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USC Liver Disease Research Center, the Division of Gastrointestinal and Liver Diseases, Department of Medicine, University of Southern California School of Medicine, Los Angeles, California 90033, USA
1Correspondence: HMR Rm. 415, Department of Medicine, USC School of Medicine, 2011 Zonal Ave., Los Angeles, CA, 90033, USA. E-mail: shellylu{at}hsc.usc.edu
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ABSTRACT |
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 TOP ABSTRACT CURRENT CONCEPTS: STRUCTURE AND...SYNTHESIS OF HEPATIC GSHAREAS OF CONTROVERSYSUMMARY AND FUTURE DIRECTIONSREFERENCES |
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-glutamylcysteine
synthetase (GCS). In the liver, major factors that determine the
availability of cysteine are diet, membrane transport activities of the
three sulfur amino acids cysteine, cystine and methionine, and the
conversion of methionine to cysteine via the
trans-sulfuration pathway. Many conditions alter GSH
level via changes in GCS activity and GCS gene expression. These
include oxidative stress, activators of Phase II detoxifying enzymes,
antioxidants, drug-resistant tumor cell lines, hormones, cell
proliferation, and diabetes mellitus. Since the molecular cloning of
GCS, much has been learned about the regulation of this enzyme. Both
transcriptional and post-transcriptional mechanisms modulate the
activity of this critical cellular enzyme.—Lu, S. C. Regulation
of hepatic glutathione synthesis: current concepts and controversies.
Key Words: -glutamylcysteine synthetase • cysteine availability • detoxification • antioxidant
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CURRENT CONCEPTS: STRUCTURE AND FUNCTIONS OF GSH |
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TOPABSTRACTCURRENT CONCEPTS: STRUCTURE AND...SYNTHESIS OF HEPATIC GSHAREAS OF CONTROVERSYSUMMARY AND FUTURE DIRECTIONSREFERENCES |
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-glutamylcysteinyl glycine, found in most
plants, microorganisms, and all mammalian tissues. Glutathione exists
in the thiol-reduced
(GSH)2
and disulfide-oxidized (GSSG) forms (1)
. Eukaryotic cells
have three major reservoirs of GSH. Almost 90% of cellular GSH are in
the cytosol, 10% in the mitochondria, and a small percentage in the
endoplasmic reticulum (2
3
4)
. In the endoplasmic
reticulum, where GSH is implicated in protein disulfide bond formation,
the GSH to GSSG ratio is 3:1 (2)
. In the cytoplasm and
mitochondria, ratios exceed 10:1 (3
, 4)
. Cytosolic GSH in
the rat liver turns over rapidly with a half-life of 2–3 h. The
-glutamyl linkage promotes intracellular stability and the
sulfhydryl group is required for GSH's functions (Fig. 1
). The peptide bond linking the amino-terminal glutamate and the
cysteine residue of GSH is through the -carboxyl group of glutamate
rather than the conventional 
-carboxyl group. This unusual
arrangement resists degradation by intracellular peptidases and is
subject to hydrolysis by only one known enzyme,
-glutamyltranspeptidase (GGT), which is on the external surfaces of
certain cell types (1
, 4)
. Furthermore, the
carboxyl-terminal glycine moiety of GSH protects the molecule against
cleavage by intracellular -glutamylcyclotransferase
(4)
. As a consequence, GSH resists intracellular
degradation and is only metabolized extracellularly.
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GSH serves several vital functions, including 1) detoxifying
electrophiles; 2) maintaining the essential thiol status of
proteins by preventing oxidation of -SH groups or by reducing disulfide
bonds induced by oxidant stress; 3) scavenging free
radicals; 4) providing a reservoir for cysteine; and
5) modulating critical cellular processes such as DNA
synthesis, microtubular-related processes, and immune function
(1
, 4
5
6)
. Some of the functions of GSH are discussed
briefly, as this review focuses on the synthesis of GSH.
Detoxifying functions of GSH
Detoxification of xenobiotics or their metabolites is one of
the major functions of GSH. These compounds are electrophiles and form
conjugates with GSH either spontaneously or enzymatically in reactions
catalyzed by GSH S-transferase (1
, 4)
. The conjugates
formed are usually excreted from the cell and, in the case of
hepatocytes, into bile. The metabolism of GSH conjugates begins with
cleavage of the -glutamyl moiety by GGT, leaving a cysteinyl-glycine
conjugate. The cysteinyl-glycine bond is cleaved by dipeptidase,
resulting in a cysteinyl conjugate. This is followed by N-acetylation
of the cysteine conjugate, forming a mercapturic acid (Fig. 2
). The metabolism of GSH conjugates to mercapturic acid begins either in
the biliary tree, intestine, or kidney, but the formation of the
N-acetylcysteine conjugate usually occurs in the kidney
(1)
. In addition to exogenous compounds, many endogenously
formed compounds also follow similar metabolic pathways. Some examples
include estradiol-17-ß, leukotrienes, and prostaglandins
(4)
. Although the majority of the conjugation reactions to
GSH result in detoxification of the compound, occasionally the product
itself is highly reactive (1)
. One such example is the GSH
conjugate of dibromoethane (1)
. GSH conjugation
irreversibly consumes intracellular GSH.
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Maintenance of essential thiol status
As the dominant nonprotein thiol in mammalian cells, GSH is
essential in maintaining the intracellular redox balance and the
essential thiol status of proteins (1
, 4)
. To achieve
this, GSH undergoes thiol-disulfide exchange in a reaction catalyzed by
thiol-transferase, as follows:
 |
. Normally,
cellular GSSG is kept extremely low (<1% of total GSH pool) so that
protein mixed disulfide formation may be limited. The thiol-disulfide
equilibrium within the cell is known to regulate a diverse number of
metabolic processes including enzyme activity, transport activity, and
gene expression via alteration of redox-sensitive
trans-activating factors (1
, 6
, 7)
.
Antioxidant function of GSH
As a consequence of aerobic metabolism, all aerobic organisms are
subject to a certain level of physiological oxidative stress. The
intermediates that are formed, such as superoxide
(O2-·) and hydrogen peroxide, can lead to
the further production of toxic oxygen radicals that can cause lipid
peroxidation and cell injury. The endogenously produced hydrogen
peroxide is reduced by GSH in the presence of selenium-dependent GSH
peroxidase (Fig. 3
). As a result, GSH is oxidized to GSSG, which in turn is reduced back
to GSH by GSSG reductase at the expense of NADPH, forming a redox
cycle. Either GSH peroxidase or GSH S-transferase can reduce organic
peroxides. Hydrogen peroxide can also be reduced by catalase, which is
present only in the peroxisome. In the mitochondria, GSH is
particularly important because there is no catalase. Recent studies
have shown that mitochondrial GSH is critical in defending against both
physiologically and pathologically generated oxidative stress (1
, 7
, 8)
. A selective reduction in the mitochondrial GSH pool has
been reported in rats fed alcohol and may play an important
pathogenetic role in the development of the liver disease (see below)
(7
, 8)
.
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Severe oxidative stress may overcome the ability of the cell to reduce
GSSG to GSH, leading to accumulation of GSSG within the cytosol. To
protect the cell from a shift in the redox equilibrium, GSSG can be
actively exported out of the cell or react with a protein sulfhydryl
group, leading to the formation of a mixed disulfide. Thus, severe
oxidative stress depletes cellular GSH (1
, 4
, 7)
.
GSH as cysteine storage and the -glutamyl cycle
One of the most important functions of GSH is to store cysteine
because cysteine is extremely unstable extracellularly and rapidly
auto-oxidizes to cystine, in a process producing potentially toxic
oxygen free radicals (4
, 7)
. The -glutamyl cycle
(Fig. 4
), first described by Meister in the early 1970s (see ref 4)
, allows GSH to serve as a continuous source of cysteine.
Here, GSH is released from the cell by carrier-mediated transporter(s)
(7)
and the ecto-enzyme GGT then transfers the
-glutamyl moiety of GSH to an amino acid (the best acceptor being
cystine), forming -glutamyl amino acid and cysteinylglycine. The
-glutamyl amino acid can then be transported back into the cell to
complete the cycle. Once inside the cell, the -glutamyl amino acid
can be further metabolized to release the amino acid and 5-oxoproline,
which can be converted to glutamate and used for resynthesis of GSH.
Cysteinylglycine is broken down by dipeptidase to generate cysteine and
glycine. Cysteine is readily taken up by most if not all cells. Once
inside the cell, the majority of cysteine is incorporated into GSH;
some is incorporated into protein, depending on the need of the cell,
and some is degraded into sulfate and taurine. For most cells, this
mechanism provides a continuous source of cysteine. Thus, the
-glutamyl cycle allows the efficient utilization of GSH as cysteine
storage.
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SYNTHESIS OF HEPATIC GSH |
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TOPABSTRACTCURRENT CONCEPTS: STRUCTURE AND...SYNTHESIS OF HEPATIC GSHAREAS OF CONTROVERSYSUMMARY AND FUTURE DIRECTIONSREFERENCES |
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, 9)
. The mitochondrial GSH transporter maintains the
GSH pool in the mitochondria, which cannot biosynthesize GSH (7
, 8)
. This transporter is selectively impaired in rats fed alcohol
and accounts for the fall in mitochondrial GSH in this liver disease
model (7
, 8)
. Pathways that consume GSH, such as formation
of GSH conjugates and mixed disulfides, account for very little of the
hepatocyte GSH utilization under normal physiological conditions. The
importance of hepatic GSH to the interorgan GSH homeostasis is
underscored by the fact that plasma GSH and cysteine levels are largely
determined by the sinusoidal efflux of hepatic GSH (9)
.
Therefore, a better understanding of the regulation of hepatic GSH
synthesis is central to understanding interorgan GSH homeostasis under
normal and pathological conditions.
GSH is synthesized from precursor amino acids in the cytosol of
virtually all cells (1
, 4
, 7)
. The synthesis of GSH from
its constituent amino acids, L-glutamate, L-cysteine, and L-glycine,
involves two ATP-requiring enzymatic steps:
[1] L-glutamate + L-cysteine + ATP 
-glutamyl-L-cysteine + ADP
+ Pi
[2] -glutamyl-L-cysteine + L-glycine + ATP GSH + ADP + Pi
The first step of GSH biosynthesis is rate-limiting and catalyzed by
-glutamylcysteine synthetase (GCS), which exhibits an absolute
requirement for either Mg2+ or
Mn2+. GCS is composed of a heavy (GCS-HS,
Mr ~ 73,000) and a light (GCS-LS,
Mr ~ 30,000) subunit, which are
encoded by different genes in both rat and human (10
11
12
13)
.
The enzyme may be dissociated under nondenaturing conditions by
treatment with dithiothreitol (14)
. The heavy subunit
obtained after this treatment exhibits all the catalytic activity of
the isolated enzyme as well as feedback inhibition by GSH
(14)
. Although the heavy subunit is active catalytically,
it has a higher Km value for glutamate (18.2 vs. 1.4
mM) and a lower Ki value for GSH (1.8 vs. 8.2 mM)
compared with the holoenzyme (15)
. Thus, the light subunit
plays an important regulatory role for the overall function of the
enzyme and allows the holoenzyme to be catalytically more efficient and
less subject to inhibition by GSH than the heavy subunit alone. The low
affinity of the heavy subunit for glutamate and the high feedback
inhibition exerted by GSH (Ki value for GSH is about
one-third of the normal physiological liver GSH level) suggest that the
heavy subunit alone is not likely to be active physiologically. This
remains to be proved.
GCS is specific for the glutamyl moiety, and Lys-38 of the heavy
subunit may be involved in binding to glutamate (16)
. GCS
in both rat and human is regulated physiologically by:
1) Feedback competitive inhibition by GSH (17
, 18)
. Inhibition by GSH is nonallosteric and involves binding of
GSH to the glutamate site and another site on the enzyme. This latter
binding appears to involve interaction with the thiol moiety of GSH but
not with a methyl group (17)
.
2) The availability of its precursor, L-cysteine (1
, 4
, 7)
. The apparent Km values of GCS for
glutamate and cysteine are 1.8 and 0.1–0.3 mM, respectively, in both
rat and human (18
, 19)
. The intracellular glutamate
concentration is several folds higher than the Km
value of GCS for glutamate, but the intracellular cysteine
concentration approximates the apparent Km value of
GCS for cysteine (7
, 19)
. Therefore, the availability of
intracellular cysteine and the activity of GCS most significantly
influence the rate of GSH synthesis.
The second step in the synthesis of GSH is catalyzed by GSH synthetase.
This enzyme has not been studied as extensively as GCS. GSH synthetase
purified from rat kidney has an Mr of
~118,000 daltons and is composed of two apparently identical subunits
(20)
. Studies of the mapping of the substrate binding
sites of the enzyme with methyl-substituted and other analogs of the
substrates indicate that the regions of the active site that bind
glycine and the cysteinyl moiety of -glutamylcysteine are highly
specific. The L--glutamyl moiety, on the other hand, can be replaced
by a variety of analogs, indicating that this binding site is not
specific (20)
. This enzyme has been cloned recently from
the rat kidney (21)
. The isolated rat kidney enzyme is
known to contain a small amount (2%) of carbohydrate and two
asparagine residues (residues 124 and 171) in the amino acid sequence,
fitting the requirement for N-linked protein glycosylation,
and one serine residue, agreeing with the general pattern for an
O-linked N-acetylglucosamine addition site (20
, 21)
.
However, the significance of glycosylation and the overall regulation
of this enzyme remain poorly understood. Early studies in hog and
pigeon liver suggested that ADP may play a regulatory role (22
, 23)
. GSH synthetase is not subject to feedback inhibition by
GSH. One recent study in the yeast Saccharomyces cerevisiae
showed that this enzyme is dispensable for growth under both normal and
oxidative stress conditions due to an accumulation of
-glutamylcysteine, which was able to protect against oxidative
stress (24)
. Overexpression of GSH synthetase failed to
increase GSH level whereas overexpression of GCS increased the GSH
level, consistent with the fact that GCS is the rate-limiting enzyme of
GSH synthesis (24)
. However, GSH synthetase deficiency in
humans can result in dramatic metabolic consequences because the
accumulated -glutamylcysteine is converted to 5-oxoproline, which
can cause severe metabolic acidosis (21)
.
Factors that determine the availability of cysteine
One of the major determinants of the rate of GSH synthesis is the
availability of cysteine. Cysteine is normally derived from the diet
and protein breakdown, and in the liver from methionine via the
transsulfuration pathway (7
, 25)
. Cysteine differs from
other amino acids because its sulfhydryl form, cysteine, is predominant
inside the cell whereas its disulfide form, cystine, is predominant
outside the cell. Cysteine readily autoxidizes to cystine in the
extracellular fluid; once it enters the cell, cystine is rapidly
reduced to cysteine (25)
. Therefore, the key factors that
regulate the hepatocellular level of cysteine other than diet include
membrane transport of cysteine, cystine, and methionine as well as the
activity of the transsulfuration pathway (25
26
27)
.
Although glutamate and glycine are also precursors of GSH, there is no
evidence to suggest that their transport influences GSH synthesis since
they are synthesized via several metabolic pathways within hepatocytes
(25)
.
Dietary influence
Studies from Tateishi and co-workers have shown that hepatic GSH
level is closely related to nutritional conditions, especially the
cysteine content of the diet (28
, 29)
. Rat liver normally
contains 7–8 µmol GSH/g tissue, mostly in the reduced state.
Starvation for 48 h results in a significant fall in liver GSH
content to between two-thirds and one-half of the normal levels. On
refeeding, the GSH level returns to normal within a few hours
(28)
. Fasting did not affect the activities of GCS and GSH
synthetase. The effects of fasting and refeeding on hepatic GSH level
were not affected by pretreatment with actinomycin D or cycloheximide,
suggesting that the amount of enzymes involved in GSH synthesis was
unaffected (28)
. This shows the strong dependency of
hepatic GSH level on food intake.
Cysteine transport
Cysteine is transported almost entirely as a neutral amino acid,
and in rat hepatocytes is transported mainly by the ASC system
(26
, 30
, 31)
. This system is Na+
dependent and especially reactive with neutral amino acids with short
to intermediate length side chains such as serine and alanine. This
system has high stereospecificity (it does not take up D-cysteine), is
pH sensitive (a change from 7.4 to 6.5 resulted in suppression of
L-cysteine transport by 30%), and is not sensitive to adaptive
regulation or insulin and glucagon stimulation under conditions
producing these effects for System A (26
, 31)
. The ASC
system mediates both inward and outward flows of its substrate amino
acids and is subject to trans-stimulation (32)
:
a System ASC amino acid at one side of the membrane stimulates the
transport of another System ASC amino acid present at the other side of
the membrane. Therefore, the intracellular cysteine concentration
depends on the intracellular and extracellular levels of not only
cysteine, but also other System ASC amino acids. Higher levels of
extracellular cysteine will raise its intracellular level, but higher
levels of other System ASC amino acids will inhibit competitively the
influx of cysteine (cis-inhibition) and stimulate the efflux
of cysteine (trans-stimulation).
Cystine transport
The transport of cystine is distinct from that of cysteine
(30
, 33
, 34)
. Cystine has four ionizable groups and is
present mostly as the tetrapolar ion at neutral pH. However, because
the pKs of the two amino groups of cystine are ~ 7.9 and 8.8, a
considerable part of cystine (~ 20% of total cystine at pH 7.4) is
present as the tripolar ion at physiological pH (25)
. The
system that transports this anionic form of cystine is System
Xc-. Glutamate also exists in anionic form at
physiological pH and is the only other significant substrate for System
Xc-. This system is Na+
independent and mediates one-to-one exchange of cystine for glutamate
(34)
. The physiologically significant flow via System
Xc- is an entry of cystine accompanied by an
exit of glutamate, because cells are usually rich in glutamate, which
is also transported by systems other than Xc-
and synthesized from its precursor, glutamine, but not in cystine
(25)
. The driving force of this exchange appears to be a
steep concentration gradient of glutamate (outside the fibroblast-1.6
mM, inside-24 mM) (25)
. Probably this high intracellular
glutamate concentration functions to stimulate the influx of cystine to
maintain an adequate balance between cysteine and glutamate inside the
cells.
Cystine transport in hepatocytes is also mediated by System
Xc-. Under normal physiological conditions,
cystine is taken up poorly by hepatocytes (27
, 30
, 35)
.
However, on culturing rat hepatocytes, the activity of System
Xc- emerges after a 12 h lag in response to
insulin and dexamethasone. The increase in System
Xc- activity is dependent on de novo
synthesis of both RNA and protein (27)
. This may be an
adaptive response of cultured cells since there is no detectable
cysteine in the culture medium (almost completely autoxidized). The
activity of System Xc- can also be induced by
treatment with electrophilic agents and O2
(25
, 35)
. In both fibroblasts and isolated hepatocytes,
treatment with electrophilic agents depletes intracellular GSH levels,
followed by enhanced cystine uptake via System
Xc- and restoration of cell GSH levels. When
cystine uptake was inhibited by glutamate or homocysteate, restoration
of cell GSH level was prevented (35)
. This suggests that
System Xc- is involved in the cell defense
mechanism against an electrophilic attack by facilitating increased
synthesis of GSH, which in turn may serve for detoxification of the
electrophiles. The exact mechanisms of this induction is unclear at
present, and it is not known whether it occurs in intact liver.
Although normal hepatocytes do not transport cystine, GSH released from
the cells can undergo thiol-disulfide exchange with cystine, liberating
cysteine, which is then available to the hepatocytes (36)
.
This phenomenon has also been observed in HepG2 cells
(37)
. In fact, this was the sole mechanism for providing
cysteine for GSH synthesis in HepG2 cells, which are not able to
convert methionine to cysteine (see below) (37)
.
Methionine transport
Methionine is transported mainly by System L in hepatocytes, as
the increase in the intracellular GSH level by methionine was almost
completely inhibited by the presence of 2-aminobicyclo-(2, 2,
1)heptane-2-carboxylic acid (BCH, a model substrate of System L), but
was scarcely affected by -methylamino isobutyrate (MeAIB, a model
substrate of System A) (26
, 38)
. System L is
Na+ independent and not responsive to either
adaptive control or hormonal stimulation. System L also exhibits
trans-stimulation. Studies from Kilberg and co-workers
(26)
of the kinetics of substrate uptake by System L
revealed two components to this system. Component I has high affinity,
low capacity (estimated Km values <200 µM)
whereas component II has low affinity and high capacity (estimated
Km values between 2 and 5 mM) (26)
. In
rat hepatocytes, methionine is transported biphasically by both
components of system L (39)
. In terms of the relative
rates of uptake of these three sulfur amino acids by rat hepatocytes
cultured for 24 h, the rate of uptake of cysteine is about 3-fold
higher than that of methionine and 13-fold higher than that of cystine
(38)
.
Transsulfuration pathway
The ability for the liver cell to convert methionine to cysteine
is important since the liver is the major site of methionine catabolism
and the major storage organ for GSH. That cysteine can be synthesized
from methionine was first demonstrated by Tarver and co-workers in 1939
(40)
. This pathway is termed the transsulfuration or the
cystathionine pathway (Fig. 5
). This pathway is active and unique to the liver cell, as it is absent
or insignificant in other GSH-synthesizing systems either in normal or
in transformed tissues (41)
. The activity of this pathway
in the liver is also markedly impaired or absent in the fetus and
newborn infant, cirrhotic patients, and patients with homocystinemia
(42
, 43)
. Liver cancer cells such as HepG2 and HuH-7 cells
have a block in the transsulfuration pathway proximal to the formation
of homocysteine. These cells are unable to form GSH from methionine but
are able to from homocysteine (37)
. The mechanism of this
block is unknown.
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Characterization of the transsulfuration pathway in the liver has
been achieved mainly through the efforts of Reed and co-workers using
isolated hepatocytes (44
, 45)
. Methionine is sequentially
converted to cysteine via several enzymatic steps. The first step
requires ATP and involves the activation of methionine to
S-adenosylmethionine in a reaction catalyzed by methionine
adenosyltransferase. In contrast to the two GSH-synthesizing enzymes,
which have a low Km for ATP (~0.1 mM), the
Km of hepatic methionine adenosyltransferase for ATP
is high (~2 mM) (46)
. Therefore, hypoxic depletion of
ATP is more likely to affect GSH synthesis from methionine than
cysteine. Subsequent demethylation and the removal of the adenosyl
moiety yields homocysteine. Homocysteine condenses with serine to form
cystathionine in a reaction catalyzed by cystathionine synthase.
Cleavage of cystathionine, catalyzed by cystathionase, then releases
free cysteine. In this pathway methionine and homocysteine are readily
interconvertible, but the subsequent step, the formation of
cystathionine, is irreversible (Fig. 5)
. Proparglyglycine is a
potent irreversible inhibitor of cystathionase; its use has been
instrumental in demonstrating the importance of this pathway in the
biosynthesis of GSH (44)
.
The main regulatory controls of the transsulfuration pathway appear to
center on keeping a fine control on the methionine level. The liver
methionine level is relatively insensitive to changes in dietary
protein intake (47)
. The control appears to be exerted at
the level of homocysteine: when methionine is needed, homocysteine is
remethylated by methionine synthase or betaine-homocysteine
methyltransferase; when methionine is in excess, catabolism of
homocysteine via the cystathionine synthase reaction is accelerated
(48)
.
Hepatic conversion of methionine to S-adenosylmethionine represents the
major catabolic pathway for methionine, accounting for nearly half of
the daily intake of methionine (43)
. Patients with liver
cirrhosis often exhibit hypermethioninemia and have impaired methionine
clearance (49
, 50)
because the activity of hepatic
methionine adenosyltransferase is significantly impaired
(43)
. This impairment has been postulated to be one of the
mechanisms of a decreased hepatic GSH level (43)
. In
support of this, administration of S-adenosylmethionine to patients
with liver cirrhosis resulted in increased hepatic GSH level
(51)
. Preliminary results showing an improved 2-year
survival in alcoholic liver disease patients treated with
S-adenosylmethionine have also been presented (52)
.
However, the protective mechanisms of S-adenosylmethionine remain ill
defined.
Regulation of GCS
The activity of GCS is the other major determinant of the rate of
GSH synthesis. GCS is a heterodimer made up of a catalytic (heavy, 73
kDa) and a regulatory (light, 30 kDa) subunit. Changes in GCS activity
can result from regulation at multiple levels affecting only the heavy
or both the heavy and light subunits. Transcriptional and
post-transcriptional regulation of both subunits have been described.
Post-transcriptional regulation includes both mRNA
stabilization/destabilization and post-translational modification. Most
of the studies examined transcriptional regulation of GCS. Table 1
lists conditions known to alter GCS activity and GCS expression
(19
, 53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96)
. Almost all of the reports published before
1997 focused on the regulation of the heavy subunit. These conditions
fall into several major categories, including oxidative stress,
drug-resistant tumor cells, hormones, and growth related. For
simplicity, Table 1
lists cytokines and GSH-conjugating agents as
separate categories, but these may also induce oxidative stress.
Inducers of Phase II detoxifying enzymes such as ß-naphthoflavone can
also induce oxidative stress. Most of these conditions [with the
exception of drug-resistant tumor cell lines, heat shock, heavy metals,
glucose, cycloheximide, and transforming growth factor
ß1 (TGFß1)] altered
GCS expression in liver cells.
|
Oxidative stress
Agents ranging from methyl mercury, quinones, hydrogen peroxide,
menadione, cytokines such as tumor necrosis factor (TNF), buthionine
sulfoximine (BSO), tert-butylhydroquinone, diethyl maleate
(DEM), okadaic acid, 4-hydroxy-2-nonenal, and ionizing radiation, which
cause oxidative stress in a variety of cells and organs, increased GCS
activity and GCS-HS transcription (60
61
62
63
64
65
, 72
, 76
, 81
82
83
, 87
88
89
90
91
92
93)
. In addition to increased gene transcription, DEM and
4-hydroxy-2-nonenal treatments have also been found to stabilize the
GCS-HS mRNA (72
, 93)
. Recently, increased transcription of
GCS-LS has also been found in response to DEM, BSO,
tert-butylhydroquinone, ß-naphthoflavone, and
4-hydroxy-2-nonenal (63
, 65
, 82
, 83
, 93
, 97)
.
4-Hydroxy-2-nonenal also stabilized the GCS-LS mRNA (93)
.
Thus, many causes of oxidative stress have similar effects on the
expression of both GCS subunits.
Recent efforts have focused on elucidating the molecular mechanism(s)
of oxidative stress induced increase in GCS expression. The 5'-flanking
regions of both human GCS subunits have been cloned and sequenced
(63
, 83
, 97
98
99)
. Putative nuclear factor kappa B
(NF-
B), Sp-1, activator protein-1 (AP-1), activator protein-2
(AP-2), metal response (MRE), and antioxidant response
(ARE)/electrophile-responsive elements (EpRE) have been identified in
the promoter of the heavy subunit (63
, 98)
. The promoter of
the light subunit also contains AP-1, AP-2, MRE and ARE/EpRE elements
(83
, 97)
. The region from -817 to +45 nucleotide sequence
of the human GCS-HS contains AP-1 or AP-1 like-responsive elements that
are critical in mediating the effects of cigarette smoke condensate
solution, menadione, hydrogen peroxide, TNF, and ionizing radiation on
GCS-HS promoter activity (64
, 76
, 77
, 87
, 88)
. In
menadione and hydrogen peroxide-mediated increase in GCS-HS gene
transcription, the most important AP-1 element was found to be located
at -269 to -263 (87)
. The same element was also found to
be responsible for induction of GCS-HS expression in BSO-resistant cell
lines (91)
. Recent studies from Sekhar et al. and Morales
et al. have further confirmed the importance of AP-1 activity in
mediating the effect of oxidative stress on GCS-HS transcription
(81
, 88)
. On the other hand, Mulcahy et al. described a
critical distal ARE element (ARE4), located ~3.1kb upstream of the
transcriptional start site, which mediated constitutive and
ß-naphthoflavone inducible expression in HepG2 cells
(63)
. ß-Naphthoflavone is known to activate Phase II
enzymes via AREs (63)
. AREs are also referred to as
electrophile-responsive elements (EpRE), which is the more appropriate
term since these elements are clearly induced by pro-oxidants
(99)
. The ARE4 element of the human GCS-HS has a consensus
ARE sequence (5'-GTGACTCAGCG-3') and an embedded
PMA-responsive element (TRE, underlined), which can bind AP-1 binding
proteins (99)
. Thus, AP-1 binding proteins may still be
involved in activating ARE4. This possibility was recently investigated
by Wild and colleagues (99)
. They demonstrated that the
AP-1 binding sequence of the AREA4-mediated constitutive expression of
the GCS-HS promoter, but not ß-naphthoflavone-induced expression, and
that other unknown protein complexes are involved (99)
. An
adjacent ARE (ARE3), located 34 bases downstream from ARE4, also was
not required for ß-naphthoflavone's effect on GCS-HS gene expression
(99)
.
EpRE and AP-1 have also been found to be important in mediating the
effect of ß-naphthoflavone on GCS-LS expression (97)
.
There is a consensus EpRE site located around 300 base pairs upstream
from the translational start site and a consensus AP-1 binding site
located 33 base pairs upstream of the EpRE. Both are capable of
supporting ß-naphthoflavone inducibility, but the EpRE is more
potent. Basal expression is influenced by the AP-1 site whereas EpRE is
not required for maximal basal expression (97)
. Thus, both
of the GCS subunits share in common the presence of functional AP-1 and
EpRE, which likely mediate the effect these various pro-oxidants have
on GCS subunit gene expression. The nature of the
trans-activating protein(s) that bind the ARE or EpRE sites
remain to be defined.
Although the effect of oxidative stress and inducers of Phase II
enzymes on the GCS subunits expression appear to be similar, different
signaling pathways are involved at least in the case of
4-hydroxy-2-nonenal (93)
. This agent increased the
transcription and stabilized the mRNA of both subunits, but de
novo protein synthesis was required only for the light subunit
(93)
. The molecular mechanisms underlying these
differences remain to be elucidated.
Drug-resistant tumor cells
Resistance to commonly used alkylating agents or platinum
chemotherapeutic drugs is often multifactorial, involving altered drug
transport, biotransformation, and enhanced detoxification capacity
(53
54
55
56
57
58
59
, 80
, 84)
. GSH has been found to be elevated in a
number of drug-resistant tumor cell lines and in tumor cells isolated
from patients whose tumors are clinically resistant to drug therapy
(53)
. The increase in cell GSH is a major contributing
factor to drug resistance by binding to or reacting with drugs,
interacting with reactive oxygen moieties, preventing damage to
proteins or DNA, or by participating in DNA repair processes. Although
increased GSH in drug-resistant tumor cells has been documented for
over 20 years, the mechanism for the increase was elucidated more
recently. Increased GCS activity, steady-state GCS-HS mRNA level, and
GCS-HS gene transcription have been found (53
54
55
56
57
58
, 80
, 84)
. Whether or not GCS-LS is also altered is controversial. MRP
(human multidrug resistance protein), a member of the superfamily of
ATP binding cassette membrane transporters, is capable of conferring
resistance to multiple classes of chemotherapeutic agents (57
, 58)
. Several studies have shown coordinated overexpression of
GCS-HS and MRP in drug-resistant tumor cell lines, human colorectal
tumors, and human lung cancer specimens after platinum drug exposure
(57
, 58)
. In contrast, no correlation was found between
MRP and GCS-LS expression (57)
. Godwin et al. found
cisplatin-resistant ovarian tumor cell lines exhibited increased levels
of GCS-HS and GCS-LS proteins (56)
. However, Yao et al.
found no difference in the mRNA level of GCS-LS among different ovarian
cancer cell lines with varying cisplatin resistance (84)
.
The reason for the discrepancy is unclear. In the latter work, AP-1
binding was highly associated with cisplatin resistance and increased
GCS-HS expression, whereas AP-2 and NF-B binding activities were not
(84)
. Using a cisplatin-resistant lung cancer cell line
SBC-3, Tomonari et al. described the 5'-flanking sequence from -192 to
+91 (especially -108 to -28 and +34 to +91 bp) to be involved in
cisplatin-induced transcriptional up-regulation of GCS-HS
(80)
. These regions contain two AP-2 sites, an Sp1 site
and a GCF binding site, but no AP-1 site (80)
. Different
mechanisms and cis-acting elements may be involved in
mediating transcriptional changes of GCS-HS in different cells.
Hormonal and cell cycle regulation of GCS
Transcriptional regulation
We and others have shown that GCS
activity and GCS-HS expression are under hormonal and growth-related
regulation (19
, 65
66
67
68
, 85)
. Specifically, in primary
cultures of rat hepatocytes the activity of GCS and the GCS-HS mRNA
level can be induced by insulin or hydrocortisone treatment or by
lowering the initial plating cell density (19
, 65
66
67)
.
The mechanism was an increase in the transcription of GCS-HS without
any change in the stability of the GCS-HS mRNA (66)
. The
effects of the hormones in cultured cells were confirmed using in
vivo models such as insulin-deficient diabetic or adrenalectomized
rats. Both exhibited lower hepatic GSH levels and GCS activity, which
were prevented with hormone replacement (19)
. However,
glucocorticoid administration to sham-operated animals did not result
in increased hepatic GSH or GCS activity (19)
. Thus, it
appears these hormones are required for maintenance of normal hepatic
GCS activity. Abnormality in GCS activity was observed only when there
was hormonal deficiency, not excess. In diabetic rats, the steady-state
hepatic GCS-HS mRNA level was decreased compared with controls, but
normal if treated with insulin (66)
. Thus, in these animal
models, a lower hepatic GSH and antioxidant defense may play a
pathogenetic role in the associated complications. It should be
stressed that these hormones may not exert similar effect in other cell
types. In fact, a recent study showed that dexamethasone actually had
the opposite effect, namely, lowering of cell GSH and GCS activity, in
a type II alveolar epithelial cell line (100)
. Thus,
results obtained from one cell type may not be applicable to another
and results obtained from cell lines may not be comparable to those
obtained from primary cultures or in vivo.
In the case of cell density, lowering the initial plating density
effectively shifts adult rat hepatocytes from the
Go to the G1 phase of the
cell cycle, analogous to liver regeneration after partial hepatectomy
or after cell death (101)
. Consistent with findings in
cultured rat hepatocytes, a doubling of the hepatic GSH level, GCS
activity, and the steady-state mRNA levels of GCS-HS were noted 12 h after two-thirds partial hepatectomy (68)
. The increase
in GSH under low cell density and after partial hepatectomy was due to
both an increase in GCS activity and cysteine availability (67
, 68)
. The increase in GSH occurred before the increase in DNA
synthesis during liver regeneration (68)
. Some
speculations can be made about the significance of the increase in
hepatic GSH and cysteine levels prior to DNA synthesis. Previous
studies involving lymphocytes and fibroblasts have shown that an
increased GSH level was associated with an early proliferative response
and was essential for the cell to enter the S phase
(102
103
104
105)
However, other studies did not find a
correlation between GSH levels and cell cycle (106
, 107)
.
The requirement for increased GSH or thiols prior to DNA synthesis may
be related to the fact that proliferating cells require increased
amounts of pentoses and thiols. DNA synthesis depends absolutely on the
formation of pentoses and on their conversion into deoxyribose by
ribonucleotide reductase (108)
. The activity of this
rate-limiting enzyme in DNA synthesis requires reduced glutaredoxin or
thioredoxin, which are maintained by GSH with concomitant oxidation to
GSSG via glutathione reductase or oxidation of NADPH via thioredoxin
reductase, respectively. Alternatively, an increase in the cellular GSH
content may change the thiol-redox status of the cell that activates
genes essential for G1 to S transition. Future
studies examining the molecular mechanism(s) of the cell-cycle effect
on GCS-HS gene expression as well as the significance of the increase
in hepatic GSH during liver regeneration will address these
possibilities.
In contrast to oxidative stress, which increased the transcription of
both heavy and light subunits of GCS, hormones and plating hepatocytes
at low cell density had no influence on the expression of GCS-LS
(65)
. Consistent with findings in cultured cells, GCS-LS
mRNA level also remained unchanged after partial hepatectomy
(68)
. Thus, at least in hepatocytes, there may be more
GCS-LS available than GCS-HS, so that a change in the GCS-HS alone
changed the GCS activity. That the two subunits are under different
regulatory controls have been suggested by others. Gipp et al. found no
correlation between the steady-state mRNA levels of the two GCS
subunits in different tissues (12)
. Huang et al. hinted
that liver might synthesize more light subunit than other tissues
relative to the amount of heavy subunit, although no data were provided
(11)
. Using HepG2 cells transfected with antisense
directed at GCS-LS, we observed a 80% reduction in the amount of
GCS-LS mRNA, accompanied by a significant reduction in the protein
level, but no change in the cell GSH level (unpublished observation).
This supports the notion that there is more GCS-LS in hepatocytes.
Kijima et al. used hammerhead ribozymes designed against the two GCS
subunits and showed that in Min-6 mouse islet cells transfected with
these ribozymes, this strategy resulted in a decrease of the GCS-HS and
GCS-LS expression to 27% and 86% of control, respectively
(109)
. Despite a minimal change in the GCS-LS expression,
cell GSH was depleted even more in the cells treated with ribozyme
against GCS-LS. The authors speculated that the low GSH level induced
the expression of GCS-LS mRNA level and reversed the decrease in GCS-LS
expression by the ribozyme. They concluded that both subunits
contributed equally to the enzyme activity. However, whether the
ribozyme against GCS-LS also affected the expression of GCS-HS is
unknown and the effect of the ribozymes on the GCS subunit protein
levels were not examined. Finally, there is likely to be cell-specific
differences in the differential regulation of the two GCS subunits.
Posttranslational regulation of GCS
GCS is also
regulated post-translationally. We and others reported that GSH
synthesis and GCS activity were acutely inhibited by hormone-mediated
activation of various signal transduction pathways (110
, 111)
. These hormones are secreted under stressful conditions,
many of which have associated lower hepatic GSH levels (7
, 112
, 113)
. The fall in hepatic GSH level can be explained by both an
increase in sinusoidal GSH efflux and an inhibition of GSH synthesis
(110
, 114)
. This may represent the hepatic stress response
by increasing the systemic delivery of GSH and cysteine to where they
are needed and channeling cysteine away from GSH synthesis to synthesis
of stress proteins, many of which are rich in cysteine
(115)
. Since the liver GSH level is normally very high
(5–10 mM), a slight fall in the GSH level would not jeopardize its
defense capacity. To explain the inhibition of GCS activity, we showed
that GCS-HS is phosphorylated directly by activation of protein kinase
A, protein kinase C or Ca2+-calmodulin kinase II
(CMK) (116)
. The degree of phosphorylation correlated with
loss of GCS activity (20% under physiological
Mg2+ concentration) and no additional inhibition
was observed when GCS was phosphorylated in the presence of all three
kinases. In addition, 2-dimensional phosphopeptide mapping studies
showed that the same five phosphopeptides were phosphorylated by these
kinases, suggesting that these kinases were acting on the same site(s).
Phosphorylation of GCS-HS was also demonstrated in cultured hepatocytes
after treatment with dibutyryl cAMP (DBcAMP) or phenylephrine using
specific antibodies that immunoprecipitated the phosphorylated
GCS-HS. There was basal GCS phosphorylation, which increased after
treatment with DBcAMP or phenylephrine, suggesting that GCS may be
under a basal inhibitory tone. The demonstration of phosphorylation of
GCS by using cultured hepatocytes suggests that
phosphorylation-dephosphorylation may be an important physiological
regulator of GCS. The inhibition of GCS activity after phosphorylation
of purified GCS was 20% at 1 mM, a physiologically relevant
Mg2+ concentration, which is similar to what we
observed when cultured rat hepatocytes or perfused rat livers were
treated with DBcAMP, glucagon, or phenylephrine (110
, 116)
. Although the degree of inhibition in GCS activity is
small, the effect on GSH synthesis and hepatic GSH turnover may be
significant. The turnover of normal hepatocyte GSH is estimated at
20%/h or 12 nmol/106 cells/h for a repleted
cell, which has 60 nmol GSH/106 cells
(36)
. This is the amount of GSH that is lost per hour,
which is normally balanced by synthesis of GSH. An adult rat liver
effluxes 12 µmol of GSH per hour (assuming 108
cells/g and 10 g=liver weight). Thus, even a 20% inhibition in GCS
activity would cause a significant reduction of GSH synthesis by the
liver (2.4 µmol per hour). Since many pathological and toxic
conditions result in sustained increase in cytosolic free
Ca2+, which can lead to activation of CMK and
phosphorylation of GCS, inhibition of GCS may theoretically further
contribute to toxicity under these conditions. The report by Lauterburg
and Mitchell that toxic doses of acetaminophen administered in
vivo suppressed hepatic GSH synthesis in rats raises this
possibility (117)
.
Post-translational regulation of the light subunit has not been well
studied. In our work on phosphorylation of the heavy subunit, no
phosphorylated light subunit was immunoprecipitated when using
anti-GCS-HS antibodies at the end of the reaction (116)
.
Thus, the light subunit is probably not phosphorylated.
Other conditions that alter GCS expression
In addition to oxidative stress, drug-resistance, hormones, and
plating density, several other conditions are also known to influence
the GCS activity and the steady-state GCS-HS mRNA level. These include
treatments with several antioxidants such as butylated hydroxyanisole,
GSH-conjugating agents such as DEM and phorone, heavy metals, heat
shock, copper deficiency, cycloheximide, TGFß1,
high glucose, and nitric oxide (NO) (60
, 70
71
72
73
74
75
, 79
, 86
, 95
, 96)
. Several of these treatments such as DEM, phorone and heavy
metals can also induce oxidative stress. All of these treatments, with
the exception of TGFß1 and high glucose
concentration, increased steady-state GCS-HS mRNA level. The mechanism
for the increase in most of these treatments was increased
transcription. In the case of cycloheximide, the increase in GCS-HS
mRNA level occurred by stabilization of the mRNA (79)
.
Finally, cellular GSH level itself may also modulate GCS expression
(see Areas of Controversy).
In addition to hormone (insulin and glucocorticoids) deficiency
(19
, 66)
, two other treatments that decreased GCS-HS
transcription are TGFß1 and high glucose
concentration (28 mM) (73
, 86)
. Arsalne et al. showed that
TGFß1 decreased GCS-HS transcription in a lung
epithelial cell line (86)
. They speculated whether
overexpression of TGFß1 in the alveolar
epithelial cells may contribute to low levels of GSH in the alveolar
epithelial lining fluid of patients with fibrotic lung disorders
(86)
. The molecular mechanism remains to be elucidated.
Regarding high glucose concentration, Urata et al. showed that GCS-HS
mRNA level and transcriptional rates in mouse endothelial cells were
increased by treatment with cytokines such as TNF and IL-1ß
(73)
. The basal GCS-HS mRNA level was decreased and the
effect of the cytokines disappeared when cells were grown in media
containing high glucose concentration (28 mM). This raises the question
of whether the decreased GCS activity and GCS-HS mRNA level observed in
diabetic rat livers is due to lack of insulin or hyperglycemia or both.
In our work, cultured hepatocytes were grown in medium containing 17.5
mM glucose ± insulin (19
, 66)
. Thus, it is likely
that insulin was involved in increasing GCS-HS gene transcription in
cultured hepatocytes. However, glucose can influence gene expression by
itself and the effect of insulin may or may not require glucose
(118
, 119)
. More work is needed to elucidate the exact
mechanism of how insulin and glucose influence GCS-HS gene expression.
The effect of NO on GCS expression is dependent on the cell type
(95
, 96)
. In rat hepatocytes, NO exerted no influence on
the basal GSH level or GCS expression (95)
. However, when
these cells were treated with interleukin-1 (IL-1), which stimulated
the expression of inducible nitric oxide synthase, the GSH level was
depleted and GCS expression decreased if NO synthesis was blocked
(95)
. This would suggest that in response to oxidative
stress induced by IL-1, rat hepatocytes up-regulate GCS expression by a
mechanism that depends on NO synthesis. In contrast, treatment of rat
aortic vascular smooth muscle cells with NO at physiological
concentrations resulted in increased cell GSH levels and expression of
both GCS subunits (96)
. This illustrates the cell
type-specific complex interactions between NO and GSH.
Finally, we showed recently that the GCS-HS mRNA level was increased in
mutant Eisai Hyperbilirubinemic rats (78)
. This along with
increased cysteine availability contributed to the high GSH levels
found in liver and many other organs (78)
. The mechanism
for the increased GCS-HS mRNA is unknown and it is speculative whether
retained GSH conjugates may have contributed to the increase.
|
AREAS OF CONTROVERSY |
|---|
|
TOPABSTRACTCURRENT CONCEPTS: STRUCTURE AND...SYNTHESIS OF HEPATIC GSHAREAS OF CONTROVERSYSUMMARY AND FUTURE DIRECTIONSREFERENCES |
|---|
B in modulation of the GCS-HS gene expression, and the role of
cell GSH itself in the modulation of GCS expression.
GSH in apoptosis
Apoptotic cell death is one of the most intensely studied research
areas. Whether GSH and/or the intracellular redox state modulate
apoptosis is controversial. Numerous studies have shown that apoptotic
cells accumulate oxidized proteins and lipids and are presumably
exposed to some degree of oxidative stress; antioxidants, including
GSH, have been shown to protect against or delay apoptosis triggered by
many different stimuli (120
121
122
123
124)
. However, apoptosis has
been shown to occur under very low oxygen environments indicating that
reactive oxygen species are unlikely to be essential mediators under
these conditions (121)
. Another study showed that the
protective effect of thiol agents may be related to down-regulation of
Fas expression on T lymphocytes rather than their antioxidative
properties (123)
. What is clear is that there is
accelerated GSH efflux from the cell stimulated to undergo apoptosis.
This was shown by several groups in several cell types (mostly
lymphocytes) with different proapoptotic stimuli (120
, 121
, 124)
. GSH loss occurred prior to chromatin fragmentation or
development of oxidative stress, required protease activation, and was
blocked by agents known to inhibit GSH transport (121)
.
Thus, increased export of GSH represents an oxygen-independent
mechanism for a fall in the cellular redox state. However, apoptotic
degradation of nuclear DNA still occurred with the same kinetics even
when GSH efflux was blocked (121)
. Similar results were
obtained by Ghibelli et al. (120)
. GSH loss occurred
concomitant with the onset of apoptosis, and modulation of
intracellular GSH did not influence the overall extent of apoptosis.
These findings seem to dispute the importance of the redox state in
modulating apoptosis. What is also unclear is the significance of the
GSH efflux during apoptosis and whether this occurs in other cell
types. One speculation is that depletion of cell GSH will facilitate
apoptosis to occur, provide antioxidants extracellularly, and possibly
stimulate phagocytic cells to engulf the apoptotic cell
(121)
.
Role of NF-B
Another area of controversy is the role of NF-B in modulation
of the GCS-HS gene expression. There is a NF-B consensus element
~1kb upstream of the transcriptional start site of the human GCS-HS
(63)
. Oxidative stress is known to induce NF-B
activity, and Urata et al. showed that blocking activation of NF-B
by antisense strategies prevented the cytokine-induced increase in
GCS-HS transcription in mouse endothelial cells (73)
. We
also showed blocking the activation of NF-B prevented the increase
in the GCS-HS mRNA level induced by BSO and
tert-butylhydroquinone in rat hepatocytes (65)
.
Iwanaga et al. also implicated the NF-B consensus element, but not
AP1 or ARE, in mediating the effect of ionizing radiation on GCS-HS
expression in a human glioblastoma cell line (90)
.
However, the NF-B consensus element was shown not to be involved in
mediating the effect of TNF, okadaic acid, and ionizing radiation on
GCS-HS expression in HepG2 cells (64
, 81
, 88)
. The
explanations for these discrepant results are unclear, and whether
there is species and/or cell type differences in mediating the effect
of these agents at the GCS-HS promoter level remains to be examined.
Role of cell GSH in GCS expression
Several lines of evidence suggest that the expression of GCS-HS is
modulated by cell GSH itself. The first is from the work of Urata and
colleagues (73)
. When mouse endothelial cell GSH was
increased by GSH ester, the steady-state mRNA level of GCS-HS
increased, regardless of the glucose concentration in the medium
(73)
. We have also observed that the expression of both
subunits of GCS was induced by BSO only after the cell's GSH level
fell below 10% (73)
. However, this may have been related
to oxidative stress. More recently, Yamane et al. showed that an
increasing GSH level of ~50% in a rat hepatoma cell line by
overexpression of human GCS-HS cDNA resulted in a decrease in the
endogenous rat GCS-HS mRNA level by ~50% (92)
. Thus, in
addition to exerting negative feedback on the GCS enzyme itself,
increased GSH may also down-regulate GCS-HS expression. These results
conflict directly with those of Urata et al. (73)
. It is
unclear whether the discrepancy is related to the cell type studied,
the method used to increase GSH, or other unknown factors. Whether the
light subunit is also affected by an increase in cell GSH is unknown.
These unresolved issues deserve further investigation.
|
SUMMARY AND FUTURE DIRECTIONS |
|---|
|
TOPABSTRACTCURRENT CONCEPTS: STRUCTURE AND...SYNTHESIS OF HEPATIC GSHAREAS OF CONTROVERSYSUMMARY AND FUTURE DIRECTIONSREFERENCES |
|---|
, methionine adenosyltransferase; 
,
transmethylation reactions; 
, S-adenosylhomocysteine hydrolase; 
,
cystathione ß-synthase; 
, 
,
, GSH synthetase; 
, methionine
synthase; 
, betaine-homocysteine methyltransferase.