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
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Abstract
Introduction
Methods
Results
Discussion
References

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
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Abstract
Introduction
Methods
Results
Discussion
References

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
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Abstract
Introduction
Methods
Results
Discussion
References

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% beta -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
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Abstract
Introduction
Methods
Results
Discussion
References

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. star  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. star  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. dagger  P <=  0.05 vs. control. star  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
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Abstract
Introduction
Methods
Results
Discussion
References

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-gamma , IL-1beta , and TNF-alpha . 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-1beta and TNF-alpha 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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Discussion
References

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Am J Physiol Lung Cell Mol Physiol 276(2):L263-L268
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