1 Pulmonary Center at Boston University School of Medicine, Boston, Massachusetts 02118; 2 Department of Pediatrics, Emory University, Atlanta, Georgia 30322; and 3 Moran Eye Center, University of Utah, Salt Lake City, Utah 84108
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ABSTRACT |
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-Glutamyl transferase (GGT) is critical
to glutathione homeostasis by providing substrates for glutathione
synthesis. We hypothesized that loss of GGT would cause oxidant stress
in the lung. We compared the lungs of GGTenu1 mice, a
genetic model of GGT deficiency, with normal mice in normoxia to study
this hypothesis. We found GGT promoter 3 (P3) alone expressed in normal
lung but GGT P3 plus P1, an oxidant-inducible GGT promoter, in
GGTenu1 lung. Glutathione content was barely decreased in
GGTenu1 lung homogenate and elevated nearly twofold in
epithelial lining fluid, but the fraction of oxidized glutathione was
increased three- and fourfold, respectively. Glutathione content in
GGTenu1 alveolar macrophages was decreased nearly sixfold,
and the oxidized glutathione fraction was increased sevenfold.
Immunohistochemical studies showed glutathione deficiency together with
an intense signal for 3-nitrotyrosine in nonciliated bronchiolar
epithelial (Clara) cells and expression of heme oxygenase-1 in the
vasculature only in GGTenu1 lung. When GGTenu1
mice were exposed to hyperoxia, survival was decreased by 25% from
control because of accelerated formation of vascular pulmonary edema,
widespread oxidant stress in the epithelium, diffuse depletion of
glutathione, and severe bronchiolar cellular injury. These data
indicate a critical role for GGT in lung glutathione homeostasis and
antioxidant defense in normoxia and hyperoxia.
glutathione; heme oxygenase-1; 3-nitrotyrosine
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INTRODUCTION |
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EXTRACELLULAR
GLUTATHIONE metabolism is initiated by the key enzyme
-glutamyl transferase (GGT) and then completed by membrane dipeptidases. The released amino acids, particularly cysteine, serve as
essential substrates for intracellular glutathione and protein
synthesis. Genetic GGT deficiency in the GGTenu1 mouse
impairs glutathione metabolism and transport and results in systemic
glutathionemia, glutathionuria, and oxidant stress in the kidney
(15, 19). GGT expression is low in the lung compared with
other tissues such as the kidney, epididymis, and pancreas (1,
37). Hence, it is not certain that GGT deficiency would produce
an oxidant stress in this organ. The low level of lung GGT activity is
believed to contribute to an abundance of glutathione in the lung
epithelial lining fluid (ELF) (4, 8).
Our previous studies have shown that the GGT gene is expressed in
distal lung epithelium and that enzymatically active GGT protein is
present in lung surfactant (22). Exposure to an inhaled oxidant (NO2) induces GGT gene expression within the
epithelium and GGT protein accumulation within surfactant, implying an
active role for GGT-mediated glutathione metabolism at the epithelial surface of the lung (36). Thus we predict that a loss of
this activity will impair glutathione homeostasis at this surface and predispose to injury by inhaled oxidants. To study this, we previously characterized the point mutation that inactivated GGT expression in the GGTenu1 mouse and developed a strategy to breed and
genotype this model of GGT deficiency. We compare normal (wild-type,
+/+) with GGTenu1 (mutant, /
) mice for the presence of
oxidant stress in 21% O2 based on the pattern of GGT mRNA
expression, lung glutathione content, and redox ratio and reactivity
for 3-nitrotyrosine residues by immunocytochemistry and then test their
ability to survive in >95% O2. Our results establish an
important role for GGT in protecting the distal lung against oxidant
stress in normoxia and hyperoxia.
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METHODS |
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Breeding. The GGTenu1 mouse was bred and housed in the Laboratory and Animal Science Center at Boston University School of Medicine according to approved guidelines. Hemizygous pairs were mated, and offspring were genotyped at 4 wk of age using tail-derived DNA and direct sequencing of PCR products as described elsewhere (19). Animals were fed Purina mouse chow and allowed access to water ad libitum.
RT-PCR. Lung RNA was obtained from wild-type and mutant mice using Tri-reagent. The RT reaction and the primary and secondary PCRs were performed as described by Joyce-Brady et al. (22). Primers used to target rat GGT promoters P1, P2, and P3 were modified for mouse GGT P1, P2, and P3 on the basis of the published mouse GGT cDNA sequences (35). Products were separated on 1.5% agarose gels along with DNA standards, visualized after staining with ethidium bromide, and photographed under ultraviolet light. Kidney RNA obtained from normal mice served as a positive GGT mRNA control, since all three GGT mRNA subtypes are expressed in this organ (31). Results were compared with normal rat lung.
Glutathione assay.
The lung was perfused with PBS, lavaged with 0.5 ml of PBS, and frozen
in liquid nitrogen. Tissue (50-100 mg) was combined with 5%
perchloric acid by trituration for 30 s on a liquid nitrogen bath.
Insoluble material was pelleted by centrifugation at 5,000 g
for 5 min at 4°C. The number of cells obtained from lung lavage was
determined on a Coulter counter. An aliquot was used for cytospin preparations to determine cellular composition after Wright stain, and
the remaining cells were collected by centrifugation at 1,000 g for 10 min at 4°C and extracted in 5% perchloric acid.
The supernatant was combined with an equal volume of ice-cold 10%
perchloric acid, and insoluble material was pelleted by centrifugation.
All samples were frozen in liquid nitrogen, stored in aluminum foil at
70°C, and assayed within 1-3 days.
Immunohistochemistry for 3-nitrotyrosine.
Peroxynitrite production was assessed as a marker of oxidant stress in
lung tissue by immunoassay for 3-nitrotyrosine. Tissue sections were
deparaffinized, and endogenous peroxides were quenched with 3%
H2O2 in methanol. Some sections were incubated
with 1 mM exogenous peroxynitrite for 10 min to serve as a positive
control. All sections were blocked with 8% BSA for 30 min before
addition of a 4 µg/ml solution of the polyclonal rabbit
-nitrotyrosine antiserum (catalog no. 06-284, Upstate Biotechnology,
Lake Placid, NY) for 1 h at room temperature. Nonimmune serum was
used in this incubation step for the negative control. Competition
experiments were performed using 10 mM nitrotyrosine to ensure
specificity of the nitrotyrosine signal. Reactivity was detected using
a goat
-rabbit IgG antibody coupled to 3,3'-diaminobenzidine (DAB)
peroxidase (Vector Laboratories) with DAB as substrate.
Exposure to hyperoxia. Mice were exposed to an atmosphere of >95% O2-balance N2 (Wesco Gases, Billerica, MA) by enclosing their cages in an airtight glove bag. O2 concentration was continuously monitored by oximetry. Crystalit absorption medium (Pharmacal, Naugatuck, CT) was added to bedding at 10 g/ft2 of cage. Drierite (Vacumed, Ventura, CA) and Sodasorb (Intertech Resources, Lincolnshire, IL) were dispersed throughout the glove bags to absorb water and CO2. Animals were checked for viability every 6 h. Lung tissues from wild-type and mutant mice were fixed with paraformaldehyde, embedded in paraffin, sectioned, stained with hematoxylin and eosin, and examined as described previously (29).
In additional experiments, normal (n = 3) and GGTenu1 (n = 3) mice were exposed to hyperoxia for 96 h and then tissues were processed for histology, heme oxygenase-1 (HO-1) expression, and glutathione immunohistochemistry. HO-1 was examined in lung tissue by immunohistochemistry using a 1:25 dilution of a mouse monoclonal antibody (IgG2b) that was raised against the intact rat HO-1 protein (catalog no. OSA-111, StressGen Biotechnologies, Victoria, BC, Canada) as the primary antibody together with the mouse-on-mouse kit (MOM kit, Vector Laboratories) and a horseradish peroxidase-conjugated secondary antibody with DAB as substrate. Primary and secondary antibodies were omitted separately and together as negative controls. HO-1 protein was also assessed on a Western blot. Lung total proteins were extracted with 150 mM NaCl, 0.5% deoxycholate, 1% NP-40, 0.1% SDS, and 50 mM Tris (pH 7.6), separated on a 10% SDS-polyacrylamide gel along with protein standards, and electroblotted onto a nitrocellulose filter (Optitran, Schlicher & Schuell, Keene, NH). The filter was probed for HO-1 protein using a 1:1,000 dilution of the same mouse monoclonal antibody and detected with a 1:2,500 dilution of a horseradish peroxidase-conjugated goatImage processing. All histology preparations were visualized on a Leitz orthoplan microscope. Photographs were obtained using the Improvision Open-Lab Users Software program (Quincy, MA).
Statistics.
Values are means ± SE (n = 3-10).
Differences were compared using a t-test, and P 0.5 was considered significant.
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RESULTS |
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Mouse GGT mRNA subtypes in GGTenu1 lung.
Primer pairs directed against the common GGT coding domain or the
unique 5'-untranslated (UT) domain of GGT P3 generated a PCR signal in
the rat lung control, the mouse kidney control, and the lungs of
wild-type and mutant GGTenu1 mice (Fig.
1). Those directed at the 5'-UT domain of
GGT P2 generated a product only in the mouse kidney control. However,
primers for the 5'-UT domain of GGT P1 generated product in the mouse
kidney control and the GGTenu1 mouse lung, whereas no
product was evident in the normal mouse lung or the rat lung. This
pattern of GGT P1 mRNA expression in GGTenu1 mouse lung
matches the pattern we previously described in the oxidant-exposed rat
lung (36) and suggests that the GGTenu1 mouse
lung is sensing an oxidant stress even in normoxia.
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Lung glutathione content.
To further explore the nature of this stress, we measured total
glutathione content in plasma, lung, lung lavage, and lung bronchoalveolar lavage (BAL) cells in GGTenu1 mice and
compared these values with values in normal control mice (Table
1). Total glutathione levels in the
plasma were elevated fourfold in GGTenu1 mice, in agreement
with the previous report in the literature (15). However,
total glutathione content of the GGTenu1 lung was only
minimally reduced from that of the normal controls, and the total
glutathione content of the lung ELF was actually elevated nearly
twofold in GGTenu1 mice. Although these two results do not
suggest oxidant stress, the fraction of GSSG was increased more than
threefold in the GGTenu1 lung and fourfold in the
GGTenu1 ELF.
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Nitrotyrosine immunoreactivity.
Because the glutathione immunohistochemical signal could not
distinguish between the relative content of reduced and oxidized glutathione, we probed for 3-nitrotyrosine as described in
METHODS to determine whether glutathione deficiency in the
GGTenu1 bronchioles was associated with cellular oxidant
stress. These modified tyrosine residues result from the interaction of
tyrosine with peroxynitrite and are stable products (18).
A 3-nitrotyrosine signal was evident in some bronchiolar cells of
normal lung, but it was weak and sparse. In contrast, this signal was
more intense and diffuse in the bronchiolar epithelium of
GGTenu1 lung (Fig. 3). A
nitrotyrosine signal was also evident in glutathione-depleted alveolar
macrophages from the GGTenu1 lung (data not shown). The
nitrotyrosine signal was inhibited by coincubation of primary antibody
with 10 mM nitrotyrosine to ensure specificity of the signal. The
decreased glutathione content in concert with the increased
nitrotyrosine signal indicates the presence of increased oxidant stress
in nonciliated bronchiolar epithelial cells in the GGTenu1
lung.
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Survival in normobaric hyperoxia.
Because the alterations in the pattern of lung GGT mRNA expression,
lung glutathione homeostasis, and 3-nitrotyrosine immunocytochemistry suggested the presence of oxidant stress even in normoxia, we hypothesized that GGTenu1 mice would be more sensitive than
their normal controls to an inhaled oxidant stress. To determine this,
we exposed them to an atmosphere of >95% O2 and
measured survival as described in METHODS. At the
outset, no major differences in the lung parenchyma were apparent
between the two groups on the basis of histological criteria.
However, survival of GGTenu1 mice was reduced ~25%
compared with their normal controls (Fig. 4).
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DISCUSSION |
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GGT is the key enzyme that initiates the metabolism and turnover of glutathione. The metabolic function of this cell surface glycoprotein is believed to protect the plasma membrane against oxidant stress and to enable the transfer of glutathione between cells and tissues. GGT is most abundant in epithelial cells and their secretions, inasmuch as these cells line surfaces that are directly exposed to environmental oxidants. We have demonstrated in the lung that GGT is expressed highly in Clara cells of the bronchioles and, to a lesser degree, in type II cells in the alveolus. We have also shown that GGT activity accumulates in surfactant secretions of the normal lung. The lung responds to oxidant gas exposure by inducing the GGT gene in epithelial cells and accumulating enzyme activity in surfactant secretions. This leads to an acceleration of glutathione turnover. Although controversy has surrounded the role of GGT in the lung, our studies have led us to propose that GGT-mediated glutathione metabolism serves a critical antioxidant function at the epithelial surface of the distal lung (22, 36).
To study this further, we initially characterized the point mutation that inactivated the GGT gene in the GGTenu1 mouse and presented evidence that the GGTenu1 kidney was under oxidant stress in normoxia (15, 19). We have compared the lungs of normal and GGTenu1 mice in normoxia, and we demonstrate oxidant stress at critical sites where GGT expression is lost. These findings correlate well with our previous results on the localization of lung GGT expression. The GGTenu1 lung exhibits a deficiency of glutathione in the bronchiolar Clara cell and a disruption of glutathione homeostasis in the ELF that lead to glutathione deficiency in the alveolar macrophage. We also found that the vascular endothelium is under oxidant stress in normoxia as HO-1 protein is induced in these cells. This stress in the vasculature may originate from the loss of GGT expression on the endothelial cell surface, inasmuch as these cells are known to express GGT at a very low level, or from the plasma, inasmuch as a pool of GGT circulates in the blood (30). The consequences of oxidant stress in the GGTenu1 lung become manifest when the mutant mice are exposed to hyperoxia. The formation of pulmonary edema and hemorrhage are accelerated as a result of vascular injury. Glutathione deficiency and oxidant stress extend widely throughout the epithelial surface. Epithelial cell injury and disruption, which predominate in the bronchioles, lead to air space edema and hemorrhage. Hence, survival of the GGTenu1 mice in hyperoxia is decreased compared with the normal control mice. The degree of O2 sensitivity that we observed in the GGTenu1 lung is similar to that which occurs with inactivation of the extracellular superoxide dismutase gene, the enzyme activity of which clears superoxide radicals from the extracellular environment (5).
Despite the presence of oxidant stress in the GGTenu1 lung, there is only a minimal decrease of total glutathione in this organ. This contrasts with the GGTm1/GGTm1 mouse, a model of GGT deficiency produced by targeted mutagenesis of the GGT gene. The GGTm1/GGTm1 mouse model differs from the GGTenu1 mouse as follows: 1) there is no detectable GGT activity in GGTm1/GGTm1 mice, 2) the degree of glutathionuria is more profound, and 3) there is an absolute deficiency of plasma cysteine. Because cysteine supply is critical for glutathione synthesis, a deficiency of glutathione is evident in many GGTm1/GGTm1 organs, including the lung (24, 34). GGTm1/GGTm1 mice are reportedly sensitive to even 80% O2, usually a sublethal level of O2, indicating that the lung glutathione deficiency is probably severe and generalized. Exogenous cysteine replacement restores glutathione content in the lung as a whole and eliminates the sensitivity to this level of hyperoxia (3).
The GGTenu1 mouse model of GGT deficiency was produced by inducing random mutations in the mouse genome with ethylnitrosourea (enu) and then selecting progeny for the presence of aminoaciduria, which was glutathionuria in the GGTenu1 mouse. GGT gene expression was inactivated; yet a small residuum of GGT activity persisted in the kidney (15). As a result, the degree of glutathionuria was less profound, and plasma cysteine levels were not decreased. Hence, the nearly normal lung glutathione content in normoxia likely reflects the adequacy of the plasma cysteine supply. However, the more than threefold rise in GSSG content in the GGTenu1 lung indicates oxidant stress. This stress likely resided within a subset of lung cells where glutathione homeostasis is dependent on GGT.
The activity of GGT P1 as well as P3 in the GGTenu1 mouse lung was our initial evidence for oxidant stress in normoxia. In our previous studies in the rat lung, we showed that GGT P3, but not P2 or P1, is active in normal lung. However, GGT P1 is induced after exposure to an inhaled oxidant gas (36). GGT P1 is also developmentally regulated in the perinatal rat lung, a time when oxidant stress is also believed to occur (29). Because the genomic organization of the GGT gene is highly conserved between the rat and the mouse (7), GGT P3, but not P2 or P1, activity in the normal mouse lung was the expected result and confirms that described in the literature (13). The activity of GGT P1 solely in the GGTenu1 lung suggests that the GGT-deficient lung is under oxidant stress. GGT P1 is the most proximal promoter in the rat and the mouse GGT gene, and its activation in response to oxidant stress appears to be a common response in both species. We suspect that GGT P1 is active in the bronchiolar Clara cell, since we have shown that this cell is the major site of GGT gene expression in the distal lung (29, 36). Current work is focused on confirming this hypothesis and determining the oxidant-associated mechanism of GGT P1 activation.
The loss of GGT gene expression in the lung correlates specifically with a cellular deficiency of glutathione in the subset of nonciliated bronchiolar epithelium (Clara cells). These cells are located in small airways that are prone to injury from inhaled particulates and oxidants, and glutathione export and GGT-mediated recycling may be important to protect their plasma membrane against oxidative damage (28). GGT deficiency could predispose to glutathione depletion in these cells in two ways. Uncoupling glutathione export from metabolism could cause glutathione depletion, inasmuch as cellular reuptake of intact glutathione is inefficient (12). Alternatively, Clara cells are also sites of active xenobiotic metabolism, and this function can deplete glutathione stores if utilization exceeds supply. Loss of GGT activity could impair the transfer of glutathione from the ELF into the Clara cell by limiting the supply of cysteine and compromising intracellular glutathione synthesis. In either case, we are examining these cells in the GGTenu1 lung to understand the full impact of GGT deficiency on Clara cell biology and function.
That these glutathione-deficient cells are sensing oxidant stress is strongly supported by their intense signal for 3-nitrotyrosine. Even sporadic Clara cells in the normal lung exhibit a signal for 3-nitrotyrosine. This suggests that glutathione stores are very dynamic in this cell. The consequence of this Clara cell glutathione depletion and oxidant stress for the GGTenu1 lung became manifest on exposure of the these mice to hyperoxia, whereupon these cells became injured and disrupted more rapidly than the normal Clara cells. This result is intriguing, given that these cells are naturally deficient in GGT at the time of birth, inasmuch as Clara cell GGT ontogeny occurs during the late postnatal period (29). We are now examining the response of the normal and GGTenu1 lung to different O2 environments at birth. Finally, the immunologic signal for glutathione in the population of other cells of the bronchiole, the ciliated epithelial cell, is the most intense that has been observed with this methodology. There is not a dramatic difference in signal intensity between ciliated cells in the normal and the GGTenu1 lung. We do not know the mechanism by which the GGTenu1 ciliated bronchiolar cells are able to maintain their glutathione content under normoxia. In hyperoxia, however, glutathione depletion still occurs more rapidly in these cells than in their normal controls.
The alveolar epithelial type II cell also expresses GGT mRNA and protein, although the level of expression is much lower than that of the Clara cell. The type II cell also has abundant glutathione (10) and appears to be the major cellular source of the glutathione that is exported into the lung ELF (17, 26). Despite this export function, our assays for glutathione and 3-nitrotyrosine did not reveal evidence of glutathione depletion or oxidant stress in the GGT-deficient type II cell. The reason for this is not clear. Experiments by others have suggested a correlation between type II cell glutathione export and the extracellular redox state, so that a more oxidized extracellular environment forces the cell to retain glutathione (38). The exact mechanism for this response is unknown, but the presence of oxidant stress in the ELF could have blocked glutathione export from the type II cell and prevented severe glutathione depletion. Alternatively, type II cell glutathione content may have been preserved by the ability of this cell to transport intact glutathione from the ELF via an Na+-dependent transport process (14).
We showed previously that the type II cell releases GGT activity along with its surfactant secretions (22). Given that the GGT specific activity in this surfactant-associated pool is about sevenfold higher than that of the whole lung, we suspected that GGT deficiency would have a greater impact on the glutathione pool within the ELF. The lung ELF is unique in its glutathione abundance, and this extracellular pool functions in antioxidant protection for the entire gas exchange surface (4). Glutathione concentration in the ELF is clearly elevated in the GGTenu1 lung, and this increase would correlate with the absence of glutathione metabolism and clearance by surfactant-associated GGT. Decreased clearance from the blood is believed to be the mechanism of glutathionemia as well (15, 24). However, there was a disproportionate rise in ELF GSSG content in the GGTenu1 lung, suggesting that GSSG clearance may have been affected to a greater degree than glutathione clearance. The mechanism by which GSSG is cleared from the alveolar space is unknown, but as a result of its accumulation, the redox state of ELF in the mutant mouse was shifted toward a more oxidized state, even in normoxia. It is known that when the normal lung is chronically exposed to an inhaled oxidant gas, GSSG levels in the ELF rise initially and then return to baseline (23). This suggests the existence of an inducible mechanism that can increase lung GSSG clearance. Studies of GGT activity in the lining fluid of the epididymis suggest that the mechanism is the preferential metabolism of GSSG by GGT protein itself and that this protein serves to regulate the redox state in testicular lining fluid (16). We believe that a similar process may occur in the lung ELF. Although the expanded ELF glutathione pool could have protected the distal lung from oxidant stress in hyperoxia, we did not observe this. Rather, there was a more pronounced depletion of glutathione and induction of HO-1 protein throughout the epithelial surface. Glutathione depletion could have resulted from a dilution by edema fluid or from increased permeability due to the loss of epithelial barrier function with injury in hyperoxia.
The alveolar macrophage is a second subset of lung cells that becomes
depleted of glutathione in the GGTenu1 lung, despite the
expansion of the ELF glutathione pool. Hence, glutathione turnover in
the ELF appears to be necessary for glutathione homeostasis within this
immune cell. In a previous study, we showed that lung alveolar
macrophages do not express GGT mRNA; yet it is known that these cells
depend on GGT activity to maintain glutathione homeostasis
(11). We proposed that this enzyme activity was derived
from lung surfactant, and our results support this hypothesis (22). The level of macrophage glutathione depletion that
we observed in the GGTenu1 lung in normoxia is severe, and
the high content of GSSG indicates intense glutathione utilization. We
do not know why there is an increase in the number of alveolar
macrophages in the GGTenu1 lung or how this severe redox
imbalance affects macrophage function. However, preliminary experiments
with expression of interleukin-1, interleukin-converting enzyme, and
inducible nitric oxide synthase in GGTenu1 macrophages
suggest that these cells are not in an activated state, despite the
presence of oxidant stress (20). The inability of these
immune cells to function optimally could have a negative impact on host defense.
A large body of literature supports the fundamental role of the glutathione system in maintaining normal lung function and protecting against oxidant-induced injury (30). The lung utilizes a large amount of glutathione. In the mouse, it utilizes more glutathione than any other organ (26). Our studies in the GGTenu1 mouse show that GGT deficiency has a dramatic impact on glutathione homeostasis and the redox state of the lung. This supports our hypothesis that GGT gene expression serves a critical role in protecting the lung against oxidant stress in normoxia and hyperoxia. The goal of future studies will be to gain a more complete understanding of the function of GGT in the lung and the role of GGT in regulating oxidant stress in specific lung cells. In this regard, we recently showed that GGT and one of its protein isoforms, which are derived from alternative splicing of GGT mRNA, can mediate an endoplasmic reticulum stress response (21). Therefore, in addition to affecting glutathione recycling and the cellular redox state, GGT may serve additional functions directly within the endoplasmic reticulum. Hence, GGT may have a number of important cellular homeostatic functions. The GGTenu1 mouse provides a model from which to gain new insight into the biological significance of GGT gene expression for cells of the normal and diseased lung.
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ACKNOWLEDGEMENTS |
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The authors thank Dr. M. C. Williams for advice regarding the studies on HO-1.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Program Project Grant PO1 HL-47049 to M. Joyce-Brady.
Address for reprint requests and other correspondence: M. Joyce-Brady, The Pulmonary Center, 715 Albany St., R304, Boston, MA 02118 (E-mail: mjbrady{at}lung.bumc.bu.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.
May 3, 2002;10.1152/ajplung.00250.2000
Received 27 July 2000; accepted in final form 30 April 2002.
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