Departments of 1Child Health and 2Physiology, University of Missouri College of Medicine, Columbia, Missouri 65212; and Departments of 3Pediatrics and 4Physiology, University of Florida College of Medicine, Gainesville, Florida 32610
Submitted 17 December 2002 ; accepted in final form 22 April 2003
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ABSTRACT |
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glucocorticoids; shock; arginine; multiple organ failure; nitric oxide; hemorrhage; hemorrhagic shock; guanosine triphosphate cyclohydrolase I; cationic amino acid transporter 2
Nitric oxide formation by iNOS requires the cofactor tetrahydrobiopterin (7, 31, 32). Tetrahydrobiopterin acts allosterically on iNOS by stabilizing high-spin heme iron conformations and by supporting L-arginine binding (45). Tetrahydrobiopterin also promotes assembly of the iNOS isoform into its active dimeric state (2). Tetrahydrobiopterin has been argued to be "absolutely" required for iNOS activity (31). Guanosine triphosphate cyclohydrolase I (GTPCH) regulates biosynthesis of tetrahydrobiopterin (21). GTPCH gene transfer has been shown to reconstitute iNOS activity in tetrahydrobiopterin-deficient vascular smooth muscle cells despite poor gene transfer efficiency (42). Interestingly, rats injected with bacterial endotoxin increase intrapulmonary transcription of GTPCH and increase intravascular concentrations of nitric oxide byproducts [nitrate (NO3-) and nitrite (NO2-)] (8, 9). Therefore, GTPCH activity can regulate the enzymatic formation of nitric oxide in some circumstances.
Cellular uptake of arginine through the high affinity cationic amino acid transporter (CAT-2) has been shown to limit nitric oxide expression (34, 37). For example, in an earlier study, we showed that cultured rat astrocytes exhibit an apparent Km that is notably higher for arginine uptake and nitric oxide formation than that which has been reported for arginine binding to purified iNOS (37). Others have shown that macrophages from mice deficient in the CAT-2 transporter exhibit dramatically impaired ability to produce nitric oxide (22). Moreover, arginine-deficient diets have been shown to limit nitric oxide production in rats (47). Conversely, selective stimulation of cellular uptake of arginine has been shown in some cell types, such as porcine endothelial cells, to augment formation of nitric oxide (27). Thus intracellular transport of arginine can also regulate the enzymatic formation of nitric oxide in some circumstances.
The mechanisms by which glucocorticoids inhibit nitric oxide expression have been previously investigated. Ordinarily, the activity of iNOS is regulated at the level of iNOS gene transcription (44, 46); however, the mechanisms by which glucocorticoids suppress iNOS activity appear more complex than simply suppression of iNOS transcription. In one study involving rat glomerular mesangial cell cultures (17), dexamethasone was shown to decrease iNOS gene transcription yet prolong the half-life of iNOS mRNA. The investigators further showed in their model that dexamethasone drastically reduces iNOS mRNA translation and increases iNOS protein degradation. In other studies, glucocorticoids were shown to inhibit iNOS activity by inhibiting arginine transport and tetrahydrobiopterin biosynthesis in cultured cells (1, 35). In a recent series of presently unpublished experiments, we found that hemorrhagic shock upregulates iNOS and CAT-2 mRNA and that it has no effect on GTPCH mRNA in rat lung tissue. We therefore sought to explore whether glucocorticoids attenuate hemorrhagic shock-induced increases in intrapulmonary nitric oxide formation and whether they might do so by inhibiting the formation of tetrahydrobiopterin, iNOS protein, and arginine transporters. To accomplish this goal, we elected to explore the effects of dexamethasone on exhaled nitric oxide concentrations and lung tissue levels of the following: iNOS protein, iNOS mRNA, GTPCH mRNA, and CAT-2 mRNA.
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MATERIALS AND METHODS |
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Ten male Sprague-Dawley rats (280350 g; Charles River Laboratory, Key Lois, FL) were used for the experiments. All rats were fed a standard laboratory chow and were provided water ad libitum until the day of the experiment. The Institutional Animal Use and Care Committee of University of Florida approved the experiments, and the care and handling of the animals were in accordance with National Institutes of Health (NIH) guidelines. Animals were anesthetized with halothane followed by an intramuscular injection of a mixture of 100 mg/kg ketamine and 10 mg/kg xylazine. A rectal temperature probe was inserted, and temperature was maintained at 37°C throughout the experiments by a heating pad and heating lamps. Polyethylene catheters (PE-50) were inserted in the right carotid artery for continuous blood pressure monitoring, in the right femoral artery for blood withdrawal, and in the left femoral vein for fluid infusion. A tracheostomy was then performed, and a 14-gauge angio-catheter was secured in the trachea and connected to a mechanical breathing circuit.
After securing the airway, we paralyzed all animals with pancuronium bromide (1 mg/kg iv). We controlled ventilation with a small animal ventilator (rodent model 683; Harvard Apparatus, South Natick, MA), using a 4-ml tidal volume of room air at 35 breaths/min. Samples of arterial blood (70 µl) were analyzed with a commercial blood gas analyzer (ISTAT, Princeton, NJ). Before starting the experiments, we adjusted the ventilation rate to ensure a PaCO2 of between 40 and 45 mmHg. The animals were then allowed to acclimate to the experimental conditions for at least 20 min before any data were collected.
Hemorrhagic Shock Protocols
Rats were randomly assigned to one of two groups, either pretreatment with dexamethasone in normal saline (2 mg/kg) or pretreatment with the normal saline vehicle. The injections were given intraperitoneally 120 min before the animals were hemorrhaged. Mean systemic blood pressure and heart rate were continuously monitored with a polygraph (model MP 100; Biopac Systems, Santa Barbara, CA) throughout the study. We induced hemorrhagic shock by withdrawing blood over a 10-min period to lower the mean systemic blood pressure to between 40 and 45 mmHg. One milliliter of the withdrawn blood was used to create a sample of plasma that was frozen and later analyzed for NO3-/NO2 content. The remaining withdrawn blood was kept in a glass syringe that had been prerinsed with 0.02 ml of heparin (1,000 U/ml). The blood pressure was maintained at a constant level by further blood withdrawal or reinfusion as needed for 60 min. Afterwards, efforts to control the blood pressure were discontinued, although the animals were continuously monitored for another 90 min before being killed.
Exhaled Nitric Oxide Measurement
The exhaled gas was collected in a 250-ml polyvinyl bag that was connected to the exhaust outlet of the ventilator. We measured the mean mixed exhaled nitric oxide concentration every 5 min by connecting the polyvinyl bag to a chemiluminescence nitrogen oxides analyzer (model 270B; Sievers, Boulder, CO). Before each experiment, the analyzer was calibrated with pure nitrogen as zero gas and certified gas (Liquid Air, Walnut Creek, CA) containing 105 ppm of nitric oxide.
Tissue Sample Collection
At the end of each experiment, 3.5 ml of arterial blood were obtained for blood gas analysis, hematocrit determination, and NO3-/NO2- determination. We used 70 µl of withdrawn arterial blood for analysis of pH, oxygen tension, and carbon dioxide tension using a commercial blood gas analyzer (ISTAT). We used 50 µl of withdrawn arterial blood to measure the spun hematocrit in heparin-coated capillary tubes. The remaining blood was spun in tubes coated with 15% EDTA so that the plasma could be separated and frozen at -20°C for subsequent analysis. Immediately after the arterial blood sample was drawn, the animals were killed. The lungs were quickly removed, snap-frozen in liquid nitrogen, and stored at -80°C for subsequent analysis.
NO3-/NO2- Assay
The sum of nitric oxide metabolites, NO3- and NO2-, was measured with a commercial colorimetric assay kit that involves conversion of NO3- to NO2- with NO3- reductase and use of the Griess reaction (Cayman Chemical, Ann Arbor, MI). Thawed plasma samples were transferred to Micro-30 filters (Millipore, Burlington, MA) and centrifuged at 13,000 g at room temperature for 20 min to remove residual hemoglobin and other large proteins. The plasma filtrates were then treated in accordance with the manufacturer's recommendations, and the absorbencies were measured at a wavelength of 450 nm.
Immunoblotting Assay
For the detection of iNOS, frozen lung samples were thawed immediately before analysis and homogenized in five volumes of boiling lysis buffer [1% sodium dodecyl sulfate (SDS), 1.0 mM sodium orthovanadate, and 10 mM Tris, pH 7.4]. After microwaving for 1015 s, we centrifuged the crude homogenates at 15°C for 5 min at 16,000 g and collected the supernatants for analysis. The protein concentration of each sample was measured with a BCA protein assay kit (Pierce Chemical, Rockford, IL). An equal volume of 2x sample buffer (250 mM Tris, pH 6.8, 4% SDS, 10% glycerol, 0.006% bromphenol blue, and 2% 2-mercaptoethanol) was added to all the samples and boiled for 35 min.
Proteins were separated by SDS-gel electrophoresis. Equal amounts of protein (65 µg) were loaded to each well of 7.5% Tris-glycine precast polyacrylamide gels (Bio-Rad Laboratories, Hercules, CA) and separated by gel electrophoresis at 50 V of constant current for 180 min using a Mini-Protean electrophoresis system (Bio-Rad). Lysate from cytokine-activated murine macrophages was used as a positive control. Then the proteins were transferred from gels to nitrocellulose membranes (Bio-Rad) at 100 V of constant current for 60 min in transfer buffer (25 mM Tris, 190 mM glycine, 20% methanol, and 0.05% SDS). The nitrocellulose membranes were then immediately placed into blocking buffer (5% nonfat dry milk, 10 mM Tris, pH 7.5, 100 mM sodium chloride, and 0.1% Tween 20) and left at room temperature for 60 min. After being blocked, the membranes were incubated overnight at 4°C in primary antibody solution (1:1,000 dilution in blocking buffer, murine monoclonal iNOS, IgG2a, antibody; Transduction Laboratories, Lexington, KY). Horseradish peroxidase-conjugated sheep anti-mouse IgG antibody (1: 1,500 dilution in blocking buffer; Amersham Pharmacia Biotech, Piscataway, NJ) was used as a secondary antibody. Bound antibody was detected by chemiluminescence (ECL plus kit, Amersham). We performed densitometric techniques to quantify the protein band densities using NIH software (Scion, Frederick, MD).
RT-PCR
RT. Total RNA was extracted from snap-frozen lung samples with TRIzol reagent (Life Technologies, Rockville, MD). The integrity of isolated total RNA was determined with 1% agarose gel electrophoresis, and RNA concentration was determined by ultraviolet (UV) light absorbance at a wavelength of 260 nm. RNA samples were incubated with RNase-free DNase 1 (Amersham) for 15 min at 37°C and extracted by a phenol-chloroform technique. Moloney murine leukemia virus RT and random hexamer primers (Ready-to-Go RTPCR Beads, Amersham) were used to reverse transcribe all mRNA species to cDNA. The reaction incubated for 30 min at 42°C in a thermocycler. The cDNA samples were then incubated at 95°C for 5 min in the thermocycler to inactivate the RT. We screened samples for genomic DNA contamination by carrying samples through the PCR procedure without adding RT.
PCR. RT-generated cDNA encoding iNOS, GTPCH, CAT-2, and
-actin were amplified by PCR.
-Actin, a housekeeping gene, was
used as an internal standard. The oligonucleotide primer sequences are shown
in Table 1 and were designed in
accordance with published rat DNA sequences for iNOS (accession no. D14051
[GenBank]
)
(26), GTPCH (accession no.
M58364
[GenBank]
) (14), CAT-2 (accession
nos. U53927
[GenBank]
and U53928
[GenBank]
) (37),
and
-actin (accession nos. V01217
[GenBank]
and J00691)
(25).
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The experimental conditions for iNOS, GTPCH, CAT-2, and -actin PCR
reactions were: initial denaturation at 95°C for 5 min followed by 32
cycles of of amplification at 94°C for 1 min and 72°C for 1.5 min. A
negative control for each set of PCR reactions contained water instead of the
DNA template. All PCR products (8 µl) were electrophoretically separated on
a 1% agarose gel and then stained with ethidium bromide. A Gel Doc 2000 Gel
Documentation System (Bio-Rad) was used to visualize the PCR products.
Densitometric techniques were then performed to quantify the DNA band
densities with NIH software (Scion).
Statistical Analysis
A one-way analysis of variance was used to analyze all data. A significance level was set as 0.05. Data were processed with SigmaStat for Windows, version 2.03 (SPSS, Chicago, IL).
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RESULTS |
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Arterial Blood Gas Measurements
The effects of dexamethasone on mean arterial blood pH, oxygen tension, and carbon dioxide tension at the end of the experiments are shown in Table 2. Of those measurements listed, only the blood pH was significantly different between the two groups. Dexamethasone inhibited the development of acidosis caused by hemorrhagic shock (P = 0.009).
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Exhaled Nitric Oxide
The mean concentrations of exhaled nitric oxide for both groups are shown
in Fig. 2. The exhaled nitric
oxide concentrations remained relatively constant throughout the experiment in
the dexamethasone-treated group yet started to increase in the control group
at 70 min after the onset of hemorrhage. The exhaled nitric oxide
concentration in the control group continued to increase throughout the
experiment. At the end of experiment, mean exhaled nitric oxide concentrations
in the control group were
21-fold higher than those in the dexamethasone
group 63.8 ± 17.4 vs. 3.3 ± 0.6 ppb (P = 0.001).
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NO3-/NO2- Concentrations
Figure 3 shows the prehemorrhage and final plasma NO3-/NO2-/concentrations of both groups. Although the initial plasma NO3-/NO2- concentrations were higher in the dexamethasone-treated group than in the control group (6.4 ± 2.7 vs. 2.7 ± 2.3 µM, respectively; P = 0.002), the final plasma NO3-/NO2- concentrations were lower in the dexamethasone-treated group than in the control group (6.2 ± 2.0 vs. 18.1 ± 15.6 µM, respectively; P = 0.021).
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Immunoblotting Assay
Figure 4 shows immunoblot analyses performed with a monoclonal anti-iNOS antibody reacted against rat lung homogenates from both groups. Densitometric analysis of iNOS protein bands indicates that there were higher levels of the protein in the lungs of the control group than in the dexamethasone group (P = 0.013).
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RT-PCR Assay
Quantitation by densitometry shows that the relative density of iNOS mRNA
bands normalized to -actin in rat lungs was higher in the control group
than the dexamethasone group (P = 0.001;
Fig. 5A). The relative
densities of GTPCH mRNA in the rat lungs were unexpectedly higher in the
dexamethasone group than in the control group (P = 0.007;
Fig. 5B). Likewise,
the relative densities of CAT-2 mRNA in the rat lungs were unexpectedly higher
in the dexamethasone group than in the control group (P = 0.006;
Fig. 5C).
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DISCUSSION |
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Previous studies have shown that the acute lung injury caused by hemorrhagic shock is manifested as infiltration of pulmonary parenchyma with polymorphonuclear neutrophils, interstitial pulmonary edema, and hypoxemia (6, 12). We have previously shown that exhaled nitric oxide concentrations are sensitive indicators of lung injury caused by hemorrhagic shock (13). This report is the first to show that glucocorticoids inhibit accumulation of nitric oxide in the airways during hemorrhagic shock. Our data support the use of this relatively noninvasive measurement, for example, to grade the severity of lung injury caused by hemorrhagic shock or to titrate the use of glucocorticoid therapies in affected patients.
In this experiment, we found that the increases in plasma NO3-/NO2- concentrations caused by hemorrhagic shock were attenuated by dexamethasone. Realizing that more blood was necessarily removed from dexamethasone-treated animals than from the control animals enhances the significance of finding an inhibition of NO3-/NO2- formation during hemorrhage. Likewise, the inhibition of acidosis caused by dexamethasone in our model is perhaps more profound in light of the relative blood losses. Our results are consistent with the idea that dexamethasone has antivasodilating (vasoconstricting) properties mediated by nitric oxide inhibition. Further experiments may be necessary to explain the prehemorrhage increase in NO3-/NO2-. Perhaps nitric oxide formation was induced as a reaction to dexamethasone's angiotensin-mediated vasoconstriction or as a consequence of its antidiuretic (mineralocorticoid) effect.
Previous studies have indicated that glucocorticoids do not affect endothelial cell NOS while inhibiting iNOS (30). We therefore sought to determine which pathways of nitric oxide formation by iNOS were being inhibited by the dexamethasone. In an endotoxin shock model, dexamethasone was shown to inhibit the formation of iNOS mRNA (8). Our findings are consistent with these data in that we found iNOS mRNA concentrations to be lower in samples from the dexamethasone-exposed animals than samples from the control animals. This fact, coupled with the findings of others who found support for regulation of iNOS by transcription, led us to believe that iNOS was being transcriptionally regulated by the glucocorticoid during hemorrhagic shock.
One speculation to explain how glucocorticoids might transcriptionally
regulate iNOS is through the transcription factor NF-B. NF-
B
exists in a complex with its inhibitor I
B in the cytoplasm in its
inactive state. When it is activated by cytokines, NF-
B translocates to
the nucleus and promotes the transcription of a variety of proinflammatory
genes including iNOS. Hemorrhagic shock has been shown in rats to increase
NF-
B activation (11,
18). Glucocorticoids have also
been shown to inhibit NF-
B function in cultured cells by interacting
with the glucocorticoid receptor and RNA polymerase II
(23). This interaction may
cause the suppression of iNOS transcription during hemorrhage in the
dexamethasone-exposed rat lungs.
To the best of our knowledge, this is the first published report to show that glucocorticoids stimulate GTPCH and CAT-2 mRNA despite decreasing iNOS mRNA in rat lungs during hemorrhagic shock. These results argue against the idea that dexamethasone suppression of iNOS activity involves inhibition of GTPCH and CAT-2 formation. This idea was perhaps most effectively argued by Simmons et al. (35), who found in cardiac microvascular cells that dexamethasone inhibited GTPCH and CAT-2 formation. They measured the effect of dexamethasone on biopterin formation directly and reversed the effect of dexamethasone on nitric oxide formation by biopterin supplementation. However, others have also found that glucocorticoids can increase GTPCH mRNA levels in whole organs and cell culture models (8, 33). Perhaps these increases in GTPCH and CAT-2 formation are associated with increased tetrahydrobiopterin and arginine metabolism caused by glucocorticoid stimulation. As with other proteins, the expression of proteins may be tissue specific and dependent on a delicate balance of formation and degradation.
Although we examined the effects of preadministered glucocorticoid on hemorrhagic shock, our results may have been different if we treated the animals with dexamethasone after the animals were bled. Although some planned surgical procedures for patients involve a high likelihood of causing hemorrhagic shock, most instances of hemorrhagic shock are unanticipated. Thus studying the effects of glucocorticoid administration after the onset of hemorrhagic shock is warranted.
Inhibition of nitric oxide production for the purpose of decreasing the inflammatory response during hemorrhagic shock is controversial. One study found that using a nitric oxide scavenger to decrease nitric oxide availability in cells increased survivorship and decreased liver damage (19), whereas another study showed that increasing nitric oxide production by giving exogenous L-arginine, but not giving a nitric oxide inhibitor, during hemorrhagic shock improves survivorship (4). Even though glucocorticoids inhibit nitric oxide synthesis, it has been shown that they can improve survivorship in a rat model of endotoxin shock (24). Therefore we cannot be certain that the therapeutic benefits of glucocorticoids in hemorrhagic shock are related to inhibition of nitric oxide formation. Our results do favor the notion that glucocorticoids protect the lungs against functional sequelae of hemorrhagic shock. Whether glucocorticoids protect against reperfusion injury such as that which might occur during resuscitation of a subject suffering from hemorrhagic shock remains unclear yet warrants investigation.
In summary, hemorrhage-induced nitric oxide formation was attenuated in a rat model by dexamethasone administration as indicated by decreased exhaled nitric oxide concentrations and decreased plasma NO3-/NO2- levels. Dexamethasone inhibits nitric oxide formation at least in part by inhibiting iNOS mRNA expression but not by inhibiting GTPCH and CAT-2 mRNA expression. The use of glucocorticoids to decrease hemorrhagic shock-induced lung injury warrants further investigation.
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ACKNOWLEDGMENTS |
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This work was supported, in part, by NIH Grant 5M01RR-000082-390655 and American Heart Association Grant 0151064B (to J. W. Skimming).
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FOOTNOTES |
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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. Section 1734 solely to indicate this fact.
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REFERENCES |
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