Nitric oxide participates in early events associated with
NNMU-induced acute lung injury in rats
Wilhelm S.
Cruz,
John A.
Corbett,
William J.
Longmore, and
Michael A.
Moxley
Edward A. Doisy Department of Biochemistry and Molecular
Biology, St. Louis University School of Medicine, St. Louis,
Missouri 63104-1079
 |
ABSTRACT |
In this study, the biochemical mechanisms by
which
N-nitroso-N-methylurethane
(NNMU) induces acute lung injury are examined. Polymorphonuclear
neutrophil infiltration into the lungs first appears in the
bronchoalveolar lavage (BAL) fluid 24 h after NNMU injection (10.58 ± 3.00% of total cells; P < 0.05 vs. control animals). However, NNMU-induced elevation of the
alveolar-arterial O2 difference requires 72 h to
develop. Daily intraperitoneal injections of the inducible nitric oxide
(· NO) synthase (iNOS)-selective inhibitor aminoguanidine
(AG) initiated 24 h after NNMU administration improve the survival of
NNMU-treated animals. However, AG administration initiated 48 or 72 h
after NNMU injection does not significantly improve the survival of
NNMU-treated animals. These results suggest that · NO
participates in events that occur early in NNMU-induced acute lung
injury. BAL cells isolated from rats 24 and 48 h after NNMU injection
produce elevated · NO and express iNOS during a 24-h ex vivo
culture. AG attenuates · NO production but does not affect
iNOS expression, whereas actinomycin D prevents iNOS expression and
attenuates · NO production by BAL cells during this ex vivo culture. These results suggest that NNMU-derived BAL cells can stimulate iNOS expression and · NO production during culture. In 48-h NNMU-exposed rats, iNOS expression is elevated in homogenates of whole lavaged lungs but not in BAL cells derived from the same lung.
These findings suggest that the pathogenic mechanism by which NNMU
induces acute lung injury involves BAL cell stimulation of iNOS
expression and · NO production in lung tissue.
N-nitroso-N-methylurethane; aminoguanidine; acute respiratory distress syndrome; bronchoalveolar
lavage; inducible nitric oxide synthase; polymorphonuclear neutrophil
 |
INTRODUCTION |
NITRIC OXIDE (· NO) is produced by a
five-electron oxidation of
L-arginine to
L-citrulline by · NO
synthase (NOS). Two general classes of NOS have been identified: the
calcium-dependent, constitutively expressed NOS and the
calcium-independent, transcriptionally inducible NOS (iNOS) (17).
Cytokines, lipopolysaccharides (18), and decreases in environmental pH
(2) stimulate iNOS expression in a variety of cell types. Cellular
sources of · NO in the lung include interstitial macrophages
(16), alveolar type II epithelial cells (21), alveolar macrophages, and
endothelium (15). In addition, these cells release a variety of
inflammatory mediators that stimulate neutrophilic infiltration, induce
iNOS expression, or stimulate and/or inhibit · NO
production (19, 20, 22).
Subcutaneous injection of the carcinogen
N-nitroso-N-methylurethane
(NNMU) results in a lung injury that displays many characteristics of
acute respiratory distress syndrome (ARDS), including severe hypoxemia
and alterations in surfactant composition, function, and metabolism
(4). We have shown (5) that aminoguanidine (AG) attenuates NNMU-induced
acute lung injury as indicated by normalization of
1) impaired
alveolar-arterial
O2 difference
(A-aDO2), 2) the influx of polymorphonuclear
neutrophils (PMNs) into the alveoli, and
3) the phospholipid-to-protein ratio
and surface tension at minimum bubble size of isolated surfactant
preparations. Recent evidence suggests that · NO is
involved in the pathogenesis of acute lung injury induced by acute
pancreatitis (24), ischemia-reperfusion (13), and
intratracheal aerosolization of ovalbumin into
ovalbumin-sensitized mice (6).
In the present study, the time-dependent effects of NNMU on acute lung
injury were examined. We show that NNMU-induced elevation of
A-aDO2
is preceded by PMN infiltration and administration of AG before the
onset of impaired gas exchange in NNMU-exposed animals increases their
survival. In addition, we show iNOS expression in whole lavaged lung
homogenates but not in freshly isolated bronchoalveolar lavage (BAL)
cells isolated from the same lung of NNMU-treated rats. These results
suggest that early events leading to acute lung injury are mediated by
pulmonary tissue-derived · NO.
 |
METHODS |
Animals and materials. In
all experiments, pathogen-free male Sprague-Dawley rats (250-350
g) obtained from Harlan Sprague Dawley
(Indianapolis, IN) were used. Extending from behind the base of the skull was a heparin-coated arterial catheter that was used
for the collection of blood samples. The rats were housed individually
in plastic cages and allowed water and normal rat chow ad libitum. NNMU
(Pfaltz and Bauer, Waterbury, CT) injections (8.0 mg/kg
body wt subcutaneously) were administered to rats anesthetized by
inhalation of halothane (Halocarbon Laboratories, River
Edge, NJ) as reported by Harris et al. (11). AG (400 mg/kg body wt ip)
was administered every 24 h to manually restrained rats, starting at
the indicated time points after NNMU exposure (5). All common chemical
reagents were obtained from Sigma (St. Louis, MO) unless otherwise noted.
A-aDO2
analysis.
Arterial blood samples (0.7 ml) were collected directly
into a heparin-flushed 1.0-ml syringe via the indwelling arterial catheter at 24, 48, 72, and 96 h after NNMU injection from
spontaneously breathing, unanesthetized, manually restrained rats.
Arterial blood gases were measured with a Radiometer ABL 520 (Copenhagen, Denmark) blood microsystem. Blood pH and
arterial partial pressures of O2
and CO2 obtained from arterial
blood gas determinations were used to calculate the
A-aDO2
(1). The respiratory quotient and the inspired
O2 fraction were assumed to be
constant at 0.8 and 0.21, respectively. Values were corrected for
animal body temperature (14).
Cellular infiltrate analysis. After
blood samples were drawn and arterial blood gases were determined, the
lungs were surgically removed and ~45 ml of BAL fluid were collected
from each isolated rat lung (5). Briefly, animals were anesthetized
with pentobarbital sodium (85 mg/kg body wt ip),
tracheotomized by insertion of a metal tube into the
trachea, and exsanguinated by severing the inferior vena cava. The
lungs were perfused through the right ventricle of the heart with a
buffered salt solution (125 mM NaCl, 5 mM KCl, 2.5 mM
Na2HPO4,
17 mM HEPES, 10 µg/ml of gentamicin, 1 mg/ml of streptomycin, 1,000 U/ml of penicillin, and 1 mg/ml of dextrose, pH 7.4) to remove blood
cells from the pulmonary vasculature. The intact lungs were then
carefully removed and lavaged with 8.0-ml aliquots of the buffered salt
solution. The BAL fluid was centrifuged at 300 g for 15 min at 4°C, the cell pellet was resuspended, the total volume was brought to 45 ml with the
buffered salt solution, and the suspension was centrifuged as noted
above. The cell pellet was resuspended in 2.0 ml of culture medium that
consisted of Ham's F-12 nutrient mixture (GIBCO BRL, Grand Island, NY)
culture medium supplemented with 10% fetal calf serum, gentamicin (50 µg/ml), streptomycin (1 mg/ml), and penicillin (1,000 U/ml). Total
viable cells were counted on a hemocytometer by trypan blue exclusion.
An aliquot of the cell suspension was centrifuged onto glass slides and
stained with Diff-Quik, a modified Wright's stain procedure (Baxter
Scientific, McGaw Park, IL), and a 300 cell/slide
sample (macrophages, lymphocytes, and PMNs) was counted.
Analysis of nitrite and nitrate in cell culture
medium. Freshly isolated BAL cells were seeded onto a
24-well tissue culture plate at 300,000 viable cells/well in 500 µl
of culture medium. To designated wells, 1 mM AG or 1 µM actinomycin D
(ActD) was added. Cells were cultured at 37°C, 85% humidity, and
10% CO2 for 4, 16, or 24 h. After
culture period, the culture medium was collected and centrifuged, and
350 µl of the supernatant were stored at
20°C for nitrite
and nitrate determination. The remaining cell pellet and the cells
adherent to the 24-well plate were combined and stored at
20°C for Western blot analysis of iNOS protein (see
Western blot analysis for
iNOS). Nitrite and nitrate determination was performed by the enzymatic conversion of nitrate to nitrite with
nitrate reductase (Aspergillus
fumigatus), followed by the removal of excess NADPH
with glutamic dehydrogenase and, subsequently, the Griess assay (8).
Total nitrite concentration was based on a sodium nitrate standard curve.
Western blot analysis for iNOS. For
0-h culture samples, 50 µl of SDS sample buffer (0.25 M
Tris · HCl, 20%
-mercaptoethanol, and 4% SDS, pH
6.6) were added to cell pellets containing 2.5 × 106 BAL cells and boiled for 5 min. Five microliters of bromphenol blue (80% glycerol and 0.05%
bromphenol blue) were added and vortexed, and the samples were stored
at
20°C. For 24-h culture samples, 75 µl of SDS sample
buffer/well were added directly onto the adherent cells of duplicate
wells of a 24-well plate, and the entire plate was set afloat in a
boiling water bath for 5 min. The SDS sample buffer was collected and
pooled into the microcentrifuge tube containing the pelleted,
nonadherent cells matching their originating wells. Total volume of SDS
sample buffer was brought up to 100 µl, 10 µl of bromphenol blue
sample dye were added and vortexed, and the samples were stored at
20°C. Whole lung homogenates were prepared from excised,
lavaged lungs as described by Cruz et al. (5).
On the basis of cell number (75,000 or 100,000) or 1.0 µg of DNA as
indicated, samples were loaded, separated by SDS-PAGE (10%), and
transferred to a nitrocellulose membrane under semidry conditions
(FisherBiotech semidry blotting unit, Fisher Scientific, Pittsburgh,
PA). Detection of iNOS protein was performed by enhanced chemiluminescence (Amersham Life Sciences, Arlington Heights, IL) with
rabbit anti-mouse iNOS (Cayman Chemical, Ann Arbor, MI) at a dilution
of 1:1,500 and horseradish peroxidase-conjugated donkey anti-rabbit
(Jackson Immunological Research, West Grove, PA) at a dilution of
1:5,000. Interleukin (IL)-1-stimulated RINm5F cells
(1.0 unit/ml, 24 h) were used as an iNOS positive control (12).
Statistics. Significance was
calculated with one-way analysis of variance with Bonferroni post hoc
analysis. Survival significance was calculated with
Kruskal-Wallis analysis. Data are means ± SE.
 |
RESULTS |
Time-dependent effects of NNMU administration on alveolar PMN
infiltration and alterations in
A-aDO2.
The acute lung injury induced by NNMU is characterized by marked
changes in normal alveolar function. The progression of acute lung
injury was examined by determining the time-dependent effects of NNMU
on
A-aDO2.
As shown in Fig.
1A,
there were no significant changes in
A-aDO2
until 72 h after NNMU injection (50.28 ± 4.38 mmHg;
P < 0.05) compared with that in
control animals. At 96 h after NNMU administration, the
A-aDO2
was also significantly higher than that in control rats (54.13 ± 7.14 mmHg; P < 0.05).

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Fig. 1.
Time course of alveolar-arterial
O2 difference
(A-aDO2;
A) and polymorphonuclear neutrophil
(PMN) influx (B) in
N-nitroso-N-methylurethane
(NNMU)-treated rats. Rats were injected with 8.0 mg/kg of NNMU
subcutaneously. At indicated time points, arterial blood samples were
taken from an indwelling arterial catheter, and blood gases were
determined. Lungs were excised and lavaged, and cells from
bronchoalveolar lavage (BAL) fluid were recovered. Cellular composition
of lavage was determined with Diff-Quik, a modified Wright's stain
procedure. Data are means ± SE; n 5 animals/time point.
P 0.05 vs.
control.
|
|
Although the NNMU-induced elevation in
A-aDO2
requires 72 h to develop, a significant increase in PMNs was present in
the BAL fluid as early as 24 h after NNMU exposure compared with that in control rats (12.24 ± 1.99 and 0.28 ± 0.10% of total BAL
cells recovered, respectively;
P < 0.05; Fig.
1B). In addition, the percentage of
infiltrating PMNs significantly increased in a time-dependent manner
(14.33 ± 2.15% at 48 h, 35.40 ± 5.87% at 72 h, and 51.70 ± 10.32% at 96 h; P < 0.05 vs.
control rats). These findings demonstrate that PMN influx precedes the
elevation in
A-aDO2
and suggest that the early influx of PMNs into the alveolus may
participate in the progression of NNMU-induced acute lung injury.
Early administration of AG improves
A-aDO2
and survival of NNMU-injected rats.
It is shown in Fig. 1 that PMN infiltration precedes
elevation in
A-aDO2.
In addition, simultaneous administration of the NOS inhibitors AG or
NG-nitro-L-arginine methyl ester to
NNMU-treated rats attenuates acute lung injury (5). Therefore, the
effects of AG injections initiated 24, 48, and 72 h after NNMU
administration on
A-aDO2 were examined. The data indicate a trend toward an improved
A-aDO2 in NNMU-exposed animals that received early AG treatment compared with
NNMU-exposed rats without AG. Although AG appeared to improve the
A-aDO2,
the difference between the NNMU-exposed rats and those that received
NNMU plus AG at the given time points did not reach significance (Fig.
2A).

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Fig. 2.
Effect of aminoguanidine (AG) treatment after NNMU exposure on
A-aDO2
(A) and percent survival
(B). Rats were injected with NNMU as
described in Fig. 1. NNMU-injected animals received no treatment or 400 mg/kg of AG intraperitoneally every 24 h starting at 24, 48, or 72 h
after subcutaneous NNMU injection. At indicated time points, arterial
blood samples were taken from an indwelling arterial catheter, and
blood gases were determined. Data are means ± SE;
n 3 and 6 animals/treatment group for A and
B, respectively.
|
|
The effects of AG injections after NNMU administration on animal
survival were examined. Figure 2B
shows that daily injections of AG starting at 24 and 48 h after NNMU
injection improved the survival rate of NNMU-exposed animals. At 96 h
after NNMU treatment, rats that received AG (400 mg · kg
1 · day
1
ip) initiated at 24, 48, and 72 h after NNMU injection displayed a
66.67 (n = 6 animals), 33.33 (n = 9 animals), and 10.00%
(n = 10 animals) survival rate,
respectively, whereas rats that did not receive AG displayed a 15.39%
(n = 13 animals) survival rate. The
administration of AG initiated at 24 and 48 h after NNMU treatment was
significant (P < 0.001 and P < 0.05 vs. control rats, respectively).
Isolated BAL cells from rats exposed to NNMU for 24, 48, and 72 h produce elevated levels of · NO in
culture. · NO production in 24-h ex vivo
cultures of BAL cells isolated from rats exposed to NNMU for 24, 48, and 72 h was investigated. Figure 3 shows that BAL cells isolated 24, 48, and 72 h after NNMU injection produced
higher levels of · NO during a 24-h culture than BAL cells
isolated from control rats. Both AG and ActD reduced · NO production during the 24-h ex vivo culture to control levels. These
findings suggest that the increased · NO production by BAL cells isolated from NNMU-treated rats during the ex vivo culture may be
due to de novo synthesis of iNOS protein.

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Fig. 3.
Nitric oxide (· NO) production from BAL cells isolated from
24-, 48-, and 72-h NNMU-exposed rats. Animals were administered NNMU as
described in Fig. 1. At indicated time points, lungs were excised and
lavaged, and recovered BAL cells were plated in a 24-well plate
(300,000 cells/well in 500 µl of culture medium) with (+) and without
( ) AG or actinomycin D (ActD). After a 24-h culture period,
culture medium was collected and centrifuged at 300 g for 15 min at 4°C. Supernatant
was collected and assayed for nitrite
(NO 2) and nitrate
(NO 3). Data are means ± SE;
n 4 animals/time point.
P 0.001 vs. control.
|
|
Time-dependent production of · NO by BAL
cells isolated from NNMU-exposed rats. The ex vivo
production of · NO by BAL cells isolated from control and
NNMU-treated rats during a 4-, 16-, and 24-h culture was examined. As
shown in Fig. 4, · NO production from BAL cells of NNMU-treated and control rats increased in a time-dependent manner during ex vivo culture. After a 4-h ex vivo incubation, the total nitrite and nitrate produced by BAL cells isolated 24 h after NNMU administration was not significantly different
from that produced by control BAL cells. However, after a 16- or 24-h
ex vivo incubation, BAL cells from NNMU-exposed rats produced threefold
higher levels of · NO compared with control BAL cells. Both
AG and ActD inhibit · NO production by BAL cells isolated
from NNMU-treated rats during the ex vivo culture to levels similar to
control BAL cells. Neither AG nor ActD had an inhibitory effect on ex
vivo · NO production from BAL cells isolated from control
animals. This result suggests that the time-dependent increase in ex
vivo · NO production from BAL cells isolated from NNMU-treated rats is due to iNOS because the increase in · NO production was 1)
inhibited by the iNOS-selective inhibitor AG and
2) required de novo protein
synthesis.

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Fig. 4.
Time course of ex vivo · NO production from
BAL cells isolated from NNMU-exposed rats. Rats were administered NNMU
as described in Fig. 1. Lungs were excised and lavaged 24 h after NNMU
injection, and recovered BAL cells were plated in a 24-well plate as
described in Fig. 3. After indicated time points in culture, culture
medium was collected, and NO 2 and
NO 3 production was determined. Data
are means ± SE; n 4 animals/time point. P 0.05 vs. control. P 0.001 vs.
control.
|
|
Ex vivo iNOS expression by BAL cells isolated from
rats exposed to NNMU for 24, 48, and 72 h. Expression
of iNOS by BAL cells placed in culture for 24 h after isolation from
control and NNMU-treated rats was examined by Western blot analysis. As
shown in Fig.
5A, freshly isolated, uncultured BAL cells from either control or NNMU-exposed rats did not express iNOS protein. However, after a 24-h
ex vivo culture, the BAL cells from 24-, 48-, and 72-h NNMU-exposed
rats expressed high levels of iNOS protein. The BAL cells isolated
from control animals did not express iNOS after a 24-h ex vivo culture.
The addition of AG to the culture medium did not reduce the iNOS
expression in NNMU-treated rats ex vivo, whereas the addition of ActD
to the culture medium prevented iNOS expression (Fig.
5B). These findings suggest that
iNOS expression in the BAL cells isolated from NNMU-exposed rats
results from ex vivo induction.

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Fig. 5.
Western blot analysis of inducible · NO synthase (iNOS)
expression by isolated BAL cells. Experiments on iNOS
expression by BAL cells from control and NNMU-exposed rats
(A) and effect of AG and ActD on
iNOS expression (B) were performed
on freshly isolated and 24-h ex vivo cultures. Rats
were administered NNMU as described in Fig. 1. At indicated time
points, lungs were excised and lavaged, and recovered BAL cells were
plated in a 24-well plate as described in Fig. 3. After 24 h in
culture, cells were isolated, lysed, and separated by SDS-PAGE, and
iNOS protein expression was determined by Western blot
analysis. Sample lanes were loaded with samples standardized to 75,000 (A) and 100,000 cells
(B). Interleukin (IL)-1-stimulated
RINm5F cells were used as iNOS-positive control. Data represent
n = 3 animals/condition.
|
|
iNOS expression in whole lavaged lung
homogenates. The results presented in Figs. 4 and
5 indicate that the BAL cells isolated from
NNMU-treated rats express iNOS and produce · NO during an ex
vivo culture, whereas freshly isolated BAL cells from either NNMU-treated or control rats do not express iNOS. In addition, we have
previously shown (5) elevated iNOS expression in homogenates of whole
unlavaged lung of NNMU-treated rats. Therefore, we examined lavaged,
whole lungs for iNOS protein to determine whether iNOS is expressed in
vivo. As shown in Fig. 6, whole lavaged
lungs of 48-h NNMU-treated rats expressed iNOS in contrast to control rat lungs. The BAL cells collected from the same lung of either NNMU-treated or control rats did not express iNOS. These findings indicate that before NNMU-induced impairment of
A-aDO2,
iNOS is expressed in the lung.

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Fig. 6.
Western blot analysis of iNOS expression from isolated, uncultured BAL
cells and whole lavaged lung from control and NNMU-treated animals.
Rats were administered NNMU as described in Fig. 1. At 48 h after NNMU
injection, lungs were excised and lavaged, and BAL cells were
recovered. BAL cells and whole lung homogenate samples were prepared
and separated by SDS-PAGE, and iNOS protein expression was determined
by Western blot analysis. Sample lanes were loaded with samples
standardized to 1.0 µg of DNA. Data represent
n 3 animals/condition.
|
|
 |
DISCUSSION |
Using the NNMU-induced acute lung injury model, we demonstrate that
daily injections of AG starting at 24 or 48 h after
NNMU injection improve the survival rate of NNMU-exposed rats,
suggesting that early events leading to NNMU-induced acute lung injury
involve · NO. We show that severe alterations in
A-aDO2
occur within a window of 48-72 h after NNMU administration. This
pathology is preceded by an influx of PMNs into the alveolar space, an
event that is detectable as early as 24 h after NNMU injection. BAL cells isolated from NNMU-treated animals
1) produce elevated levels of
· NO during ex vivo culture compared with BAL cells isolated from control rats and 2) express
iNOS after 24 h in culture, which correlates with an elevation in
· NO production. Both AG and ActD attenuate · NO
production by BAL cells isolated from NNMU-treated rats to levels
comparable with those produced by BAL cells isolated from control rats,
indicating that the increase in · NO production is due to the
activity of newly synthesized NOS. Freshly isolated BAL cells from
NNMU-treated rats do not express iNOS, indicating that iNOS expression
and · NO production are induced ex vivo. Moreover, expression
of iNOS is found in whole lavaged lung homogenates of 48-h NNMU-exposed
animals, suggesting that · NO production in vivo is derived
from lung tissues and not from the cells infiltrating the alveolus.
Collectively, the data presented suggest that BAL cells derived from
NNMU-treated animals are capable of stimulating iNOS expression and
· NO production in lung tissue.
The NNMU-induced acute lung injury is characterized by severe damage to
the alveolar epithelium, resulting in the characteristics of ARDS (4).
NNMU-induced alveolar damage may involve · NO because the
administration of NOS inhibitors attenuates the ARDS-like characteristics of PMN infiltration, alteration of surfactant activity,
and elevation of
A-aDO2
(5). In vitro evidence suggests that · NO
inhibits the activities of surfactant proteins and lipids (9) and
decreases surfactant synthesis and cellular ATP levels (10). In
addition, alveolar type II pneumocytes undergo apoptosis in the
presence of · NO donor compounds (7). Our findings presented here support a role for the involvement of · NO in the
pathogenesis of ARDS.
Recent evidence suggests that tumor necrosis factor (TNF),
· NO (23), and IL-1 (3) mediate acute lung injury induced by
ischemia-reperfusion. Alveolar macrophages (18) and
type II pneumocytes (21) can be stimulated to express iNOS and produce elevated levels of · NO with cytokines such as
interferon-
, IL-1
, and TNF-
. Furthermore, alveolar
macrophage-derived · NO has been shown to mediate lung injury
in the acute pancreatitis model (24). In the present study, inhibition
of · NO production from isolated BAL cells ex vivo by AG and
ActD suggests that inflammatory cell-derived cytokines such as IL-1
and TNF-
may participate in stimulating iNOS expression and
· NO production.
In conclusion, we have shown in NNMU-exposed rats
1) an increased percentage of PMNs
in the BAL fluid before elevation of A-aDO2,
2) an increased survival rate with
early administration of an iNOS-selective inhibitor,
3) iNOS expression and elevated · NO production from BAL cells only after ex vivo culture,
and 4) elevated iNOS levels in whole
lavaged lungs. Our findings suggest that inflammatory cells infiltrate
the alveolus and stimulate iNOS expression and · NO
production in lung tissue, resulting in NNMU-induced lung injury.
 |
ACKNOWLEDGEMENTS |
We acknowledge the help of Dr. George A. Vogler (Department of
Comparative Medicine) in the placement of the indwelling arterial catheters, Dr. Randy S. Sprague for inspiring discussions concerning this manuscript, Tracey Baird for excellent technical assistance, and
the Pulmonary Function Laboratory for use of the Radiometer ABL 520.
 |
FOOTNOTES |
This work was supported by National Heart, Lung, and Blood Institute
Grant HL-13405 (to W. J. Longmore); National Institute of Diabetes and
Digestive and Kidney Diseases Grant DK-52194 (to J. A. Corbett); a research grant from Alteon Inc., Ramsey, NJ (to J. A. Corbett); and a career development award from the Juvenile Diabetes Foundation Institute (to J. A. Corbett).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: M. A. Moxley, 1402 South Grand Blvd., St.
Louis Univ. School of Medicine, Edward A. Doisy Dept. of Biochemistry
and Molecular Biology, St. Louis, MO 63104-1079.
Received 24 July 1998; accepted in final form 20 October
1998.
 |
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