activity and lipid aggregation (15, 17). Our in vivo data support this finding by demonstrating the attenuation of surfactant impairment with treatment of NOS inhibitors (Fig. 3). On the other hand, this attenuation may be a result of the normalization of the PL/Pr of the CSP with treatment of NOS inhibitors (Fig. 2). Future in vivo investigations into the effects of NO ·-mediated lung damage (e.g., nitrosylation of surfactant proteins) will demonstrate the cause(s) of the attenuation of surfactant impairment in the NNMU-induced acute lung injury.
Our data clearly show that, at the same NOS inhibitor dose for either L-NAME or AG, there are different degrees of attenuation of the injury depending on the parameter investigated. For example, the (A-a)DO2 is affected more than the PMN infiltration with either NOS inhibitor administration in NNMU-treated animals. This finding suggests that NO · may participate in the recruitment of PMNs into the alveoli by modulating the synthesis and/or degradation of chemokines and intracellular adhesion molecules. More importantly, the data suggest that after PMN influx into the alveolar space, the production of elevated levels of NO · from a variety of sources may directly or indirectly (e.g., forming peroxynitrite) damage the alveolar epithelium and surfactant system. Therefore, by administration of NOS inhibitors, its activity could be attenuating PMN influx and/or alveolar damage.
The data also demonstrate that less
L-NAME
(mg · kg1 · injection
1)
is sufficient for producing effects similar to those of AG when assessing a particular parameter. One possible reason for the difference in effective dosage would be the half-life of each inhibitor
in vivo. The half-life of AG in vivo is estimated at 6-8 h (8),
whereas N
-nitro-L-arginine
(L-NNA) has been shown to have a
significant effect in vivo on neuronal NOS for 5 days at a
dosage of 50 mg/kg (11). A recent publication from Pfeiffer et al. (32)
has shown that the inhibitory activity of
L-NAME corresponds to its rate of hydrolysis to L-NNA in vivo.
Therefore, it can be predicted that the inhibitory activity of
L-NAME exceeds AG in vivo.
Another reason for the different dose responses may be due in part to the selectivity of the NOS inhibitors. The two NOS inhibitors used in
this study were L-NAME and AG,
the latter having greater selectivity for iNOS (8). Because more AG is
required to visualize effects similar to those of
L-NAME, it can be hypothesized
that cNOS may play the critical role in the development of the
NNMU-induced injury. However, this assumes that AG and
L-NAME have equal stability in
vivo, which, as discussed previously, is not the case. In addition, Western blot analysis demonstrates that iNOS expression correlates with
NNMU administration with or without NOS inhibitor injections, which is
suggestive of a role for iNOS activity in the progression of acute lung
injury. It is quite possible that both isoforms are involved in
NNMU-induced lung injury.
In conclusion, the data presented in this paper suggest that the progression of acute lung injury may be mediated in part by NO ·. In the inflamed lung, NO · produced by iNOS from alveolar macrophages, neutrophils, and type II cells may inhibit type II cell function and damage surfactant phospholipids and proteins. These effects culminate in acute lung inflammation, a decreased PL/Pr of surfactant, surfactant impairment, and an elevated (A-a)DO2, all of which have been seen in ARDS and the NNMU-injured lung. More importantly, these effects can be attenuated by the administration of NOS inhibitors, further indicating the probable role of NO · in lung injury.
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ACKNOWLEDGEMENTS |
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We acknowledge the help of Dr. George A. Vogler, Department of Comparative Medicine, in the placement of the indwelling arterial catheters, Tracey Baird for technical assistance in the laboratory, and the Pulmonary Function Laboratory for use of the Radiometer ABL 520.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grant HL-13405 (to W. J. Longmore), a research grant from Alteon (to J. A. Corbett), and a Career Development award from the Juvenile Diabetes Foundation International (to J. A. Corbett).
Address for reprint requests: W. J. Longmore, 1402 S. Grand Blvd., Edward A. Doisy Dept. of Biochemistry and Molecular Biology, St. Louis Univ. School of Medicine, St. Louis, MO 63104-1004.
Received 15 May 1997; accepted in final form 4 September 1997.
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