Regulation of lung manganese superoxide dismutase: species variation in response to lipopolysaccharide

Ghenima Dirami1, Donald Massaro1, and Linda Biadasz Clerch2

Lung Biology Laboratory, Departments of 1 Medicine and 2 Pediatrics, Georgetown University School of Medicine, Washington, District of Columbia 20007


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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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Lipopolysaccharide (LPS) treatment increases survival of rats, but not of mice, during hyperoxia. Manganese superoxide dismutase (Mn SOD) in the lung plays a critical role in LPS-induced tolerance to hyperoxia in rats. Therefore, we now compared the response of lung Mn SOD with treatment of mice and rats with LPS. LPS treatment of rats increased Mn SOD activity and protein concentration, did not change its specific activity, increased Mn SOD mRNA concentration 35-fold, and elevated Mn SOD synthesis 50% without changing general protein synthesis. LPS treatment of mice did not alter any of these parameters except for a 16-fold increase in Mn SOD mRNA concentration. Mn SOD translational efficiency (synthesis/mRNA concentration) was diminished 93% in rat lung and 76% in mouse lung by treatment with LPS. However, the absolute translational efficiency was twofold higher in lungs of LPS-treated rats than in lungs of LPS-treated mice. The failure of LPS to raise Mn SOD activity in mouse lungs is due, at least in part, to a smaller increase in Mn SOD mRNA and lower translational efficiency in LPS-treated mice than in LPS-treated rats.

endotoxin; protein synthesis; translational efficiency; rat; mouse


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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EXPOSURE OF ORGANISMS to O2 at pressures higher than normally experienced causes damage to molecular components of cells, resulting in cellular dysfunction. Death occurs in animals and humans if the O2 tension is sufficiently high and the exposure is prolonged (3, 18). The damaging effects of O2 are believed to be mediated in part by superoxide and H2O2, moieties the cellular production of which increases during hyperoxia (1, 2, 10). Among the cellular defenses against superoxide and H2O2 are four major antioxidant enzymes: copper-zinc superoxide dismutase (Cu/Zn SOD), manganese (Mn) SOD, catalase, and glutathione peroxidase (11). The SODs each catalyze the conversion of superoxide to H2O2. Catalase and glutathione peroxidase each convert H2O2 to water (11).

At present, there are no widely effective pharmacological means of protecting against the harmful effects of hyperoxia. However, treatment of rats with lipopolysaccharide (LPS) from gram-negative organisms results in almost 100% survival with little lung damage during a 72-h exposure to >95% O2; diluent-treated rats exhibit <40% survival (8, 9). The survival of LPS-treated O2-exposed rats is associated with an increased activity in the lung of the four major antioxidant enzymes (4, 8, 9). In contrast, treatment of mice with LPS fails to induce an increase in antioxidant enzyme activity in the lung during exposure to hyperoxia and fails to confer tolerance to mice (8). Treatment of sheep with LPS is also ineffective in preventing lung damage during hyperoxia (15).

The ability of LPS treatment to confer almost complete tolerance to >95% O2 in rats (8, 9) and its inability to confer tolerance to similarly treated mice (8) provide useful and important animal models to explore the induction of tolerance to O2 by LPS. We have now used these models to study the ability of LPS treatment to increase expression of Mn SOD in lungs of rats and mice. We elected to examine Mn SOD because, among the antioxidant enzymes, it appears most important for LPS-induced tolerance to hyperoxia (4). Treatment with LPS resulted in a substantially diminished translational efficiency in Mn SOD synthesis in both species. In rats, but not in mice, there were increases in synthesis, concentration, and activity of Mn SOD associated with a 35-fold increase in Mn SOD mRNA in rat lungs compared with a 15-fold increase in Mn SOD mRNA in mouse lungs. The basis for the loss of translational efficiency is not known.


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Animals and treatments. Specific pathogen-free Sprague-Dawley rats were purchased from Taconic Farms. CD-1 mice were purchased from Charles River. All animals were maintained in the animal care facility at Georgetown University (Washington, DC) Medical Center on a 12:12-h light-dark cycle and were allowed food (rodent laboratory chow 5001, Ralston-Purina, St. Louis, MO) and water ad libitum. All animal care procedures were done according to the National Research Council's Guide for the Care and Use of Laboratory Animals and were approved by the Animal Care and Use Committee of Georgetown University.

LPS-treated animals were injected intraperitoneally with 500 µg/kg of LPS from Salmonella typhimurium (Sigma) in 0.15 M NaCl (diluent). Control animals were given an equal volume of 0.15 M NaCl. At various times after treatment, the animals were anesthetized with an intraperitoneal injection of ketamine (~100 mg/kg) and xylazine (~12 mg/kg) and killed by exsanguination.

Measurement of Mn SOD activity, concentration, and protein synthesis. To estimate the activity of Mn SOD, we disrupted the lung tissue with a Polytron homogenizer with a 10-mm-diameter generator (Brinkmann Instruments, Westburg, NY) operated at its highest speed. We centrifuged the homogenate at 27,000 g for 45 min at 4°C and dialyzed the supernatant fraction overnight at 0-4°C against three changes of 40 volumes of 5 mM potassium phosphate buffer with 0.1 mM EDTA. The xanthine oxidase-cytochrome c method was used for all SOD assays (7), and sodium cyanide (0.015 mM) was used to inhibit cytochrome oxidase. To measure Mn SOD, we used diethyldithiocarbamate in a procedure that inhibits Cu/Zn SOD activity (13). One unit of SOD activity was the amount that halves the rate of reduction of cytochrome c.

We quantitated Mn SOD protein by Western analysis as previously described (5). The quantity of Mn SOD protein was determined by densitometric analysis of the immunoreactive band and is expressed in densitometric units. Pure Mn SOD protein was also assayed to ensure that we were working in a range within which density was proportional to the amount of Mn SOD. Densitometry was performed on a Molecular Dynamics laser densitometer with ImageQuant software (Sunnyvale, CA). Specific activity of Mn SOD was calculated by dividing Mn SOD activity by the concentration of Mn SOD protein and is expressed in units per densitometric units.

To measure the rate of synthesis of Mn SOD protein and trichloroacetic acid-precipitable proteins (general proteins), 1.0-mm-thick lung slices were incubated in 10 ml of Krebs-Ringer bicarbonate buffer containing 5.5 mM glucose, adult rat plasma concentrations of 19 amino acids (14), and 0.7 mM L-[3H]phenylalanine. At this concentration of phenylalanine, the specific activity of tRNA-bound phenylalanine equals that of phenylalanine in the medium within 15 min of the start of the incubation. This allows the use of medium phenylalanine specific activity in the calculation of absolute rates of protein synthesis (6, 12). At the end of the incubation, the rate of incorporation of phenylalanine into general proteins and Mn SOD was measured as previously described (5).

Measurement of Mn SOD mRNA. Mn SOD mRNA was analyzed by Northern blot assay. Lung total RNA was isolated with TRI Reagent according to the protocol outlined by the manufacturer (Molecular Research Center, Cincinnati, OH). RNA was quantitated by absorbance at 260 nm and with an extinction coefficient of 0.025 (µg/ml)-1 · cm-1. To perform Northern analysis, we fractionated the RNA by electrophoresis in a 1.5% agarose-0.66 M formaldehyde gel and transferred it to Nytran Plus nylon membranes (Schleicher & Schuell, Keene, NH). RNA ladders of 0.24-9.5 kb of RNA and 0.16-1.77 kb of RNA (GIBCO BRL) were used as size markers. The RNA was covalently cross-linked to the membranes by ultraviolet irradiation with a Stratalinker (Stratagene). To detect Mn SOD RNA, the membranes were hybridized with a double-stranded radiolabeled Mn SOD cDNA probe generated with the random-primer DNA labeling system (GIBCO BRL), [alpha -32P]dCTP, and a 533-bp EcoR I-Hind III fragment of the rat liver Mn SOD cDNA obtained from Dr. Ye Shih Ho (Wayne State University, Detroit, MI). Hybridization and washes were done with standard procedures (16). For quantitation, the autoradiographs were scanned by laser densitometry (Molecular Dynamics) with ImageQuant software. Mn SOD RNA data are expressed as relative densitometric units of the 1.0-kb and 1.4-kb Mn SOD RNA transcripts per unit of 18S rRNA (Ambion) used as an internal standard.

Statistical analysis. The values for individual animals were averaged per experimental group, and the SEs of the means were calculated. The significance of difference between two groups was obtained with the Mann-Whitney test (17). The Kruskal-Wallis test was used when more than two groups were compared, and the Mann-Whitney test was then used to compare two populations at a time. The Bonferroni adjustment was used to adjust the significance level to the number of tests performed (17).


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Treatment with LPS did not alter Mn SOD activity, specific activity, or concentration in mouse lungs (Fig. 1, Table 1). In contrast, treatment with LPS resulted in an ~2-fold increase in Mn SOD activity in rat lungs (Fig. 1). The increase in Mn SOD activity in rat lungs was due to an increased concentration of the protein without a change in the specific activity of Mn SOD (Table 1).


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Fig. 1.   Manganese superoxide dismutase (Mn SOD) activity in mouse and rat lungs. Animals were injected intraperitoneally with lipopolysaccharide (LPS; 500 µg/kg body wt) or an equal volume of diluent (DIL). Animals were killed 24 h later, and their lungs were assayed for Mn SOD activity. Data are means ± SE; nos. in parentheses, no. of animals. * P < 0.005 compared with DIL-treated rats.


                              
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Table 1.   LPS treatment increases Mn SOD concentration in rat but not in mouse lung and does not alter Mn SOD specific activity in rat or mouse lung

Although treatment with LPS did not alter Mn SOD activity or concentration in mouse lungs, it resulted in an ~15-fold increase in the mouse lung concentration of Mn SOD mRNA (Table 2). In rats, where LPS treatment resulted in a 1.6-fold increase in the concentration of Mn SOD protein (Table 1), the same treatment caused a 35-fold rise in the lung concentration of Mn SOD mRNA (Table 2). Thus LPS induced an increased concentration of Mn SOD mRNA in both species, and in both, the increase in the mRNA was vastly greater than the change (rat) or lack of change (mouse) in the concentration of Mn SOD protein.

                              
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Table 2.   LPS treatment causes a greater increase in Mn SOD mRNA in rat lung than in mouse lung

To determine whether the disparity in both species between the induction of an increased concentration of Mn SOD mRNA and the concentration of the protein was due, at least in part, to impaired translation, we measured the absolute rate of Mn SOD synthesis (12) by lung slices from diluent- and LPS-treated mice and rats. To determine whether any effect of LPS treatment on Mn SOD synthesis might have some specificity, we also measured its effect on general protein synthesis. LPS treatment of mice did not alter the synthesis rate of Mn SOD protein or of general proteins (Fig. 2). Treatment of rats with LPS resulted in an ~2-fold increase in the rate of Mn SOD synthesis. This marked increased rate of Mn SOD synthesis by the lung was not part of an overall elevation of lung protein synthesis (Fig. 3).


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Fig. 2.   Synthesis of general proteins and Mn SOD protein in LPS- and DIL-treated mice. Mice were injected intraperitoneally with LPS (500 µg/kg body wt) or an equal volume of DIL. Animals were killed 6 h later, and rates of synthesis of general proteins (nmol Phe incorporated · mg DNA-1 · h-1) and Mn SOD (pmol Phe incorporated · mg DNA-1 · h-1) by lung slices were measured. Data are means ± SE; nos. in parentheses, no. of mice. P > 0.05 between DIL- and LPS-treated mice for both general protein and Mn SOD synthesis.



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Fig. 3.   Synthesis of general proteins and Mn SOD protein by rat lungs. Rats were injected intraperitoneally with LPS (500 µg/kg body wt) or an equal volume of DIL. Animals were killed 6 h later, and rates of synthesis of general proteins (nmol Phe incorporated · mg DNA-1 · h-1) and Mn SOD (pmol Phe incorporated · mg DNA-1 · h-1) by lung slices were measured. Data are means ± SE; nos. in parentheses, no. of rats. * P = 0.01 compared with DIL-treated rats.

Measurement of the rate of Mn SOD protein synthesis and the concentration of Mn SOD mRNA allows calculation of translational efficiency (Mn SOD protein synthesis rate/Mn SOD mRNA concentration). Treatment with LPS resulted in a 76% decline in translational efficiency in mouse lungs and a 93% decrease in rat lungs (Fig. 4). However, on an absolute basis, translational efficiency for Mn SOD was twofold higher in lungs from LPS-treated rats compared with lungs from LPS-treated mice (13.4 ± 4.7 and 5.8 ± 1.3, respectively; P = 0.008). Thus it appears that in rats and mice, treatment with LPS results in a marked loss of translational efficiency for the synthesis of Mn SOD. The failure of LPS to raise Mn SOD activity in mouse lungs compared with rat lungs is associated with a smaller increase in Mn SOD mRNA in response to LPS and to an overall lower translational efficiency in LPS-treated mice than in LPS-treated rats. We believe these observations are important because if the molecular basis for the low translational efficiency of Mn SOD in lungs of LPS-treated rats and mice can be explained and the low translational efficiency corrected, the possibility of using LPS to protect against hyperoxia-induced lung damage in humans can be considered.


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Fig. 4.   Translational efficiency (Mn SOD protein synthesis rate/Mn SOD mRNA concentration) for synthesis of Mn SOD by mouse and rat lungs. Animals were injected with LPS (500 µg/kg body wt) or an equal volume of DIL. Animals were killed 6 h later, and measurements of Mn SOD synthesis and concentration of Mn SOD mRNA were made on lungs. Data are means ± SE; nos. in parentheses, no. of animals. * P = 0.006 compared with DIL-treated mice. ** P = 0.0002 compared with DIL-treated rats.


    ACKNOWLEDGEMENTS

We thank Alla Berkovich for expert technical assistance.


    FOOTNOTES

This work was supported in part by National Heart, Lung, and Blood Institute Grants HL-20366 and HL-47413 and a Career Investigator Award from the American Lung Association (to L. B. Clerch).

D. Massaro is Cohen Professor of Pulmonary Research at Georgetown University (Washington, DC) and a Senior Fellow of the Lovelace Respiratory Research Institute (Albuquerque, NM).

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 and other correspondence: L. B. Clerch, Lung Biology Laboratory, Georgetown Univ. Medical Center, Preclinical Science Bldg., GM12, 3900 Reservoir Rd. NW, Washington, DC 20007 (E-mail: CLERCHLB{at}gusun.georgetown.edu).

Received 2 September 1998; accepted in final form 22 January 1999.


    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

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Am J Physiol Lung Cell Mol Physiol 276(5):L705-L708
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