Hypothermia attenuates iNOS, CAT-1, CAT-2, and nitric oxide expression in lungs of endotoxemic rats

Philip O. Scumpia1, Paul J. Sarcia1, Vincent G. DeMarco1,2,3, Bruce R. Stevens4, and Jeffrey W. Skimming1,2,3

Departments of 1 Pediatrics and 4 Physiology and Functional Genomics, University of Florida, Gainesville, Florida 32610; and Departments of 2 Child Health and 3 Physiology, University of Missouri, Columbia, Missouri 65211


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Endotoxemia stimulates endogenous nitric oxide formation, induces transcription of arginine transporters, and causes lung injury. Hypothermia inhibits nitric oxide formation and is used as a means of organ preservation. We hypothesized that hypothermia inhibits endotoxin-induced intrapulmonary nitric oxide formation and that this inhibition is associated with attenuated transcription of enzymes that regulate nitric oxide formation, such as inducible nitric oxide synthase (iNOS) and the cationic amino acid transporters 1 (CAT-1) and 2 (CAT-2). Rats were anesthetized and randomized to treatment with hypothermia (18-24°C) or normothermia (36-38°C). Endotoxin was administered intravascularly. Concentrations of iNOS, CAT-1, CAT-2 mRNA, iNOS protein, and nitrosylated proteins were measured in lung tissue homogenates. We found that hypothermia abrogated the endotoxin-induced increase in exhaled nitric oxide and lung tissue nitrotyrosine concentrations. Western blot analyses revealed that hypothermia inhibited iNOS, but not endothelial nitric oxide synthase, protein expression in lung tissues. CAT-1, CAT-2, and iNOS mRNA concentrations were lower in the lungs of hypothermic animals. These findings suggest that hypothermia protects against intrapulmonary nitric oxide overproduction and nitric oxide-mediated lung injury by inhibiting transcription of iNOS, CAT-1, and CAT-2.

lipopolysaccharide; nitrotyrosine; arginine transport


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

INDUCIBLE NITRIC OXIDE SYNTHASE (iNOS) has been shown in rat models of endotoxemia (47), gram-positive sepsis (15), and ischemia-reperfusion injury (41, 42) to be primarily responsible for endogenous intrapulmonary nitric oxide overproduction. Endotoxins stimulate various cell types within the lung, including bronchial epithelia (7), pulmonary artery smooth muscle cells (30), macrophages (47), and neutrophils (52), to overexpress iNOS. Increases in exhaled concentrations of nitric oxide have been shown to be a noninvasive indicator of lung injury during endotoxemia (38).

Recently, inhibition of nitric oxide synthase (NOS) has been considered as a possible treatment for endotoxemia. Selective inhibition of iNOS has been shown to decrease exhaled concentrations of nitric oxide and attenuate lung injury (45). Endotoxin infused into iNOS-knockout mice fails to induce intrapulmonary nitric oxide formation and causes less lung injury than it does in wild-type mice (17). Interestingly, one group of investigators found that nonselective inhibition of NOS during endotoxemia decreases exhaled nitric oxide concentrations yet promotes lung injury (1). Whether nitric oxide mediates or simply indicates endotoxin-induced lung injury remains controversial.

As with all NOS isoforms, iNOS catalyzes the formation of nitric oxide and citrulline from arginine. In macrophages (50) and other cell types (37), cytosolic access to extracellular arginine is regulated by membrane-bound cationic amino acid transporters, such as CAT-1 and CAT-2. CAT-1 has been found to transport arginine in the lung as well as a variety of other tissues (9, 23, 37). CAT-2 has a higher affinity for arginine than does CAT-1 (22) and has been found in alveolar macrophages (8) and pulmonary artery endothelial cells (27). Using a silica-exposed rat model, a group of investigators recently showed that increases in intrapulmonary arginine uptake correspond with coinduction of CAT-1 and CAT-2 (26). Macrophages isolated from mice deficient in CAT-2 exhibit dramatically impaired ability to shuttle arginine intracellularly and produce nitric oxide in response to endotoxin (28). Interestingly, arginine-deficient diets have been shown to limit nitric oxide production in rats (50). We therefore believe that the cationic amino acid transporters are key regulators of intrapulmonary overproduction of nitric oxide by endotoxin.

Albeit controversial, hypothermia has been used clinically and experimentally as an anti-inflammatory therapy for injury associated with increases in systemic nitric oxide production such as cardiopulmonary bypass (4, 11, 43), brain trauma (3), and severe bacterial meningitis (2, 13, 33, 46). Hypothermia was shown to decrease circulating concentrations of nitrates and nitrites in all these models (13, 31, 34, 53). The effect of hypothermia on intrapulmonary nitric oxide formation remains unclear. Moreover, the effect of temperature regulation on intrapulmonary nitric oxide by CAT-1 and CAT-2 has not been previously studied.

We tested the hypothesis that hypothermia inhibits iNOS, CAT-1, and CAT-2 mRNA and decreases exhaled nitric oxide in the lungs of endotoxemic rats. We also sought to determine whether hypothermia could attenuate nitric oxide-mediated injury as indicated by decreased intrapulmonary nitrotyrosine concentrations. We speculate that exhaled nitric oxide concentrations could eventually serve as a useful indicator of temperature-mediated influences on lung inflammation. We also speculate that dissecting the nitric oxide-related mechanisms affected by hypothermia could lead to improved organ preservation strategies.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Surgical Preparation and Endotoxin Administration

Fifteen male Sprague-Dawley rats (Charles River Laboratory, Key Lois, FL; 280-350 g) were fed a standard laboratory chow and provided water ad libitum until the day of the experiment. The Institutional Animal Use and Care Committee of the 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 ketamine and xylazine (100 and 10 mg/kg body wt, respectively). Three rats were sham instrumented to control for the effects of instrumentation. The other rats were randomized into two groups: hypothermia (n = 6, 18-24°C) and normothermia (n = 6, 36-38°C). The rats in the hypothermia group were placed on an ice pack; the rats in the normothermia and sham groups were placed on a heating pad. A rectal temperature probe was inserted, and temperature was maintained within the temperature range for the group. Polyethylene (PE-50) catheters were inserted in the right carotid artery for continuous blood pressure monitoring using a polygraph (model MP 100, Biopac, Santa Barbara, CA) and for fluid infusion. A tracheostomy was then performed, and a 14-gauge catheter was secured in the trachea and connected to a mechanical breathing circuit.

After the airway was secured, all animals were paralyzed with pancuronium bromide (1 mg/kg iv). Ventilation was controlled 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 using a commercial blood gas analyzer (I-STAT, Princeton, NJ). Before the experiments were started, the ventilation rate was adjusted to ensure arterial PCO2 between 40 and 45 mmHg. The animals were then allowed to acclimate to the experimental conditions for >= 20 min before collection of data. When the animals had acclimated to the conditions, Escherichia coli lipopolysaccharide (15 mg/kg; Sigma-Aldridge, St. Louis, MO) was given intravascularly to the normothermic and hypothermic rats to induce endotoxemia. An identical volume of normal saline (1 ml) was given intravascularly to the sham group. Animals were monitored for 150 min after the infusion of endotoxin.

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. The exhaled nitric oxide concentration was measured every 30 min by connection of the polyvinyl bag to a chemiluminescence nitric oxide analyzer (model 280, Sievers, Boulder, CO). Before each experiment, the analyzer was calibrated using nitrogen stock passed through a charcoal filter and a 1,300-ppb nitric oxide stock tested against standards traceable to the National Institute of Standards and Technology (Air Liquide, Houston, TX).

Tissue Sample Collection

At the end of the experiment, the animals were killed. The lungs were quickly removed, snap-frozen in liquid nitrogen, and stored at -80°C for subsequent analysis.

RT-PCR

RT. Total RNA was extracted from snap-frozen lung samples using TRIzol Reagent (Life Technologies, Rockville, MD). The integrity of isolated total RNA was determined using 1% agarose gel electrophoresis, and RNA concentrations were determined by ultraviolet light absorbance at a wavelength of 260 nm. RNA samples were incubated with RNase-free DNase 1 (Amersham Pharmacia Biotech, Piscataway, NJ) for 15 min at 37°C and extracted by a phenol-chloroform technique. Moloney murine leukemia virus reverse transcriptase and random hexamer primers (Ready-to-Go RT-PCR beads, Amersham) were used to reverse transcribe all mRNA species to cDNA. The reaction was 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 reverse transcriptase. Samples were screened for genomic DNA contamination by carrying samples through the PCR procedure without addition of reverse transcriptase.

PCR. Reverse transcriptase-generated cDNA encoding iNOS, CAT-2, and 18S rRNA were amplified using PCR. 18S rRNA was used as an internal control. cDNA from 18S rRNA was amplified with a QuantumRNA primer-competimer set (Ambion, Austin, TX) to allow semiquantitation of ethidium bromide bands. Because 18S rRNA was far more abundant than the mRNA studied in this experiment, the 18S rRNA amplification reaction was inhibited by addition of competimers. Pilot experiments were performed to optimize the ratio of primers to competimers, the cycle number, and the amount of cDNA to yield multiplex PCR products that were in the linear range of amplification. The PCR cocktail consisted of 5 µl of an 18S rRNA primer-competimer (4:6) mix, 2.5 µl of 10× high-fidelity PCR buffer (Invitrogen, Carlsbad, CA), 0.5 µl of 10 mM dNTP mixture, 5 µl of 1 µM forward target primer, 5 µl of 1 µM reverse target primer, 1 µl of 50 mM MgSO4, 0.25 µl of platinum Taq high-fidelity (Invitrogen), 4 µl of template DNA, and 1.75 µl of H2O. PCR was performed in a thermocycler (MJ Research, Watertown, MA). The PCR procedure consisted of 2 min at 94°C followed by 34 cycles of denaturing for 40 s at 94°C, annealing for 40 s at 55°C, and extension for 40 s at 68°C. The oligonucleotide primer sequences are shown in Table 1 and were designed from published rat DNA sequences from the nucleotide database of the National Library of BioInformatics for iNOS, CAT-1, and CAT-2. A negative control for each set of PCR contained water instead of the DNA template. All PCR products were electrophoretically separated on a 1% agarose gel and then stained with ethidium bromide. A gel documentation system (Gel Doc 2000, Bio-Rad Laboratories, Hercules, CA) was used to visualize the PCR products. Densitometric techniques were then performed to quantify the DNA band densities using National Institutes of Health software (Scion, Frederick, MD).

                              
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Table 1.   Oligonucleotide primers for RT-PCR

Real-Time PCR

Quantitative real-time PCR was performed using the GeneAmp 5700 sequence detection system (Applied Biosystems), SYBR green core reagents (catalog no. 4304886, Applied Biosystems), and Ampli-Taq gold polymerase. Primer sequences for each real-time PCR are listed in Table 2. Varied concentrations of cDNA were used to generate the calibration curve for each primer set. Forty cycles of PCR were performed twice for each primer set using 25-µl reactions, and cDNA loading was normalized to rat 18S rRNA according to the Applied Biosystems protocol. The amount of gene transcript was measured using the comparative (2<UP><SUB>T</SUB><SUP>−&Dgr;&Dgr;C</SUP></UP>) method described by Applied Biosystems. Values obtained from the sham-instrumented rat lung samples were used for comparison.

                              
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Table 2.   Oligonucleotide primers for real-time PCR

Immunoblotting Assay

For the detection of iNOS and endothelial NOS (eNOS), frozen lung samples were thawed immediately before analysis and homogenized in 5 vol of boiling lysis buffer (1% SDS, 1.0 mM sodium orthovanadate, and 10 mM Tris, pH 7.4). After the crude homogenates were microwaved for 10-15 s, they were centrifuged at 15°C for 5 min at 16,000 g, and the supernatants were collected for analysis. The protein concentration of each sample was measured using a bicinchoninic acid protein assay kit (Pierce Chemical, Rockford, IL). An equal volume of 2× sample buffer (250 mM Tris, pH 6.8, 4% SDS, 10% glycerol, 0.006% bromphenol blue, and 2% beta -mercaptoethanol) was added to all the samples and boiled for 3-5 min.

Proteins were separated by SDS-gel electrophoresis. Equal amounts of protein (65 µg) were loaded onto each well of 7.5% Tris-glycine precast polyacrylamide gels (Bio-Rad) and separated by gel electrophoresis at 50 V of constant voltage for 180 min using a Mini-Protean electrophoresis system (Bio-Rad). The proteins were then transferred from gels to nitrocellulose membranes (Bio-Rad) at 100-V constant voltage 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 the membranes were blocked, they 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; or 1:1,000 dilution in blocking buffer, murine monoclonal eNOS, IgG2a, antibody; Transduction Laboratories). According to the manufacturer, neither antibody cross-reacts with other isoforms of NOS. Horseradish peroxidase-conjugated sheep anti-mouse IgG antibody (1:2,000 dilution in blocking buffer; Amersham) was used as a secondary antibody. Bound antibody was detected by chemiluminescence (ECL plus kit, Amersham). The bands were expected at a size of 130 and 140 kDa for iNOS and eNOS, respectively. Densitometric techniques were performed to quantify the protein band densities using NIH software (Scion).

Nitrotyrosine Enzyme-Linked Immune Assay

Lung tissue was prepared for enzyme-linked immunosorbent assay by adding potassium phosphate-EDTA buffer (PE buffer) at 15 times the weight of the tissue. PE buffer was made by adding 1.36 g of KH2PO4 and 0.37 g of Na2EDTA to 1 liter of H2O as the pH was adjusted to 7.4. The tissue was homogenized on ice for 1 min and spun in a centrifuge at 10,000 g for 10 min. The pellet was discarded, and the supernatant was stored at -80°C until the assay was run. We followed the protocol provided by Cayman Chemicals for the nitrotyrosine enzyme-linked immune assay kit. Briefly, 50 µl of serially diluted standards were added in duplicate to a precoated (mouse anti-rabbit IgG) 96-well plate, giving a range of 0-260 pg/ml. Fifty microliters of a 1:5 dilution of the sample supernatants were added to the remaining wells to adjust the nitrotyrosine concentrations of the samples within the standard curve. Then, 50 µl of nitrotyrosine acetylcholinesterase tracer and nitrotyrosine enzyme-linked immune assay antiserum were added to the samples and standards. The plate was incubated overnight at room temperature. On the following day, the plate was washed five times with wash buffer, and 200 µl of Ellman's reagent were added to each well, including the blanks. Then 5 µl of tracer were added to the total activity well, and the plate was left to develop for ~60 min. Optical density was measured at a wavelength of 420 nm. Concentrations of nitrotyrosine in the diluted sample (pg/ml) were then converted to nanograms per gram of tissue wet weight.

Statistical Analysis

The one-way analysis of variance was used to analyze all data. The Student-Newman-Keuls test was used to determine whether differences between the normothermia and hypothermia results existed when the analysis of variance yielded a significant result. A significance level was set as 0.05. Data were processed using SigmaStat for Windows (version 2.03, SPSS, Chicago, IL) and are shown as means ± SD.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Exhaled Nitric Oxide

The mean concentrations of exhaled nitric oxide for both groups are shown in Fig. 1. In the normothermia group, exhaled nitric oxide concentrations began increasing ~60 min after infusion of the endotoxin. At the end of the experiment (at 150 min), exhaled nitric oxide concentration was 73.5 ± 12.4 ppb in this group. In the hypothermia group, exhaled nitric oxide concentrations remained unchanged throughout the experiment. The mean exhaled nitric oxide concentration at the end of the experiment in the hypothermia group was 2.4 ± 1.2 ppb. At the end of the experiment, exhaled nitric oxide concentrations were dramatically lower in the hypothermia than in the normothermia group (P < 0.001). Also, at the end of the experiment, exhaled nitric oxide concentration for three sham-treated controls was 4.6 ± 1.2 ppb.


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Fig. 1.   Average exhaled nitric oxide concentrations in sham, normothermic, and hypothermic rats. Exhaled nitric oxide concentrations were measured every 30 min for 150 min. In normothermic rats (n = 6), exhaled nitric oxide increased abruptly after 60 min. Exhaled nitric oxide in hypothermic (n = 6) and sham-instrumented (n = 3) rats remained unchanged throughout the entire 150 min. At 150 min, there was a significant difference between exhaled nitric oxide concentrations in normothermic and hypothermic rats (P < 0.001).

RT-PCR Assay

Quantitation using densitometry showed that the relative densities of iNOS mRNA bands normalized to 18S rRNA in rat lungs were lower in the hypothermia than the normothermia group (P < 0.001; Fig. 2). Likewise, the relative densities of CAT-1 and CAT-2 mRNA in the rat lungs were lower in the hypothermia than in the normothermia group [P < 0.001 (Fig. 3) and P < 0.001 (Fig. 4)].


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Fig. 2.   Analysis of rat lung inducible nitric oxide synthase (iNOS) mRNA using conventional multiplex RT-PCR and real-time PCR. Top: blots from conventional RT-PCR gel. Middle: densitometric analysis of all bands in conventional PCR gel. Bottom: real-time PCR results for all groups. Conventional (P < 0.001) and real-time (P < 0.001) PCR methods revealed that hypothermia attenuated mRNA induction caused by endotoxin. For both PCR methods, 18S rRNA was used as an internal control for the amount of cDNA in each sample.



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Fig. 3.   Analysis of rat lung cationic amino acid transporter 1 (CAT-1) mRNA using conventional multiplex RT-PCR and real-time PCR. Top: blots from conventional RT-PCR gel. Middle: densitometric analysis of all bands in conventional PCR gel. Bottom: real-time PCR results for all groups. Conventional (P = 0.001) and real-time (P = 0.038) PCR methods revealed that hypothermia attenuated mRNA induction caused by endotoxin. For both PCR methods, 18S rRNA was used as an internal control for the amount of cDNA present in each sample.



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Fig. 4.   Analysis of rat lung CAT-2 mRNA using conventional multiplex RT-PCR and real-time PCR. Top: blots from conventional RT-PCR gel. Middle: densitometric analysis of all bands in conventional PCR gel. Bottom: real-time PCR results for all groups. Conventional (P = 0.001) and real-time (P = 0.002) PCR methods revealed that hypothermia attenuated mRNA induction caused by endotoxin. For both PCR methods, 18S rRNA was used as an internal control for the amount of cDNA present in each sample.

Real-Time PCR

Analysis with quantitative PCR using the manufacturer's protocol showed that hypothermia decreased iNOS, CAT-1, and CAT-2 mRNA levels in the rat lungs. The sham-instrumented animals served as baseline for the calculation of mRNA expression for each gene of interest. Quantitation utilizing the 2<UP><SUB>T</SUB><SUP>−&Dgr;&Dgr;C</SUP></UP> method revealed that hypothermia decreased the mRNA expression levels of iNOS (1.1 ± 1.1 vs. 17.1 ± 8.3, P < 0.001; Fig. 2), CAT-1 (0.09 ± 0.04 vs. 5.2 ± 4.9, P = 0.038; Fig. 3), and CAT-2 (1.8 ± 0.7 vs. 59.6 ± 38.4, P = 0.002; Fig. 4). These values were first adjusted for variability in the amount of cDNA in each sample using the cDNA signal reflecting 18S rRNA. The data were then converted to ratios that were related to the mean values from sham-operated animals. In other words, the data reflect unitless "fold changes" from sham values.

Immunoblotting Assay

Figure 5 shows Western blots performed with a monoclonal anti-iNOS antibody reacted against rat lung homogenates from the normothermia and hypothermia groups. Densitometric analysis of iNOS protein bands indicated higher levels of the protein in the lungs of the normothermia than the hypothermia group (P = 0.003). Figure 6 shows Western blots performed with a monoclonal anti-eNOS antibody against the same rat lung homogenates. Densitometric analysis of eNOS protein bands showed no significant difference between the two groups (P = 0.062).


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Fig. 5.   Western immunoblot of whole lung homogenates visualized using monoclonal anti-iNOS antibody. Densities of iNOS protein bands in rat lungs were higher in normothermia than in hypothermia (P = 0.003).



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Fig. 6.   Western immunoblot of whole lung homogenates visualized using monoclonal anti-endothelial nitric oxide synthase antibody. No effect of hypothermia on endothelial nitric oxide synthase (P = 0.062) was detected.

Intrapulmonary Concentrations of Nitrotyrosine

Figure 7 illustrates the results for the enzyme-linked immunosorbent assay for nitrotyrosine. The normothermia group exhibited a high concentration of intrapulmonary nitrotyrosine, and the hypothermia therapy attenuated this formation of nitrotyrosine (8.1 ± 2.8 vs. 2.19 ± 0.50 ng/g wet wt, P < 0.001). The mean concentration of nitrotyrosine in three sham-instrumented animals was 0.169 ± 0.054 ng/g wet wt.


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Fig. 7.   Enzyme-linked immunosorbent assay using a monoclonal antibody revealed that hypothermia attenuated the effect of endotoxin on intrapulmonary nitrotyrosine concentrations (P < 0.001). Lung tissue from sham-instrumented animals was also tested and found to contain 0.169 ± 0.054 ng/g wet wt (gww) nitrotyrosine.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

This study demonstrates in a whole animal model that hypothermia attenuates the induction of intrapulmonary iNOS, CAT-1, and CAT-2 transcription caused by endotoxemia. Decreased formation of iNOS mRNA was associated with a decrease in iNOS expression in the hypothermic rat lungs. Hypothermia also attenuated overall enzymatic formation of intrapulmonary nitric oxide, as demonstrated by decreased concentrations of nitric oxide in exhaled gas and nitrosylated tyrosine residues on lung tissue proteins.

Because the regulation of endogenous nitric oxide production varies with each isoform of NOS, selective isoform inhibition strategies may provide therapeutic advantages over nonselective isoform inhibition strategies. Nonselective inhibition with Nomega -nitro-L-arginine methyl ester (L-NAME) has been shown to inhibit the hypotension (45) associated with sepsis in various animal models. However, inhibition of NOS using L-NAME has been shown to increase mortality (25) and worsen lung (1), heart (20, 49), and liver (20, 21) injury during endotoxemia. This phenomenon has been attributed to L-NAME's inhibition of eNOS-derived nitric oxide, which preserves endothelium vasodilation (6) and prevents leukocyte adhesion (32). Selective inhibition of iNOS, on the other hand, has been shown to inhibit nitric oxide-dependent hypotension (16, 39, 48) and to ameliorate lung (29), heart (49), and liver damage (20) during septic shock. Selective iNOS inhibition has also been shown to decrease mortality in endotoxin-treated mice and rats (19, 20, 40). Our results reveal that the effects of hypothermia on nitric oxide formation mimic those of selective iNOS inhibition.

The downregulation of CAT-1 and CAT-2 in hypothermic lungs is an important finding of this study. We (37) and others (9) have demonstrated in cell culture models that these transporters regulate nitric oxide production by iNOS. One group of investigators found that intranasal instillation of the fungus Fusarium kyushuense induces intrapulmonary mRNA expression of CAT-2 mRNA and argininosuccinate synthetase, an enzyme responsible for regeneration of arginine from citrulline (23). Another group of investigators found that exposure to silica induced an eightfold increase in CAT-1 and CAT-2 mRNA in rat lungs (26). This increase in mRNA expression of arginine transporters was associated with an increase in arginine uptake. Realizing that several studies (9, 24, 27), including our own (37), have shown that changes in arginine uptake ordinarily parallel changes in CAT-1 and/or CAT-2 mRNA expression, we believe that our data support the idea that hypothermia inhibits endotoxin-induced intrapulmonary arginine uptake.

Although we found that hypothermia can decrease endotoxin-induced nitric oxide production by inhibiting transcription of iNOS and CAT-2, the exact mechanism remains unknown. Several studies suggest the involvement of IL-10, which increases during hypothermia (18, 43). IL-10 has been shown to induce apoptosis in neutrophils (5) and monocytes (35), two of the cells responsible for nitric oxide production. We have shown that IL-10 directly attenuates nitric oxide production in murine macrophages by downregulating iNOS and CAT-2 (12). Others have shown that IL-10 inhibits nitric oxide production by inhibiting nuclear factor-kappa B (10, 51), the transcription factor responsible for iNOS and CAT-2 expression. Thus the effects of hypothermia on IL-10 and nuclear factor-kappa B warrant further investigation.

The inhibition of nitric oxide formation caused by hypothermia may offer important insight into mechanisms controlling the well-recognized adaptive advantages regarding vasomotor control of heat preservation. Previous studies have shown that formation of nitric oxide mediates cutaneous vasorelaxation caused by hyperthermia (14). Other studies have shown that inhibition of nitric oxide formation mediates vasoconstriction in the brain (36, 44). We speculate that arginine transporters and nitric oxide may be involved in regulating heat preservation through their effects on vascular tone.

Several important limitations to our study exist. One limitation relates to interspecies differences suggested by the finding that our rats continued to have a normal cardiac rhythm at a core temperature of 18-24°C. At these temperatures, human subjects ordinarily spontaneously arrest their cardiac function. Another limitation is that the effects of rewarming on nitric oxide formation in our model are unknown; however, we intentionally designed the study to reveal the effects of hypothermia on nitric oxide formation, rather than provide rationale for hypothermia therapy.

In summary, hypothermia inhibits the formation of iNOS during endotoxemia at the transcriptional level. This inhibition of iNOS abrogates intrapulmonary nitric oxide formation and the resultant oxidative protein injury. The inhibition of nitric oxide expression by hypothermia may explain some of the therapeutic benefits of hypothermia that have been observed in patients who have meningitis and who undergo cardiac surgery. Elucidating the therapeutic mechanisms of hypothermia may lead to improved means of organ preservation.


    ACKNOWLEDGEMENTS

We thank K. M. Kelly for assisting in performing the PCR assays.


    FOOTNOTES

This work was supported, in part, by NIH Grant 5M01RR000082-390655 and American Heart Association Grant 0151064B awarded to J. W. Skimming.

Address for reprint requests and other correspondence: J. W. Skimming, Dept. of Child Health, One Hospital Dr., Columbia, MO 65211 (E-mail: skimmingj{at}missouri.edu).

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.

August 2, 2002;10.1152/ajplung.00102.2002

Received 5 April 2002; accepted in final form 16 July 2002.


    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Aaron, SD, Valenza F, Volgyesi G, Mullen JB, Slutsky AS, and Stewart TE. Inhibition of exhaled nitric oxide production during sepsis does not prevent lung inflammation. Crit Care Med 26: 309-314, 1998[ISI][Medline].

2.   Angstwurm, K, Reuss S, Freyer D, Arnold G, Dirnagl U, Schumann RR, and Weber JR. Induced hypothermia in experimental pneumococcal meningitis. J Cereb Blood Flow Metab 20: 834-838, 2000[ISI][Medline].

3.   Biagas, KV, and Gaeta ML. Treatment of traumatic brain injury with hypothermia. Curr Opin Pediatr 10: 271-277, 1998[Medline].

4.   Conroy, BP, Grafe MR, Jenkins LW, Vela AH, Lin CY, DeWitt DS, and Johnston WE. Histopathologic consequences of hyperglycemic cerebral ischemia during hypothermic cardiopulmonary bypass in pigs. Ann Thorac Surg 71: 1325-1334, 2001[Abstract/Free Full Text].

5.   Cox, G. IL-10 enhances resolution of pulmonary inflammation in vivo by promoting apoptosis of neutrophils. Am J Physiol Lung Cell Mol Physiol 271: L566-L571, 1996[Abstract/Free Full Text].

6.   Fischer, LG, Horstman DJ, Hahnenkamp K, Kechner NE, and Rich GF. Selective iNOS inhibition attenuates acetylcholine- and bradykinin-induced vasoconstriction in lipopolysaccharide-exposed rat lungs. Anesthesiology 91: 1724-1732, 1999[ISI][Medline].

7.   Gutierrez, HH, Pitt BR, Schwarz M, Watkins SC, Lowenstein C, Caniggia I, Chumley P, and Freeman BA. Pulmonary alveolar epithelial inducible NO synthase gene expression: regulation by inflammatory mediators. Am J Physiol Lung Cell Mol Physiol 268: L501-L508, 1995[Abstract/Free Full Text].

8.   Hammermann, R, Dreissig MD, Mossner J, Fuhrmann M, Berrino L, Gothert M, and Racke K. Nuclear factor-kappa B mediates simultaneous induction of inducible nitric-oxide synthase and upregulation of the cationic amino acid transporter CAT-2B in rat alveolar macrophages. Mol Pharmacol 58: 1294-1302, 2000[Abstract/Free Full Text].

9.   Hattori, Y, Kasai K, and Gross SS. Cationic amino acid transporter gene expression in cultured vascular smooth muscle cells and in rats. Am J Physiol Heart Circ Physiol 276: H2020-H2028, 1999[Abstract/Free Full Text].

10.   Heyen, JR, Ye S, Finck BN, and Johnson RW. Interleukin (IL)-10 inhibits IL-6 production in microglia by preventing activation of NF-kappa B. Brain Res Mol Brain Res 77: 138-147, 2000[ISI][Medline].

11.   Honore, PM, Jacquet LM, Beale RJ, Renauld JC, Valadi D, Noirhomme P, and Goenen M. Effects of normothermia versus hypothermia on extravascular lung water and serum cytokines during cardiopulmonary bypass: a randomized, controlled trial. Crit Care Med 29: 1903-1909, 2001[ISI][Medline].

12.   Huang, CJ, Stevens BR, Nielsen RB, Slovin PN, Fang X, Nelson DR, and Skimming JW. Interleukin-10 inhibition of nitric oxide biosynthesis involves suppression of CAT-2 transcription. Nitric Oxide 6: 79-84, 2002[ISI][Medline].

13.   Irazuzta, JE, Pretzlaff R, Rowin M, Milam K, Zemlan FP, and Zingarelli B. Hypothermia as an adjunctive treatment for severe bacterial meningitis. Brain Res 881: 88-97, 2000[ISI][Medline].

14.   Kellogg, DL, Jr, Crandall CG, Liu Y, Charkoudian N, and Johnson JM. Nitric oxide and cutaneous active vasodilation during heat stress in humans. J Appl Physiol 85: 824-829, 1998[Abstract/Free Full Text].

15.   Kengatharan, KM, De Kimpe SJ, and Thiemermann C. Role of nitric oxide in the circulatory failure and organ injury in a rodent model of gram-positive shock. Br J Pharmacol 119: 1411-1421, 1996[Abstract].

16.   Kilbourn, RG, Jubran A, Gross SS, Griffith OW, Levi R, Adams J, and Lodato RF. Reversal of endotoxin-mediated shock by NG-methyl-L-arginine, an inhibitor of nitric oxide synthesis. Biochem Biophys Res Commun 172: 1132-1138, 1990[ISI][Medline].

17.   Kristof, AS, Goldberg P, Laubach V, and Hussain SN. Role of inducible nitric oxide synthase in endotoxin-induced acute lung injury. Am J Respir Crit Care Med 158: 1883-1889, 1998[Abstract/Free Full Text].

18.   Lee, SL, Battistella FD, and Go K. Hypothermia induces T-cell production of immunosuppressive cytokines. J Surg Res 100: 150-153, 2001[ISI][Medline].

19.   Liaudet, L, Feihl F, Rosselet A, Markert M, Hurni JM, and Perret C. Beneficial effects of L-canavanine, a selective inhibitor of inducible nitric oxide synthase, during rodent endotoxaemia. Clin Sci (Lond) 90: 369-377, 1996[ISI][Medline].

20.   Liaudet, L, Rosselet A, Schaller MD, Markert M, Perret C, and Feihl F. Nonselective versus selective inhibition of inducible nitric oxide synthase in experimental endotoxic shock. J Infect Dis 177: 127-132, 1998[ISI][Medline].

21.   Liu, P, Yin K, Yue G, and Wong PY. Role of nitric oxide in hepatic ischemia-reperfusion with endotoxemia. J Inflamm 46: 144-154, 1995[ISI][Medline].

22.   MacLeod, CL, Finley KD, and Kakuda DK. y+-Type cationic amino acid transport: expression and regulation of the mCAT genes. J Exp Biol 196: 109-121, 1994[Abstract].

23.   Mahmoud, YA, Harada K, Nagasaki A, Gotoh T, Takeya M, Salimuddin Ueda A, and Mori M. Expression of inducible nitric oxide synthase and enzymes of arginine metabolism in Fusarium kyushuense-exposed mouse lung. Nitric Oxide 3: 302-311, 1999[ISI][Medline].

24.   Messeri, D, Hammermann R, Mossner J, Gothert M, and Racke K. In rat alveolar macrophages lipopolysaccharides exert divergent effects on the transport of the cationic amino acids L-arginine and L-ornithine. Naunyn Schmiedebergs Arch Pharmacol 361: 621-628, 2000[ISI][Medline].

25.   Minnard, EA, Shou J, Naama H, Cech A, Gallagher H, and Daly JM. Inhibition of nitric oxide synthesis is detrimental during endotoxemia. Arch Surg 129: 142-147, 1994[Abstract].

26.   Nelin, LD, Krenz GS, Chicoine LG, Dawson CA, and Schapira RM. L-Arginine uptake and metabolism following in vivo silica exposure in rat lungs. Am J Respir Cell Mol Biol 26: 348-355, 2002[Abstract/Free Full Text].

27.   Nelin, LD, Nash HE, and Chicoine LG. Cytokine treatment increases arginine metabolism and uptake in bovine pulmonary arterial endothelial cells. Am J Physiol Lung Cell Mol Physiol 281: L1232-L1239, 2001[Abstract/Free Full Text].

28.   Nicholson, B, Manner CK, Kleeman J, and MacLeod CL. Sustained nitric oxide production in macrophages requires the arginine transporter CAT2. J Biol Chem 276: 15881-15885, 2001[Abstract/Free Full Text].

29.   Numata, M, Suzuki S, Miyazawa N, Miyashita A, Nagashima Y, Inoue S, Kaneko T, and Okubo T. Inhibition of inducible nitric oxide synthase prevents LPS-induced acute lung injury in dogs. J Immunol 160: 3031-3037, 1998[Abstract/Free Full Text].

30.   Nunokawa, Y, Ishida N, and Tanaka S. Cloning of inducible nitric oxide synthase in rat vascular smooth muscle cells. Biochem Biophys Res Commun 191: 89-94, 1993[ISI][Medline].

31.   Ohata, T, Sawa Y, Kadoba K, Kagisaki K, Suzuki K, and Matsuda H. Role of nitric oxide in a temperature-dependent regulation of systemic vascular resistance in cardiopulmonary bypass. Eur J Cardiothorac Surg 18: 342-347, 2000[Abstract/Free Full Text].

32.   Ou, J, Carlos TM, Watkins SC, Saavedra JE, Keefer LK, Kim YM, Harbrecht BG, and Billiar TR. Differential effects of nonselective nitric oxide synthase (NOS) and selective inducible NOS inhibition on hepatic necrosis, apoptosis, ICAM-1 expression, and neutrophil accumulation during endotoxemia. Nitric Oxide 1: 404-416, 1997[ISI][Medline].

33.   Rowin, ME, Xue V, and Irazuzta J. Hypothermia attenuates beta 1-integrin expression on extravasated neutrophils in an animal model of meningitis. Inflammation 25: 137-144, 2001[ISI][Medline].

34.   Sakamoto, KI, Fujisawa H, Koizumi H, Tsuchida E, Ito H, Sadamitsu D, and Maekawa T. Effects of mild hypothermia on nitric oxide synthesis following contusion trauma in the rat. J Neurotrauma 14: 349-353, 1997[ISI][Medline].

35.   Schmidt, M, Lugering N, Pauels HG, Schulze-Osthoff K, Domschke W, and Kucharzik T. IL-10 induces apoptosis in human monocytes involving the CD95 receptor/ligand pathway. Eur J Immunol 30: 1769-1777, 2000[ISI][Medline].

36.   Speziali, G, Russo P, Davis DA, and Wagerle LC. Hypothermia enhances contractility in cerebral arteries of newborn lambs. J Surg Res 57: 80-84, 1994[ISI][Medline].

37.   Stevens, BR, Kakuda DK, Yu K, Waters M, Vo CB, and Raizada MK. Induced nitric oxide synthesis is dependent on induced alternatively spliced CAT-2 encoding L-arginine transport in brain astrocytes. J Biol Chem 271: 24017-24022, 1996[Abstract/Free Full Text].

38.   Stewart, TE, Valenza F, Ribeiro SP, Wener AD, Volgyesi G, Mullen JB, and Slutsky AS. Increased nitric oxide in exhaled gas as an early marker of lung inflammation in a model of sepsis. Am J Respir Crit Care Med 151: 713-718, 1995[Abstract].

39.   Strunk, V, Hahnenkamp K, Schneuing M, Fischer LG, and Rich GF. Selective iNOS inhibition prevents hypotension in septic rats while preserving endothelium-dependent vasodilation. Anesth Analg 92: 681-687, 2001[Abstract/Free Full Text].

40.   Szabo, C, Southan GJ, and Thiemermann C. Beneficial effects and improved survival in rodent models of septic shock with S-methylisothiourea sulfate, a potent and selective inhibitor of inducible nitric oxide synthase. Proc Natl Acad Sci USA 91: 12472-12476, 1994[Abstract/Free Full Text].

41.   Tassiopoulos, AK, Carlin RE, Gao Y, Pedoto A, Finck CM, Landas SK, Tice DG, Marx W, Hakim TS, and McGraw DJ. Role of nitric oxide and tumor necrosis factor on lung injury caused by ischemia/reperfusion of the lower extremities. J Vasc Surg 26: 647-656, 1997[ISI][Medline].

42.   Tassiopoulos, AK, Hakim TS, Finck CM, Pedoto A, Hodell MG, Landas SK, and McGraw DJ. Neutrophil sequestration in the lung following acute aortic occlusion starts during ischaemia and can be attenuated by tumour necrosis factor and nitric oxide blockade. Eur J Vasc Endovasc Surg 16: 36-42, 1998[ISI][Medline].

43.   Vazquez-Jimenez, JF, Qing M, Hermanns B, Klosterhalfen B, Woltje M, Chakupurakal R, Schumacher K, Messmer BJ, von Bernuth G, and Seghaye MC. Moderate hypothermia during cardiopulmonary bypass reduces myocardial cell damage and myocardial cell death related to cardiac surgery. J Am Coll Cardiol 38: 1216-1223, 2001[ISI][Medline].

44.   Wagerle, LC, Russo P, Dahdah NS, Kapadia N, and Davis DA. Endothelial dysfunction in cerebral microcirculation during hypothermic cardiopulmonary bypass in newborn lambs. J Thorac Cardiovasc Surg 115: 1047-1054, 1998[Abstract/Free Full Text].

45.   Wang, D, Wei J, Hsu K, Jau J, Lieu MW, Chao TJ, and Chen HI. Effects of nitric oxide synthase inhibitors on systemic hypotension, cytokines and inducible nitric oxide synthase expression and lung injury following endotoxin administration in rats. J Biomed Sci 6: 28-35, 1999[ISI][Medline].

46.   Whalen, MJ, Carlos TM, Clark RS, Marion DW, DeKosky MS, Heineman S, Schiding JK, Memarzadeh F, Dixon CE, and Kochanek PM. The relationship between brain temperature and neutrophil accumulation after traumatic brain injury in rats. Acta Neurochir Suppl (Wien) 70: 260-261, 1997[Medline].

47.   Wizemann, TM, Gardner CR, Laskin JD, Quinones S, Durham SK, Goller NL, Ohnishi ST, and Laskin DL. Production of nitric oxide and peroxynitrite in the lung during acute endotoxemia. J Leukoc Biol 56: 759-768, 1994[Abstract].

48.   Wolfard, A, Kaszaki J, Szabo C, Balogh Z, Nagy S, and Boros M. Effects of selective nitric oxide synthase inhibition in hyperdynamic endotoxemia in dogs. Eur Surg Res 31: 314-323, 1999[ISI][Medline].

49.   Wolfard, A, Kaszaki J, Szabo C, and Boros M. The role of nitric oxide synthase, and of granulocytes, in endotoxin-induced early myocardial depression. Magy Seb 53: 247-252, 2000[Medline].

50.   Wu, G, Flynn NE, Flynn SP, Jolly CA, and Davis PK. Dietary protein or arginine deficiency impairs constitutive and inducible nitric oxide synthesis by young rats. J Nutr 129: 1347-1354, 1999[Abstract/Free Full Text].

51.   Yoshidome, H, Kato A, Edwards MJ, and Lentsch AB. Interleukin-10 inhibits pulmonary NF-kappa B activation and lung injury induced by hepatic ischemia-reperfusion. Am J Physiol Lung Cell Mol Physiol 277: L919-L923, 1999[Abstract/Free Full Text].

52.   Yui, Y, Hattori R, Kosuga K, Eizawa H, Hiki K, Ohkawa S, Ohnishi K, Terao S, and Kawai C. Calmodulin-independent nitric oxide synthase from rat polymorphonuclear neutrophils. J Biol Chem 266: 3369-3371, 1991[Abstract/Free Full Text].

53.   Zhang, Y, Wong KC, and Zhang Z. The effect of intraischemic mild hypothermia on focal cerebral ischemia/reperfusion injury. Acta Anaesthesiol Sin 39: 65-69, 2001[Medline].


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