Laboratory of Pulmonary Pathobiology, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709
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ABSTRACT |
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Glutathione
plays an essential role in protecting the pulmonary system from toxic
insults. -Glutamyl transpeptidase-related enzyme (GGT-rel) is a
novel protein capable of cleaving the
-glutamyl peptide bond of
glutathione and of converting leukotriene
C4 to leukotriene
D4. A rat homologue of GGT-rel was
identified and was found to be highly expressed in cultures of
differentiating rat tracheal epithelial (RTE) cells. The 2.6-kb cDNA
predicts a 572-amino acid protein with 79% identity to
human GGT-rel. GGT-rel was weakly expressed in normal trachea but was
strongly induced by epidermal growth factor in cultures of RTE cells.
GGT-rel was also highly expressed in lung tumors induced by inhalation
of isobutyl nitrite. These results demonstrate that GGT-rel
1) is expressed in normal tracheal
cells, 2) can be induced by
epidermal growth factor, and 3) is
elevated after chemical exposure. The induction of high levels of
GGT-rel may play an important role in protecting the lung from
oxidative stress or other toxic insults.
glutathione; oxidative stress; pulmonary epithelium
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INTRODUCTION |
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GLUTATHIONE (-glutamylcysteinylglycine; GSH), the
most abundant intracellular nonprotein thiol, is the major reductant of cellular thiol groups and participates in many important biological processes, including the
-glutamyl cycle and the conjugation of
carcinogens and toxins. The membrane-bound enzyme
-glutamyl transpeptidase (GGT) catalyzes the first step in the extracellular breakdown of GSH into its constituent amino acids, which can then be
transported into the cell and used to maintain the level of intracellular GSH (24). GGT is abundant in tissues with a secretory or
absorptive function, such as kidney and pancreas (6). Mice deficient in
GGT appear normal at birth but grow slowly and suffer premature death,
demonstrating the importance of GGT to normal homeostasis (17).
Increased GGT activity is also widely used as a marker of preneoplastic
lesions in the liver during chemical carcinogenesis (12).
In addition to GGT, a second enzyme capable of cleaving GSH has been described by Heisterkamp et al. (13) and Morris et al. (19). This enzyme, referred to as GGT-related enzyme (GGT-rel), was cloned from a human placental cDNA library. GGT-rel, when transfected into NIH/3T3 cells, stimulated the degradation of GSH and the conversion of leukotriene C4 into leukotriene D4. However, GGT-rel expression was not demonstrated in any mouse tissue examined, including embryo and placenta, and its biological role is unknown.
The role of GSH and the enzymes involved in GSH metabolism have been
the focus of several recent studies of pulmonary cells. For example,
exogenous GSH has been shown to protect cultured bovine pulmonary
artery endothelial cells from menadione toxicity (3), and oxidative
stress increases -glutamylcysteine synthetase and GSH (23). In
alveolar epithelial cells, oxidative stress increases GGT transcription
and activity (15), and increased levels of GGT and
-glutamylcysteine
synthetase have been shown to provide increased resistance to oxidative
stress (18). In addition, increased GSH levels may be part of an
adaptive response to long-term ozone exposure (8). Glutathione
S-transferases (GSTs), which catalyze
the addition of the thiol group of GSH to many xenobiotics, may also
protect the airway from toxic compounds. A lack of the GST-M1 isoenzyme
has been associated with a higher risk of lung cancer (7). Thus GSH and
the enzymes responsible for its synthesis and metabolism are of
significant importance to the protective mechanisms of the airways in
response to a variety of chemical insults.
In our laboratory, we have been investigating the regulation of mucociliary differentiation of rat tracheal epithelial (RTE) cells using a model system in which primary RTE cells undergo many of the processes that occur during injury and repair (14). We have previously shown that the pathway of differentiation can be altered by changing the concentrations of growth factors and hormones used to culture RTE cells. For example, epidermal growth factor (EGF) stimulates mucous cell differentiation and mucus production (11), whereas the removal of EGF increases ciliated cell differentiation (5).
In this work, differential display was used to identify genes for which
expression was altered during differentiation of RTE cells under
specific conditions. We have identified and cloned the rat homologue of
GGT-rel and have demonstrated that its expression can be strongly
upregulated by EGF. Additionally, GGT-rel expression was observed to be
elevated in lung adenomas and carcinomas from rats chronically exposed
to isobutyl nitrite. The increased levels of GGT-rel expression suggest
that GGT-rel plays an important role in the metabolism of -glutamyl
compounds in the airways after injury. Induction of GGT-rel expression
may be a protective mechanism in the airways after exposure to toxic
insults.
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MATERIALS AND METHODS |
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Materials. Oligonucleotides were obtained from Genosys (The Woodlands, TX) or Research Genetics (Huntsville, AL). All radioactive materials were obtained from Amersham (Arlington Heights, IL) except 35Sequetide, which was obtained from NEN (Boston, MA). All amplifications were performed in a GeneAmp polymerase chain reaction (PCR) system 2400 or 9600 (Perkin-Elmer, Branchburg, NJ). All tissue culture media and reagents were obtained from Sigma Chemical (St. Louis, MO) with the exception of EGF and rat tail collagen, which were purchased from Collaborative Biomedical Products (Bedford, MA), and bovine pituitaries, which were purchased from Pel-Freeze (Rogers, AR).
Cell culture.
Primary RTE cells were cultured using the method originally described
by Kaartinen et al. (14) with modifications described in detail
previously (22). Briefly, tracheas were obtained from 10- to 14-wk-old
male Fischer 344 rats, filled with 1% Pronase, and incubated overnight
at 4°C. Primary RTE cells were plated at a density of 2.4 × 104
cells/cm2 on collagen-coated
Transwell clear membranes (Costar, Cambridge, MA; see Ref. 2). For all
experiments reported here, the cells were submerged in growth
factor-supplemented Dulbecco's modified Eagle's medium + F-12 medium
[complete medium (CM)] until they reached confluency
(day
8). On
day
8, parallel cultures were divided into
the different treatment groups as described for the individual experiments. Cultures were refed daily beginning on
day
5. Previous studies have demonstrated
that, under these conditions, RTE cells grow predominantly as a
pseudostratified mucociliary epithelium with no obvious fibroblast
contamination (5, 11, 14). Recent studies from our laboratory have
shown that submersion of RTE cells in CM (SUB cultures) inhibits
ciliogenesis (21), whereas withdrawal of EGF and cholera toxin (CT)
from the media (EGF/
CT) promotes ciliated cell
differentiation (5). Cells were harvested for RNA at different time
points, according to the experimental design.
RNA isolation. Cultured cells were collected by scraping with a rubber policeman directly into guanidinium thiocyanate solution and processed for RNA isolation by the method of Chomczynski and Sacchi (4). The same procedure was used to isolate RNA from rat tissues, except the frozen tissues were ground to a powder in liquid N2, then added to the guanidinium thiocyanate solution. RNA from tracheal epithelium was obtained by simply flushing excised tracheas with the guanidinium thiocyanate solution as described by Ostrowski et al. (20). To remove DNA contamination, total cellular RNA (15-30 µg) was incubated for 30 min at 37°C with 10 units of RNasin ribonuclease inhibitor (BRL, Gaithersburg, MD) and 10 units of ribonuclease free deoxyribonuclease I (Stratagene, La Jolla, CA). After extraction with phenol-chloroform, the supernatant was ethanol precipitated in the presence of 0.3 M sodium acetate. The precipitated RNA was dissolved in diethyl pyrocarbonate-treated water and was used for differential display and reverse transcriptase (RT)-PCR analysis.
Lung tumors. Frozen adenomas, carcinomas, and nonneoplastic lung tissue from Fischer 344 rats treated with isobutyl nitrite were obtained from the National Toxicology Program study, courtesy of Dr. R. Maronpot (National Institute of Environmental Health Sciences). Animals used for this study were exposed by inhalation to 75 or 150 parts/million of isobutyl nitrite for 6 h/day, 5 days/wk for 2 yr. Samples were homogenized in guanidinium thiocyanate solution and were processed as above.
Differential display.
Differential display (16) was performed using a Delta RNA
Fingerprinting Kit (Clontech, Palo Alto, CA). Two micrograms of purified RNA (days
14-16)
from SUB cultures, air-liquid interface (ALI) cultures grown in CM, and
ALI cultures grown in EGF/
CT were compared. These
conditions were shown previously to result in differential expression
of ciliated and mucous cell-specific genes (1, 11). Aliquots
of cDNA were amplified with an arbitrary primer,
5'-ATTAACCCTCACTAAATGCTGGTGG-3' (P3), and an oligo(dT) primer, 5'-CATTATGCTGAGTGATATCTTTTTTTTAG-3' (T3),
using Advantage KlenTaq Polymerase (Clontech). The PCR cycling
conditions were as described by the manufacturer. The amplified cDNAs
were then separated on a denaturing 6% polyacrylamide gel. The gel was
dried and exposed to film. Differentially expressed bands of interest were cut from the gel and were reamplified using the same set of
primers, according to the manufacturer's instructions. The products of
the PCR reaction were separated on a 3% NuSieve/Seakem GTC agarose
gel (FMC Bioproducts, Rockland, ME). The isolated band was
used as a probe for initial confirmation of the differential expression
of these genes by Northern analysis and for cloning into the pCRII
vector using the TA Cloning Kit (Invitrogen, San Diego, CA).
Cloning of rat GGT-rel. Rapid amplification of cDNA ends (RACE) was performed using a rat brain Marathon-Ready cDNA library (Clontech) essentially as described by the manufacturer. GGT-rel specific primers were 5'-GCTTCATTTCAACGTGCTGAAAGG-3' and 5'-AAGTTCGCAGTAGGGCAGAGGTGG-3' for 3'-RACE and 5'-GGCAAACTTGAGCGTCTCTACAAG-3' and 5'-TCAATCTGCTGGCGGATGTG CT-3' for 5'-RACE. Products were cloned into the pCRII vector as above.
Sequence analysis. Plasmid DNA was isolated using the Wizard Plus Minipreps DNA Purification System (Promega, Madison, WI) and was sequenced using a Sequenase Kit (Amersham) or using an ABI PRISM 377 DNA sequencer and Dye Terminator Cycle Sequencing Kit (Perkin-Elmer). The final sequence was analyzed using the Genetic Computer Group (GCG) sequence-analysis package (version 8; GCG, Madison, WI). Sequences were also analyzed using the MacVector and AssemblyLIGN programs (International Biotechnologies, New Haven, CT).
Northern analysis.
Northern analysis was performed using standard procedures essentially
as previously described (20). Ten to twenty micrograms of RNA were
electrophoresed through a 1.2% agarose-1.0 M formaldehyde gel.
Ethidium bromide staining demonstrated equal loading before transfer to
the membrane. Gels were immersed in 50 mM NaOH for 15 min and were
washed two times in 10× sodium chloride-sodium phosphate-EDTA
(SSPE) buffer for 15 min, and RNA was transferred to
Nytran membranes (Schleicher & Schuell, Keene, NH) by capillary blotting overnight with 10× SSPE buffer. RNA was cross-linked to
the membrane with a Stratalinker (Stratagene) and then was prehybridized and hybridized in QuikHyb hybridization solution (Stratagene), according to the manufacturer's instructions.
Nonspecifically bound radioactivity was removed by washing the membrane
in 2× standard sodium citrate (SSC) and 0.1% sodium
dodecyl sulfate (SDS) two times for 15 min at room temperature followed
by a 30-min wash in 0.1× SSC and 0.1% SDS at 58°C. The blots
were exposed to Kodak film at 70°C with intensifying
screens. For estimation of changes in the expression level of GGT and
GGT-rel, Northern blots were analyzed using a Phosphorimager (Molecular
Dynamics, Sunnyvale, CA). To avoid any cross-hybridization between GGT
and GGT-rel, duplicate Northern blots were prepared and probed
separately for each gene. Blots were also probed with a
-actin cDNA.
Results for GGT and GGT-rel were then normalized to the expression
level of
-actin as a control for loading and transfer. For the
results shown, two completely independent RTE cultures were analyzed
with reproducible results.
Labeling of probes. A rat GGT probe was prepared by RT-PCR of RNA isolated from RTE cell cultures with primers specific for rat GGT. The upstream primer was 5'-AGCCAGGTAAGCAACCGCTTTC-3' and the downstream primer was 5'-CCGCCTTTTCTGGAATCTGAG-3' (bases 1319-1679 of rat GGT). An annealing temperature of 58°C was used. The 361-bp product was cloned and was verified by sequencing. Probes for GGT-rel and GGT were generated by digestion of plasmid DNA to remove the insert. The cDNA insert was isolated from a low melting point agarose gel, and 25 ng of insert DNA were labeled with the Rediprime DNA Labeling System (Amersham). Probes were purified on NucTrap columns (Stratagene).
RT-PCR analysis. One microgram of RNA from rat brain, heart, kidney, liver, lung, spleen, testis, trachea, or cultured RTE cells was reverse transcribed and amplified with an RNA PCR kit (Perkin-Elmer). After reverse transcription, the cDNA samples were amplified using specific primers for rat GGT (as above) or GGT-rel. Primers used for rat GGT-rel were 5'-TGAAGGGAGGGTGAACGTGTAC-3' (upstream) and 5'-TCAATCTGCTGGCGGATGTGCT-3' (downstream). Typical PCR cycling conditions were 95°C for 1 min followed by 35-45 cycles of 95°C for 30 s, 59°C for 30 s, and 72°C for 30 s, followed by 72°C for 7 min. An aliquot of each reaction was electrophoresed through a 3% NuSieve-Seakem GTC agarose gel and was visualized by ethidium bromide staining.
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RESULTS |
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Isolation of a differentially expressed cDNA during RTE cell
differentiation.
To isolate genes for which the expression changes during mucous or
ciliated cell differentiation, differential display was performed using
RNA isolated from RTE cells cultured under three different conditions
(described in MATERIALS AND METHODS)
that have previously been shown to influence the extent of mucous and ciliated cell differentiation (5, 11, 21). Samples from two independent
cultures (days
14-16)
were analyzed in parallel. Overall, the differential display banding
pattern was highly reproducible between the cultures and between these
three conditions (Fig. 1A).
With the use of various primer combinations, several differentially expressed bands were identified. One cDNA, which was abundantly expressed in RTE cells cultured in CM at an ALI and in SUB cultures but
was not expressed in RTE cells cultured at an ALI in
EGF/
CT (Fig. 1A),
was chosen for further characterization in this study.
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Cloning and sequencing of rat GGT-rel cDNA. The reamplified cDNA was cloned, and several independent clones were sequenced. The sequence data indicated that during the differential display procedure the cDNA was primed with the arbitrary primer (P3) at both ends. A search of GenBank revealed a high degree of homology between the cloned 373-bp cDNA fragment and human GGT-rel (amino acids 247-270). No significant homology with any other gene was found. To determine the full-length sequence for rat GGT-rel, primers were designed based on the partial sequence, and 5'- and 3'-RACE was performed using a rat brain cDNA library. Several clones for both the 5'- and 3'-ends were obtained and sequenced. The composite 2.6-kb cDNA contained a 1,716-nucleotide open reading frame and codes for a predicted protein of 61,600 kDa. An alignment of the predicted rat and human GGT-rel protein sequences (Fig. 2) demonstrated 79% identity and 82% similarity. For comparison, an alignment of rat and human GGT also showed 79% identity (83% similarity). Rat GGT-rel contains eight potential N-linked glycosylation sites (Fig. 2), four of which are conserved between the rat and human protein. Rat GGT-rel also contains a two-amino acid insertion (positions 533 and 534) and a 16-amino acid deletion (after position 443) compared with the human protein. Overall, these results indicate that the differentially expressed cDNA is the rat homologue of GGT-rel.
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Expression of GGT-rel in rat tissues. To examine the expression of GGT-rel in different tissues, cDNA was used to probe a Northern blot of RNA isolated from rat tissues and RTE cell cultures. As expected, RNA from RTE cells cultured in CM demonstrated a strong hybridization signal (Fig. 3A). A faint signal was also observed in RNA isolated from tracheal epithelium by flushing the lumen with lysis solution. However, no message was detected by Northern analysis in all other tissues (brain, heart, kidney, liver, lung, spleen, and testis) tested. To examine the expression of GGT-rel at a more sensitive level, the same tissues were analyzed by RT-PCR using primers specific for GGT-rel. After 45 cycles of amplification using the GGT-rel specific primers, a clear product was visible in all tissues tested (brain, heart, kidney, liver, lung, spleen, testis, and trachea; Fig. 3B). Amplifications performed without RT or without RNA template were routinely negative, indicating the product obtained was dependent on the presence of GGT-rel mRNA. These results indicate that GGT-rel is expressed in many tissues of the adult rat, although at low levels.
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Effect of EGF and CT depletion on GGT-rel expression.
As shown above, GGT-rel is strongly expressed in cultures grown in CM
but was not detected in cells cultured 7 days in the absence of EGF and
CT. To examine the regulation of GGT-rel by EGF and CT, RTE cells were
cultured for 7 days in CM, media without EGF, media without CT, or
media without EGF and CT. Northern analysis showed that removal of CT
from the media had no effect on the expression of GGT-rel (Fig.
4A),
whereas the removal of EGF alone reduced GGT-rel expression
substantially (72% reduction when normalized to the expression of
-actin). The removal of both EGF and CT caused a further decrease in
the expression of GGT-rel. In contrast, the removal of EGF or EGF and
CT only slightly reduced GGT expression (
EGF, 36% reduction,
EGF/
CT, 30% reduction; Fig.
4B). These results indicate that EGF
has a major effect on the expression of GGT-rel in RTE cells.
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Expression of GGT-rel during differentiation in the presence and
absence of EGF.
To examine the level of expression of GGT-rel during mucociliary
differentiation and its regulation by EGF, RTE cells were cultured in
media with or without EGF for 7 days after the formation of the ALI.
RNA was isolated from parallel cultures at daily intervals and was
analyzed for the level of GGT-rel, GGT, and -actin transcripts by
Northern blotting (Fig. 5,
A and
B). Removal of EGF from the media
resulted in a substantial decrease in the level of GGT-rel expression.
When normalized to
-actin expression and compared with RTE
cells cultured in CM, RTE cells cultured in the absence of EGF showed a
57% reduction in GGT-rel expression by
day
13 (Fig.
5C). By
day
15, the level of GGT-rel expression in
cultures grown in the absence of EGF was 3.9-fold less than in the CM
cultures. The level of GGT expression also decreased after the removal
of EGF, although the magnitude of the reduction was <50% on
day
15 (Fig.
5C). These results show that GGT-rel
is expressed throughout differentiation of RTE cells in vitro and that
the removal of EGF causes a substantial reduction in the expression of
GGT-rel.
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Induction of GGT-rel by EGF. To determine if EGF could induce GGT-rel expression directly, RTE cells were grown in media without EGF from days 8 to 13. On days 13 and 14, some of the EGF-depleted cultures were refed with media containing 25 ng/ml EGF. Cells were harvested from the different groups 8, 24, and 48 h after the addition of EGF to the EGF-depleted cultures. Northern analysis demonstrated a 2.4-fold increase (average of 2 experiments) in GGT-rel expression 24 h after addition of EGF (Fig. 6A). This induction was maintained for at least 48 h (2.2-fold, average of 2 experiments), although the level of GGT-rel expression in cultures treated with EGF did not reach the level in parallel cultures grown continuously in CM. GGT expression was also increased after addition of EGF, although not as strongly (1.7-fold at 24 h and 1.4-fold at 48 h, average of 2 experiments; Fig. 6B). EGF is therefore capable of rapidly inducing the expression of GGT-rel in RTE cells.
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Expression of GGT-rel in lung tumors.
As shown above, EGF induced GGT-rel expression in RTE cells.
Transforming growth factor (TGF)-, which activates the EGF receptor, is overexpressed by transformed RTE cells (9) and also by many other
transformed cell lines and tumors. Therefore, GGT-rel expression was
examined in several lung tumor samples. RNA was isolated from normal
lung, adenomas, and carcinomas from rats treated with isobutyl nitrite
and also from normal untreated lung. Northern analysis demonstrated a
strong signal for GGT-rel in both the carcinomas and adenomas compared
with normal untreated lung (Fig. 7). The normal lung tissue obtained from an animal treated with isobutyl nitrite also showed a high level of GGT-rel expression. GGT-rel expression was particularly high in one of the carcinomas (Fig. 7,
lane
5). These results show that GGT-rel
is expressed at high levels in lung tissue and tumors from animals
chronically exposed to isobutyl nitrite.
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DISCUSSION |
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Using differential display to analyze changes in gene expression during mucociliary differentiation of RTE cells, we have identified a rat homologue of human GGT-rel. This is the first evidence that GGT-rel is expressed in airway epithelial cells. Heisterkamp et al. (13) were unable to demonstrate GGT-rel expression in any of the mouse tissues they analyzed and only observed a very low level of expression in two human cancer cell lines. In contrast, our studies have demonstrated a high level of GGT-rel expression by normal RTE cells in primary culture. In agreement with the results of Heisterkamp et al. (13), GGT-rel expression was not detected by Northern analysis using whole lung or other rat tissues as a source of RNA. However, GGT-rel expression was detectable by RT-PCR in all rat tissues examined, including brain, heart, kidney, liver, lung, spleen, testis, and trachea. This suggests that, similarly to GGT, GGT-rel is widely distributed, although constitutively expressed at much lower levels. Alternatively, it is possible that only a small number of specialized cells express GGT-rel in any particular tissue or that GGT-rel is only highly expressed under certain conditions. In support of the latter hypothesis, our results show that GGT-rel expression can be induced by EGF. Treatment of EGF-deprived RTE cultures with 25 ng/ml of EGF caused a rapid and sustained increase in GGT-rel expression. This demonstrates that GGT-rel expression can be regulated by growth factors and may link the expression of GGT-rel to increased levels of proliferation. In addition, high levels of GGT-rel expression were observed in lung carcinomas, adenomas, and normal lung tissue from rats exposed by inhalation to isobutyl nitrite, a chemical that causes hyperplasia of the respiratory airway epithelium. Increased levels of GGT expression have been observed in many tumor types, and its expression is frequently used as an early marker for hepatocarcinogenesis (12). Whether GGT-rel expression is induced in tumors as a result of chemical exposure or as a direct result of the neoplastic process is unclear. Additional experiments will be needed to elucidate the possible roles of GGT-rel in these conditions.
GGT-rel has many potential functions in airway epithelial cells. For
example, GGT-rel could function in the defense mechanisms of the
airways by supplying reducing equivalents to the cell via GSH. Forman
and co-workers (3, 15, 18, 23) have demonstrated that GSH can protect
pulmonary cells from oxidative stress and that oxidative stress can
induce GGT and -glutamylcysteine synthetase. Because GGT-rel has
been shown to catalyze some of the same reactions as GGT (13), high
levels of GGT-rel could also provide protection from oxidative stress.
GGT-rel may also play a role in the secretory functions of RTE cells.
RTE cell cultures grown in media containing EGF show a high percentage
of mucous cells and actively produce and secrete mucus and other
products. The withdrawal of EGF decreases mucus production and also
reduces the level of GGT-rel expression. Thus GGT-rel may play a role
in the secretory pathway, perhaps by providing essential precursors for
the synthesis of secreted molecules. In addition, GGT-rel could
function in the metabolism of important biological mediators. Human
GGT-rel was shown to catalyze the conversion of leukotriene
C4 into leukotriene
D4, and leukotrienes can cause
bronchoconstriction and changes in vascular permeability in the lung
(10). At present, the physiological function(s) of GGT-rel in the
airways is unknown, but the present work indicates that further studies
are warranted.
In summary, the experiments presented here have identified a rat
homologue of GGT-rel and demonstrated that GGT-rel is expressed in a
wide variety of rat tissues. GGT-rel was shown to be inducible to high
levels in cultures of primary RTE cells by EGF. High levels of GGT-rel
expression were also observed in several lung tumors and in lung tissue
chronically exposed to a carcinogen. Under these conditions, GGT-rel,
in addition to GGT, may be important to the metabolism of -glutamyl
compounds, and future studies will need to distinguish between these
two enzymes.
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ACKNOWLEDGEMENTS |
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We thank Dr. Bob Maronpot for supplying the rat lung tumors used in this study, Daniel Hart for assistance with automated sequencing, and Drs. Doug Bell and Tom Eling for reviewing the manuscript. We also acknowledge Dr. John Groffen for helpful discussions of this work.
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FOOTNOTES |
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P. D. Potdar was funded by Rameshwardas Birla Smarak Kosh, Mumbai India; the National Cancer Institute short-term scientist exchange program; and National Institute of Environmental Health Sciences.
Present address of P. D. Potdar: Cancer Research Institute, Tata Memorial Centre, Parel, Bombay 400012, India
Address for reprint requests: L. E. Ostrowski, Laboratory of Pulmonary Pathobiology, National Institute of Environmental Health Sciences, P.O. Box 12233, Research Triangle Park, NC 27709.
Received 9 June 1997; accepted in final form 14 August 1997.
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