Glutamine synthetase gene expression in the lungs of endotoxin-treated and adrenalectomized rats

Gregory Lukaszewicz, Steve F. Abcouwer, Brian I. Labow, and Wiley W. Souba

Department of Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

During sepsis, the lung responds by exporting increased amounts of the amino acid glutamine. This response is accompanied by increased enzymatic activity of glutamine synthetase (GS), which catalyzes the synthesis of glutamine from glutamate and ammonia. It is also known that GS expression in the rat lung can be induced by glucocorticoid hormones. To determine whether the septic response and the response to glucocorticoids are related, we have characterized the induction of GS expression during lipopolysaccharide (LPS)-induced endotoxemia in normal, neutropenic, and adrenalectomized rats. Normal rats exhibited a time- and dose-dependent induction of GS mRNA levels after a single intraperitoneal dose of LPS. Responses to LPS were maximal at doses of 0.1 mg/kg body wt and above. A single 10 mg/kg body wt dose of LPS led to a rapid, transient sevenfold increase in GS mRNA (P <=  0.1) and a twofold increase in GS protein level 8 h postinjection. Induction of lung GS mRNA 4 h after LPS injection was approximately fivefold in neutropenic (P <=  0.1) and fourfold in nonneutropenic control rats (P <=  0.1), suggesting that infiltrating neutrophils or neutrophil-derived factors are not required for GS induction. In response to high-dose, short-term endotoxemia, adrenalectomized rat lung GS mRNA increased twofold (P <=  0.02) compared with sixfold in sham-operated control rats (P <=  0.02). However, in response to low-dose, long-term endotoxemia, adrenalectomized rat lung GS mRNA increased threefold (P <=  0.02) compared with fourfold in sham-operated control rats (P <=  0.02). Adrenalectomy did not affect the elevation of lung GS mRNA levels in response to dexamethasone. In addition, GS mRNA was induced four- and sixfold in rat microvascular pulmonary endothelial cells exposed to plasma from control and septic rats, respectively. The addition of a glucocorticoid antagonist, RU-38486, completely blocked GS gene induction in the presence of control plasma but only attenuated the response to plasma from septic animals by 30%. These results suggest that GS gene induction during sepsis is only partially mediated by adrenal-derived glucocorticoid hormones.

glutamine; pulmonary; sepsis

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

CATABOLIC ILLNESSES are characterized by a number of alterations in protein and amino acid metabolism, including increased muscle proteolysis, negative nitrogen balance, and increased amino acid flux from the periphery to the intestine, liver, and kidney. Glutamine, the most abundant amino acid in the body, plays a critical role in this interorgan exchange. Although skeletal muscle has been considered the principal organ of glutamine synthesis and export, regional flux studies have demonstrated that glutamine release by the lungs contributes significantly to glutamine homeostasis (24). Other studies (6-8) in rats have demonstrated that glutamine release by the lungs increases in response to endotoxemia, sepsis, injury, and glucocorticoid treatment. In addition, septic surgical patients exhibit a similar increase in lung glutamine release (18). Although the total lung mass is small compared with skeletal muscle mass, the lungs do contain the necessary enzyme, glutamine synthetase (GS; EC 6.3.1.2), for de novo glutamine synthesis (3, 4, 7).

Previous studies with a rat model have shown that glucocorticoids increase glutamine efflux by the lung (22), increase GS enzyme activity (6), and increase GS mRNA expression (1, 21). We have also examined GS gene regulation in response to a host of inflammatory mediators using two rat lung cell types, an epithelial cell line (L2 cells) and a microvascular endothelial cell (MPEC) line. These studies demonstrated that, of the various mediators examined, including glucocorticoids, interleukin (IL)-1, -2, and -6, tumor necrosis factor (TNF), C5a, and lipopolysaccharide (LPS), only dexamethasone caused a marked induction in GS mRNA levels (3, 4). Given the central role of glucocorticoids in the septic response, as well as their stimulatory effect on GS mRNA expression in the lung, we investigated whether increased GS mRNA expression in the rat lung during endotoxemia and in response to glucocorticoid hormones were related.

The relationship among endotoxemia, glucocorticoids, and GS gene induction in lung tissue was investigated by measuring GS mRNA levels in whole lungs from normal, neutropenic, and adrenalectomized rats subjected to intraperitoneal injection of Escherichia coli LPS. In addition, RU-38486 was used to block the induction of GS expression in rat lung endothelial cells treated with plasma derived from control and endotoxemic rats. The results obtained show that endotoxemia causes a marked increase in rat lung GS mRNA expression, which is largely, but not entirely, mediated through glucocorticoids.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Reagents. Chemicals were purchased from Sigma Chemical (St. Louis, MO) and Fisher Scientific (Pittsburgh, PA). [32P]dCTP was purchased from Amersham (Arlington Heights, IL). RU-38486 was obtained from Roussel-UCLAF (Romaineville, France).

Animal studies. Male Sprague-Dawley rats weighing between 150 and 200 g were obtained from Charles River Laboratories (Wilmington, MA). Adrenalectomies and sham operations were performed by Charles River Laboratories, and all adrenalectomized and sham-operated rats were received 5-7 days after surgery. All rats were allowed to acclimate for 3-4 days on a 12:12-h light-dark cycle. Normal rats (those that had no surgical procedure) were fed standard rat chow and water ad libitum, whereas adrenalectomized and sham-operated animals received a 0.85% saline solution and standard rat chow ad libitum.

In all experiments, tissue was harvested after the rats were anesthetized with ketamine (100 mg/kg ip) and acepromazine (1.25 mg/kg ip). When adequate anesthesia had been obtained, a midline sternotomy and laparotomy were performed, and the incision was extended to expose the left hindquarter; both lungs as well as the skeletal muscle from the left hindquarter were removed. All tissue samples were immediately frozen in liquid nitrogen and stored at -80°C until they were processed. Plasma samples were obtained by open cardiac puncture with a heparinized syringe and centrifuging whole blood samples at 423 g for 10 min at 4°C. All plasma corticosterone levels were measured by Ani Lytics (Gaithersburg, MD). Experimental procedures were performed in accordance with the National Institutes of Health guidelines on the use of experimental animals and were approved by the Massachusetts General Hospital Subcommittee on Research Animal Care.

Lung GS mRNA induction after LPS injection. Normal rats were injected intraperitoneally with E. coli LPS serotype O127:B8 [10 mg/kg body wt diluted in phosphate-buffered saline (PBS); Sigma Chemical]. Control rats received intraperitoneal injections of PBS alone. Animals injected with LPS or PBS carrier were killed between 1 and 48 h after the intraperitoneal injection, and lung tissue was harvested as described in Animal studies. Lung tissue harvested from rats immediately before injection was used for the t = 0 time points in both the LPS- and PBS-treated groups.

Lung GS mRNA induction vs. LPS concentration. Normal rats received intraperitoneal injections of E. coli LPS serotype O127:B8 diluted in PBS at doses ranging between 0 and 10 mg/kg body wt. Four hours after the intraperitoneal injection, all animals were killed, and the lung tissue was harvested as described in Animal studies.

Effect of neutropenia on lung GS mRNA induction after LPS injection. Normal rats were made neutropenic by a single intraperitoneal injection of cyclophosphamide (100 mg/kg body wt; n = 6) 5 days before experimentation. Control rats received intraperitoneal injections of PBS alone (n = 6). At the time of experimentation, one-half of the neutropenic and control rats received either a single LPS injection of 10 mg/kg body wt or PBS carrier intraperitoneally. All animals were killed 4 h after injection, and the lung tissue was harvested as described in Animal studies.

Effect of dexamethasone on adrenalectomized rat lung GS mRNA. Adrenalectomized and sham-operated rats were obtained and acclimatized as described previously. At the time of experimentation, adrenalectomized (n = 6) and sham-operated animals (n = 6) received intraperitoneal injections with either 0.5 mg/kg body wt of dexamethasone (Dex) or an equal volume of absolute ethanol (EtOH) carrier. The animals were killed, and the lung tissue was harvested 4 h after injection.

Effect of high- and low-dose LPS on adrenalectomized rat lung GS mRNA. Adrenalectomized and sham-operated rats were injected with either 10 (high-dose) or 0.5 mg/kg body wt (low-dose) LPS or PBS carrier. All rats in the "high-dose" group (n = 9) and their PBS-treated counterparts (n = 8) were killed 3 h postinjection. All rats in the "low-dose" group (n = 9) and their PBS controls (n = 8) were killed 5.5 h postinjection. All lung tissue was harvested and stored as described in Animal studies.

Effect of septic rat plasma on MPEC GS mRNA expression. A rat MPEC line (19) was obtained from Dr. Una S. Ryan (T-Cell Sciences, Boston, MA). MPECs were maintained on Dulbecco's modified Eagle's medium (DMEM; GIBCO Life Technologies, Grand Island, NY) supplemented with 10% heat-inactivated fetal bovine serum (FBS; GIBCO), 4 mmol/l of glutamine, 50 µg/ml of heparin (Calbiochem, La Jolla, CA), and 50 µg/ml of gentamicin. Cells were passed two times weekly with trypsin and seeded in a 1:8 dilution. Before experimentation, MPECs were seeded onto sterile 10-cm tissue culture plates (Falcon, Bedford, MA) at a 1:8 dilution and grown to confluence. One day before experimentation, all cells were washed and fed with DMEM supplemented with 0.1% heat-inactivated FBS, 4 mmol/l of glutamine, 50 µg/ml of heparin, and 50 µg/ml of gentamicin. Eighteen hours later, MPECs were refed with DMEM containing 25% rat plasma from from one of three rat groups and either 10 µM RU-38486 or EtOH carrier. All plasma samples were obtained from normal rats 4 h postinjection with either LPS (10 mg/kg body wt) or PBS (10 ml/kg body wt) or from noninjected rats via cardiac puncture (as described in Animal studies). MPECs refed with standard DMEM (containing 10% FBS) containing 1 µM Dex or EtOH carrier and 10 µM RU-38486 or carrier were used as control cells. RNA was harvested for Northern blot analysis (as described in Northern blotting) 4 h after refeeding.

Northern blotting. Northern blotting was performed as previously described in detail (1). For RNA isolation from skeletal muscle, 0.1 g of frozen tissue was rapidly thawed and homogenized in 1 ml of Trisolve with an Omnimixer homogenizer (Omni International, Warrenton, VA). Total RNA was isolated by the one-step acid-phenol guanidinium procedure (10) with Trisolve reagent (Biotex, Woodlands, TX) according to the manufacturer's protocol followed by an additional acid-phenol, phenol-chloroform-isoamyl alcohol, and chloroform extraction and EtOH precipitation in the presence of sodium acetate. Total RNA (~10 µg each) was fractionated by electrophoresis through denaturing agarose gels containing 0.2 M formaldehyde, stained with 10 µg/ml of ethidium bromide in 50 mM NaOH-10 mM NaCl, destained in 200 mM sodium acetate, pH 5.2, ultraviolet transilluminated, and photographed. The RNA was transferred to a nylon membrane (Micron Separations, Westboro, MA) by capillary action and cross-linked to the membrane with a Fisher Biotech ultraviolet cross-linker. An 800-base pair segment of rat GS cDNA containing primarily a coding sequence (a gift from Dr. John Mill and Dr. Steven Max, National Institutes of Health, Bethesda, MD) was used as a template to generate a [32P]dCTP-labeled probe with a random-prime labeling kit (Megaprime, Amersham) according to the manufacturer's protocol. Hybridization with radiolabeled probe was performed overnight at 65°C as described previously (20). After the nylon membrane was washed with a high-stringency buffer [0.1 × 15 mM NaCl-1 mM NaH2PO4-0.1 mM EDTA buffer and 1% sodium dodecyl sulfate (vol/vol) in distilled water treated with a ribonuclease inhibitor] at 65°C, autoradiographic detection of the hybridized probe was performed by exposing it to Fuji XAR film for 12-24 h at -80°C. Quantitation of autoradiograph bands was accomplished with a laser densitometer (Molecular Dynamics, Sunnyvale CA). Membranes were stripped of GS cDNA probe by boiling in 0.1% sodium dodecyl sulfate and were rehybridized with a random-primed cDNA probe for a constitutively expressed mRNA. Although beta -actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were present in both lung and MPEC extracts, the levels of beta -actin were relatively low in MPEC extracts. Therefore, rat GAPDH was used as an internal control for all tissue culture experiments.

Statistical analysis. Given the sample populations within each segment of this study (n = 3 or 4 rats), a normal distribution could not be assumed. Therefore, the two-tailed Mann-Whitney U-test, a nonparametric method of statistical analysis, was utilized to estimate the significance of differences between groups.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

GS gene induction in response to endotoxemia was first examined in normal rat lung to define the temporal and dose-dependent nature of this response. To evaluate the temporal response to a single dose of LPS, normal rats were treated with a single intraperitoneal injection of LPS (10 mg/kg body wt) or PBS carrier control. Within 3-4 h after LPS injection, animals exhibited characteristic signs of sepsis, including piloerection, diarrhea, hyperventilation, and lethargy. The animals were killed at various time points after injection, and the lung tissue was removed and analyzed by Northern blot analysis as described in Northern blotting. A typical Northern blot and graph from one such series of animals are shown in Fig. 1. A rat GS cDNA probe detected 2.8- and 1.4-kb transcripts in both control and experimental animals in all experiments (GS mRNA was taken as the sum of both transcripts). Laser densitometry of these autoradiographs and normalization of GS mRNA signals to beta -actin reveals that rats injected with a single dose of LPS exhibited a threefold increase in lung GS mRNA by 3 h, a maximal sevenfold induction at 8 h, and a return to basal levels by 24 h. Control animals receiving PBS injections did not demonstrate an appreciable change in GS mRNA levels.


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Fig. 1.   Effect of a single intraperitoneal injection of Escherichia coli lipopolysaccharide (LPS) on lung glutamine synthetase (GS) mRNA levels. Normal adult male Sprague-Dawley rats received a single 10 mg/kg body wt dose of LPS or phosphate-buffered saline (PBS) carrier and were killed at time 0 and at indicated times after injection. Total cellular RNA was purified from whole lung and analyzed as outlined in MATERIALS AND METHODS. A: Northern blot of lung RNA probed with GS and beta -actin cDNA probes from a single series of rats (similar results were obtained in 3 additional series of rats). rRNA, ribosomal RNA. B: relative GS mRNA induction in whole rat lung. Relative GS mRNA levels were obtained by densitometric analysis of GS and beta -actin signals, GS mRNA signals were normalized to beta -actin, and normalized values are presented as a ratio relative to control (time 0) levels.

To determine the effect of LPS dosage on GS gene induction in the rat lung, normal rats were treated with a single intraperitoneal dose of LPS ranging from 0.05 to 10 mg/kg body wt. Four hours after the injection, the lung tissue was excised, and Northern blot analysis was performed as described in Northern blotting. A Northern blot and graph from one such series of rats are shown in Fig. 2. GS mRNA levels in the normal rat lung were increased approximately twofold by a dose of LPS as low as 0.05 mg/kg body wt and approximately four- to fivefold by LPS dosages of 0.1-10 mg/kg body wt. Therefore, GS expression was significantly induced by relatively low doses of LPS.


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Fig. 2.   Effect of LPS dose on GS mRNA induction in whole rat lung. Adult male Sprague-Dawley rats received a single intraperitoneal injection of E. coli LPS ranging in dose from 0 to 10 mg/kg body wt (BW) and were killed 4 h after injection. Whole lung RNA was purified and analyzed as outlined in MATERIALS AND METHODS. A: Northern blot of lung RNA probed with GS and beta -actin probes from a single series of rats (similar results were obtained in 3 additional series of rats). B: relative GS mRNA induction in whole rat lung. Relative GS mRNA levels were obtained by densitometric analysis of GS and beta -actin signals, GS mRNA signals were normalized to beta -actin, and normalized values are presented as a ratio relative to control (time 0) levels.

Intraperitoneal LPS injection is accompanied by a rapid and marked pulmonary neutrophil infiltrate (25). It is conceivable that these infiltrating cells influence GS mRNA induction observed in the whole lung after LPS injection either by contributing their own GS mRNA content to the total or by releasing mediators that stimulate GS expression by lung cells. To evaluate the contribution of infiltrating neutrophils on GS mRNA induction in the lung, the effects of LPS injection in normal and neutropenic rats were examined. Neutropenia was induced with intraperitoneal injections of cyclophosphamide 5 days before the time of experimentation when leukocyte and neutrophil counts would be at a nadir (9). Control rats were injected with PBS carrier without cyclophosphamide. Total leukocyte counts were decreased by an average of 80% (neutrophils were ~1% of the total leukocyte count) in cyclophosphamide-treated compared with control rats. Neutropenic and respective control rats were given a single injection of LPS (10 mg/kg body wt) or PBS and were killed 4 h later, and lung GS mRNA levels were measured. Neutropenia did not attenuate, but rather seemed to augment, lung GS mRNA expression in response to endotoxemia (Fig. 3). Northern blot analysis demonstrated induction of lung GS mRNA by LPS to be approximately fivefold in neutropenic rats (P <=  0.1) and fourfold in nonneutropenic control rats (P <=  0.1). Although part of this apparent accentuation seemed to be due to reduced beta -actin mRNA content in the lungs of LPS-treated neutropenic rats, this result suggests that the elevation in whole lung GS mRNA content in LPS-treated rats was not dependent on infiltrating leukocytes.


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Fig. 3.   Effect of neutropenia (Neutro) on GS mRNA induction after LPS injection. Adult male Sprague-Dawley rats received a single intraperitoneal injection of 100 mg/kg body wt of cyclophosphamide or PBS carrier. Five days after cyclophosphamide injection, rats were injected with a single intraperitoneal dose of LPS (10 mg/kg body wt) and were killed 4 h after injection. Lung RNA was purified and analyzed as outlined in MATERIALS AND METHODS. A: Northern blot of lung RNA probed with GS and beta -actin probes. B: relative GS mRNA induction in whole rat lung. Relative GS mRNA levels were obtained by densitometric analysis of GS and beta -actin signals, GS mRNA signals were normalized to beta -actin, and normalized values are presented as means ± SD of a ratio relative to control (PBS) levels.

It is known that an LPS injection increases glucocorticoid levels in rats (11, 23). Furthermore, previous studies have demonstrated that Dex itself can induce GS expression in both rat lung and lung-derived cells. To determine whether adrenalectomy affects the capacity of the lung to respond to glucocorticoids or whether other adrenal gland-derived hormones (e.g., aldosterone, sex steroids, epinephrine, norepinephrine) are necessary for GS gene induction, adrenalectomized and sham-operated rats were given a single intraperitoneal dose of Dex (0.5 mg/kg body wt) and killed 4 h later. Northern blot analysis of lung tissue taken from these animals demonstrated an eightfold induction in GS mRNA levels in both adrenalectomized (P <=  0.1) and sham-operated animals (P <=  0.1) after Dex injection (Fig. 4). Furthermore, adrenalectomy had no significant effect on baseline levels of GS mRNA compared with those in sham-operated control rats.


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Fig. 4.   Effect of adrenalectomy on GS mRNA induction after dexamethasone (Dex) administration. Adult male Sprague-Dawley rats that had undergone previous adrenalectomy (ADX) or a sham operation (Sham) received a single 0.5 mg/kg body wt dose of Dex or ethanol (EtOH) carrier control and were killed 6 h after injection. Whole rat lung was purified and analyzed as outlined in MATERIALS AND METHODS. A: Northern blot of lung RNA probed with GS and beta -actin probes. B: relative GS mRNA induction in sham-operated and adrenalectomized rat lung. Relative GS mRNA levels were obtained by densitometric analysis of GS and beta -actin signals, GS mRNA signals were normalized to beta -actin, and normalized values are presented as means ± SD of a ratio relative to mean control level (Sham/EtOH).

The effect of LPS injection on adrenalectomized rats was studied to evaluate the importance of adrenal hormones on GS gene induction during sepsis. Initially, the same dose of LPS (10 mg/kg body wt) was used in adrenalectomized and sham-operated rats as had been used in previous experiments with normal rats. However, the adrenalectomized animals rapidly developed signs of severe distress and were therefore killed 3 h after LPS injection. Northern blot analysis revealed that lung GS mRNA was induced twofold by LPS in adrenalectomized animals (P <=  0.02) compared with sixfold in the sham-operated group (P <=  0.02; Fig. 5).


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Fig. 5.   Effect of ADX on GS mRNA induction after a single high dose of E. coli LPS. Adult male Sprague-Dawley rats that had undergone previous ADX or Sham received a single 10 mg/kg body wt dose of LPS or PBS carrier and were killed 3 h after injection. Whole rat lung was purified and analyzed as outlined in MATERIALS AND METHODS. A: Northern blot of lung RNA probed with GS and beta -actin probes. B: relative GS mRNA induction in lungs of Sham and ADX rats. Relative GS mRNA levels were obtained by densitometric analysis of GS and beta -actin signals, GS mRNA signals were normalized to beta -actin, and normalized values are presented as means ± SD of a ratio relative to mean control value (Sham/PBS).

In a second experiment with a lower dose of LPS (0.5 mg/kg body wt), adrenalectomized rats were maintained for 5.5 h after injection. At this later time point, lung GS mRNA was elevated threefold in adrenalectomized rats (P <=  0.02) and fourfold in sham-operated animals (P <=  0.02) after LPS administration (Fig. 6). Therefore, although adrenalectomy markedly reduced lung GS expression 3 h after a high dose of endotoxin, this effect was less pronounced when a relatively low dose of LPS was administered and lung GS mRNA was quantified at a later time point (i.e., 5.5 h).


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Fig. 6.   Effect of ADX on GS mRNA induction after a single low dose of E. coli LPS. Adult male Sprague-Dawley rats that had undergone previous ADX or Sham received a single 0.5 mg/kg body wt dose of LPS or PBS carrier and were killed 5.5 h after injection. Whole rat lung was purified and analyzed as outlined in MATERIALS AND METHODS. A: Northern blot of lung RNA probed with GS and beta -actin probes. B: relative GS mRNA induction in Sham and ADX rat lungs. Relative GS mRNA levels were obtained by densitometric analysis of GS and beta -actin signals, GS mRNA signals were normalized to beta -actin, and normalized values are presented as means ± SD of a ratio relative to mean control value (Sham/PBS).

Although adrenalectomized rats were readily distinguishable by a heightened response to LPS administration and drinking behavior, adrenalectomy was confirmed by analysis of plasma corticosterone levels. Corticosterone, rather than cortisol, is the primary glucocorticoid hormone present in rat plasma. Plasma samples from LPS- and PBS-injected sham-operated and adrenalectomized animals were obtained as described in Animal studies and pooled according to treatment group. Plasma corticosterone levels from PBS- and LPS-injected (0.5 mg/kg body wt) sham-operated animals were 242 ± 4 and 330 ± 4 ng/ml, respectively. However, in PBS- and LPS-injected (0.5 mg/kg body wt) adrenalectomized rats, corticosterone levels were 6.1 ± 0.2 and 5.6 ± 0.8 ng/ml, respectively (normal reference range for plasma corticosterone in adult male Sprague-Dawley rats is 10-230 ng/ml). Therefore, GS induction in adrenalectomized animals is able to occur in the near absence of corticosterone.

To determine whether plasma from septic rats contained a factor other than glucocorticoid hormones that could induce GS mRNA expression, rat MPECs in culture were exposed to medium containing septic rat plasma with and without the glucocorticoid-receptor antagonist RU-38486. Plasma was isolated from normal rats that had received no treatment and from rats that were injected intraperitoneally with either PBS (10 ml/kg body wt) or LPS (10 mg/kg body wt). MPECs were grown to confluence, serum starved for 18 h, and then exposed for 4 h to medium containing 1 µM Dex (as a positive control) or 25% plasma from untreated, PBS-injected, or LPS-treated rats in the presence and absence of 10 µM RU-38486. Northern blotting was used to compare GS mRNA levels in these cells. An example of the GS mRNA induction seen under these conditions is represented in the Northen blot and graph in Fig. 7. Dex added directly to the tissue culture medium caused a 25-fold induction in relative GS mRNA levels, which was completely blocked by 10 µM RU-38486. Plasma from untreated and PBS-treated rats caused a fourfold induction in GS mRNA levels, whereas plasma from LPS-treated rats caused a sixfold induction. GS mRNA levels were not appreciably induced in MPECs by plasma from untreated and PBS-treated animals when RU-38486 was present. However, relative GS mRNA levels in MPECs were still elevated nearly threefold by plasma from septic rats in the presence of RU-38486, suggesting that other, nonadrenal mediators are able to induce GS expression as well. Similar results were obtained with a rat lung cell line of epithelial origin and with plasma isolated from rats at other times after LPS injection (data not shown).


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Fig. 7.   Effect of sera from untreated (No Inj), control (PBS), and septic (LPS) rats on GS gene induction in microvascular pulmonary endothelial cells (MPECs). Sera were obtained from normal and LPS-treated rats as described in MATERIALS AND METHODS and added to confluent MPECs in presence or absence of 10 µM RU-38486 (RU). MPECs exposed to 1 µM Dex or EtOH each with and without 10 µM RU were used as control cells. A: Northern blot of MPEC RNA probed with GS and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe. B: relative GS mRNA levels after normalization to GAPDH mRNA and after normalized values were set relative to EtOH-treated (without RU) control value.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

GS, the principle enzyme of glutamine synthesis, is elevated in the rat lung in a number of catabolic disease states including endotoxemia (2, 5, 7, 8). The physiological response to endotoxemia is complex and involves a wide array of hormonal and metabolic alterations including cytokine, prostaglandin, and glucocorticoid release. Glucocorticoid hormones alone have been shown to increase glutamine efflux from rat lung and induce GS expression in lung tissue (1, 6, 21, 22). In addition, our laboratory (3, 4) has previously shown that glucocorticoids cause GS gene induction in two rat lung cell lines via a direct, glucocorticoid receptor-mediated mechanism, whereas other mediators of the septic response such as IL-1, -2, and -6, TNF, and C5a had little or no effect on GS gene expression. These results led to the hypothesis that glucocorticoid hormones play a vital role in GS induction in the septic lung. Therefore, adrenalectomized rats and an LPS injection model of sepsis were used to test this hypothesis.

The time course of induction and the dose-dependent relationship between endotoxin challenge and GS mRNA level in the lungs of normal rats were examined. Endotoxin caused a rapid, transient induction in lung GS mRNA levels. After a single dose of LPS (10 mg/kg body wt), GS mRNA levels were elevated within 3 h and had a maximal sevenfold induction compared with control levels by 8 h. GS mRNA levels returned to their baseline values by 24 h after LPS injection. In addition, GS mRNA expression in normal rat lung responded to LPS injection in a dose-dependent fashion. When animals were killed 4 h after injection with LPS, GS mRNA levels were augmented twofold in response to a dose as low as 0.05 mg/kg body wt, with a maximal fourfold induction observed at doses of 0.1 mg/kg body wt or greater. It can be concluded from these experiments that relatively low levels of endotoxin induce lung GS mRNA within hours of exposure. This brisk and substantial upregulation suggests that GS and, by extension, glutamine play an important role during endotoxemia either locally in the lung or systemically.

In contrast to GS mRNA levels, lung GS protein levels in rats injected with LPS (10 mg/kg body wt) underwent a maximal twofold induction and returned to basal levels by 24 h postinjection (data not shown). This increase in GS protein is comparable to the elevation in lung GS activity observed in other sepsis models reported in the literature (5, 6, 8, 21). In addition, although one in vivo study showed that Dex injection caused a sevenfold increase in GS mRNA in lungs, GS protein levels increased by only twofold. Although the significance of this disparity between GS mRNA induction and GS protein expression is not known, one possible explanation comes from in vitro data from hepatoma and astrocyte cultures that show that GS activity is inversely related to ambient glutamine concentration (12, 13, 15, 16). Similarly, although lung GS mRNA levels increase rapidly in response to endotoxemia, lung GS protein levels may only rise when glutamine levels fall. The brief period of endotoxemia analyzed in this study may be insufficient to significantly affect glutamine levels. Indeed, other work in our laboratory has shown that GS protein levels do rise in type II pneumocyte cultures when the medium is depleted of glutamine (Labow, Abcouwer, and Souba, unpublished data).

Endotoxemia is associated with a marked neutrophil influx into the lung (25). It is possible that these infiltrating cells contribute to GS mRNA levels in the whole lung either as expressors of GS themselves or as inducers of GS expression by lung cells. To eliminate the confounding effect of neutrophil influx into the lung, we used a well-proven method of inducing neutropenia utilizing a single intraperitoneal dose of cyclophosphamide ~5 days before experimentation (9). At the nadir of their leukocyte counts, animals received a single 10 mg/kg body wt intraperitoneal injection of LPS and were killed 4 h after injection. GS mRNA induction was actually slightly greater in neutropenic rats compared with nonneutropenic animals. Therefore, the increase in lung GS mRNA levels observed does not depend on infiltrating neutrophils or other leukocyte-derived factors.

Adrenalectomized and sham-operated rats were exposed to endotoxin to determine the role that endogenous glucocorticoids play in lung GS expression during sepsis. Adrenalectomized rats exhibited the same level of induction in lung GS in response to Dex injection as sham-operated control rats, suggesting that no other adrenal-derived factors other than glucocorticoid hormones are needed for this response. When adrenalectomized rats were injected with 10 mg/kg body wt of LPS, they quickly developed signs of severe distress such as hyperventilation, increased lethargy, and piloerection and had to be killed only 3 h after injection. This exaggerated response to endotoxemia has been reported previously and may be partly due to the extreme cytokine levels that result when glucocorticoids are absent (17). In response to this high-dose, short-term endotoxemia, adrenalectomized rat lung GS mRNA increased twofold compared with sixfold in sham-operated control rats. Corticosterone was virtually absent in both adrenalectomized groups, confirming successful adrenalectomy and demonstrating that GS expression can be induced in the absence of this hormone. This result suggests that adrenal-derived factors play a significant, but not exclusive, role in regulating lung GS mRNA induction during sepsis. However, when a lower dose of endotoxin was used (0.1 mg/kg body wt of LPS) so that GS induction could be studied at a later time point (5.5 h), the difference in lung GS expression between adrenalectomized and sham-operated rats was less pronounced. In this experiment, adrenalectomized rat lung GS mRNA increased threefold compared with fourfold in sham-operated control rats. Unlike the results obtained with high-dose LPS, these results suggest that adrenal-derived factors play a limited role in GS induction in the septic rat lung.

How can one account for this differential response between low-dose and high-dose LPS challenge? It is conceivable that adrenalectomized rats have a slower response to endotoxin challenge compared with sham-operated and normal control groups. In adrenalectomized animals, extra-adrenal steroid release might occur, albeit at a slower rate or in a lesser amount. Thus, when lung GS mRNA levels are measured at 3 h, the difference between adrenalectomized and sham-operated rats is more pronounced than at 5.5 h. However, plasma analysis in this study, along with other published reports, does not support this hypothesis. In fact, corticosterone levels are low and do not rise in response to LPS in adrenalectomized rats (11). Although corticosterone is the major glucocorticoid hormone in rats, these data do not exclude other glucocorticoid hormones. Alternatively, other nonglucocorticoid mediators may also stimulate lung GS mRNA expression. If such mediators do exist and if, like cytokines, their levels are suppressed in the presence of glucocorticoids, this could also account for the apparent discrepancy between GS mRNA levels at 5.5 and 3 h in adrenalectomized and sham-operated animals.

Because of the complex interactions that occur in vivo in response to endotoxemia, we examined the role of glucocorticoids in lung GS expression using an in vitro model as well. A rat MPEC line was exposed to medium containing plasma from normal rats 4 h after injection with endotoxin (10 mg/kg body wt of LPS), from rats injected with PBS, or from untreated rats. GS mRNA levels were induced sixfold in cells treated with plasma from LPS-injected rats and fourfold in cells treated with plasma from PBS-injected rats or untreated rats. When the cells were exposed to medium containing 1 µM Dex, there was a 25-fold increase in GS mRNA. We also used the glucocorticoid-receptor antagonist RU-38486 to block the induction of GS mRNA in cells treated with plasma from LPS- or PBS-injected rats and in Dex-treated cells. Whereas RU-38486 completely blocked induction of GS mRNA by Dex or plasma from PBS-injected rats, the glucocorticoid-receptor antagonist still allowed a threefold induction by plasma from LPS-treated rats. This result also suggests that glucocorticoids play a significant role but are not entirely responsible for GS upregulation during sepsis.

In summary, glucocorticoids remain the only mediators released during sepsis that have a demonstrated ability to upregulate GS expression in the lung and in lung cells. Indeed, we found that adrenalectomy attenuates the expression of lung GS in response to endotoxemia. However, lungs of adrenalectomized animals do respond to endotoxemia by augmenting GS expression. In addition, plasma from septic animals can induce GS expression in lung cells in the presence of an effective dose of a glucocorticoid-receptor antagonist. These results suggest that a mediator(s) other than a glucocorticoid is capable of enhancing GS expression in the septic lung.

    ACKNOWLEDGEMENTS

We acknowledge the fine technical assistance of Raymond J. Lustig.

    FOOTNOTES

This work was funded by National Heart, Lung, and Blood Institute Grant HL-44986 (to W. W. Souba).

Address for reprint requests: W. W. Souba, Massachusetts General Hospital, Cox Bldg., Rm. 626, 100 Blossom St., Boston, MA 02114.

Received 3 March 1997; accepted in final form 1 September 1997.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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AJP Lung Cell Mol Physiol 273(6):L1182-L1190
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