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FORUM ORIGINAL RESEARCH COMMUNICATION
Interaction of Methylglyoxal and Hydrogen Sulfide
in Rat Vascular Smooth Muscle Cells
Tuanjie Chang,*Ashley Untereiner,*Jianghai Liu, and Lingyun Wu
Abstract
Hydrogen sulfide (H
2
S) is a gasotransmitter with multifaceted physiological functions, including the regulation
of glucose metabolism. Methylglyoxal (MG) is an intermediate of glucose metabolism and plays an important
role in the pathogenesis of insulin resistance syndromes. In the present study, we investigated the effect of MG
on H
2
S synthesis and the interaction between these two endogenous substances. In cultured vascular smooth
muscle cells (VSMCs), MG (10, 30, and 50 mM) significantly decreased cellular H
2
S levels in a concentration-
dependent manner, while H
2
S donor, NaHS (30, 60, and 90 mM), significantly decreased cellular MG levels. The
expression level and activity of H
2
S-producing enzyme, cystathionine g-lyase (CSE), were significantly decreased
by MG treatment. NaHS (30–90 mM) significantly inhibited MG (10 or 30 mM)-induced ROS production. Cellular
levels of GSH, cysteine, and homocysteine were also increased by MG or NaHS treatment. Furthermore, direct
reaction of H
2
S with MG in both concentration- and time-dependent manners were observed in in vitro incu-
bations. In conclusion, MG regulates H
2
S level in VSMCs by downregulating CSE protein expression and directly
reacting with H
2
S molecule. Interaction of MG with H
2
S may be one of future directions for the studies on glucose
metabolism and the development of insulin resistance syndromes. Antioxid. Redox Signal. 12, 1093–1100.
Introduction
Hydrogen sulfide (H
2
S) is the third gasotransmitter
with multifaceted physiological functions (5, 19). Two
pyridoxal-5’-phosphate-dependent enzymes, cystathionine
b-synthase (CBS) and cystathionine g-lyase (CSE), are re-
sponsible for the majority of endogenous H
2
S production in
mammalian tissues using L-cysteine as the substrate (1, 16).
The expression of CSE and CBS is tissue specific. For instance,
CBS is the major H
2
S producing enzyme in the nervous sys-
tem, whereas CSE is mainly expressed in vascular and
nonvascular smooth muscle cells (5, 19, 29). Another less
important endogenous source of H
2
S is the nonenzymatic
reduction of elemental sulfur to H
2
S using reducing equiva-
lents obtained from the oxidation of glucose (15).
H
2
S exerts a host of biological effects on various types of
cells and tissues. At micromolar concentrations, H
2
S can have
cytoprotective effects (26), while at millimolar concentrations
it has been shown to be cytotoxic (6, 27, 28). Previous studies
have also proved that H
2
S upregulates the expression of anti-
inflammatory and cytoprotective genes, including heme
oxygenase-1 in pulmonary artery smooth muscle cells (14)
and macrophages (13). The vascular relaxation effect of H
2
S
was proved largely due to the opening of K
ATP
channels (26,
30). In line with its vasorelaxant effect, a H
2
S donor was
shown to induce a transient hypotensive response in animals
(19, 30). In patients with coronary heart disease, plasma
H
2
S level was reduced from *50 to *25 mM(9). We recently
showed that CSE deficiency and reduced endogenous H
2
S
production in vascular tissues resulted in the development of
hypertension in CSE gene knockout mice (29).
Methylglyoxal (MG) is a metabolite of sugar, protein, and
fatty acid, formed in virtually all mammalian cells, including
vascular smooth muscle cells (VSMCs) (10). Increased MG
production has been reported in human red blood cells,
bovine endothelial cells, and VSMCs under hyperglycemic
conditions or with increased availability of MG precursors
such as fructose (18). We recently discovered that hyperten-
sion in spontaneously hypertensive rats was related to
increased MG levels in plasma and vascular tissues in an age-
dependent fashion (20, 21). It has been reported that an ele-
vated MG level is associated with oxidative stress in vascular
tissues (22, 25). MG can induce the production of reactive
oxygen species (ROS), including peroxynitrite (ONOO
-
), hy-
drogen peroxide (H
2
O
2
), and superoxide anion (O
2.-
) in cul-
tured VSMCs (2). Moreover, as a highly reactive dicarbonyl
molecule, MG can interact with the side chains of arginine,
lysine, and cysteine residues in proteins to yield different
Department of Pharmacology, College of Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada.
*These authors contributed equally to this work.
ANTIOXIDANTS & REDOX SIGNALING
Volume 12, Number 9, 2010
ªMary Ann Liebert, Inc.
DOI: 10.1089=ars.2009.2918
1093
types of advanced glycation endproducts (3). In the present
study, we investigated the interaction of MG and H
2
Sin
VSMCs and the cellular effects of this interaction.
Materials and Methods
VSMC preparation
Rat thoracic aortic smooth muscle cell line (A-10 cells) was
obtained from American Type Culture Collection (Manassas,
VA) and cultured in Dulbecco’s modified Eagle’s medium
(DMEM) containing 10% bovine serum, 100 U=ml penicillin,
and 100 mg=ml streptomycin at 378C in a humidified atmo-
sphere of 95% air and 5% CO
2
, as described in our previous
study (2). Cultured cells were grown to 60*80% of confluence
before starved in serum-free DMEM for 24 h and then exposed
to MG or H
2
S treatments for 24 h. Treated and untreated cells
were washed with ice-cold phosphate-buffered saline (PBS),
and then harvested by trypsinization. For the determination
of oxidized DCF production, cells were seeded in 96-well
plates with equal amount of cells in each well (*410
4
cells)
and treated as indicated above.
Measurement of cellular H
2
S levels
H
2
S was measured using a microelectrode as previously
described (7, 25). Briefly, harvested cells were resuspended
in 400 ml of cell lysis buffer (20 mMTris-HCl at pH 7.5,
150 mMNaCl, 1 mMEDTA, 1 mMEGTA, 1% Triton X-100,
2.5 mMsodium pyrophosphate, 1 mMb-glycerolphosphate,
1mMNa
3
VO
4
,1mMphenylmethylsulfonyl fluoride, and a
proteinase inhibitor cocktail). H
2
S in cell lysis (200 ml) was
released by adding 5 ml of 80% sulfuric acid in a sealed fil-
tering flask bubbled with N
2
gas for 15 min. Released H
2
S was
carried by N
2
gas to an absorber container (15 ml test tube),
containing 1 ml of 1 MNaOH into which H
2
S was absorbed.
H
2
S level in NaOH was measured with a microelectrode
specific for sulfide (Lazar Research Laboratories Inc., Los
Angeles, CA). H
2
S concentration was calculated using a
standard curve of NaHS at different concentrations and is
expressed in nmol=mg protein.
Measurement of cellular MG level
Collected cells were lysed in a cell lysis buffer containing
a proteinase inhibitor cocktail. MG in the supernatant of cell
lysis was measured as previously reported (7,21). Briefly,
samples were incubated with 10 mMo-phenylenediamine
(o-PD, derivatizing agent) for 3 h at room temperature and
protected from light. The quinoxaline formed between di-
carbonyl compounds and o-PD, as well as the internal stan-
dard (5-methylquinoxaline) were measured using a Hitachi
D-7000 high-performance liquid chromatography (HPLC)
system (Hitachi Ltd., Mississauga, Ontario, Canada) (21). A
Nova-Pak C18 column was used (Waters, MA). The mobile
phase was composed of 8% (v=v) of 50 mMNaH
2
PO
4
(pH
4.5), 17% (v=v) of HPLC grade acetonitrile, and 75% of water.
Samples were measured in triplicates.
Measurement of CSE activity
CSE enzyme activity was determined by measuring the
production rate of H
2
S as reported (25). Briefly, collected cells
were suspended in 400 ml of ice-cold potassium phosphate
buffer (50 mM, pH 6.8) supplemented with proteinase inhib-
itor cocktail and lysed by sonication on ice. Supernatant
(100 ml) was added to 1 ml of reaction mixture containing
(mM): 100 potassium phosphate buffer (pH 7.4), 10 L-cysteine,
and 2 pyridoxal-5’-phosphate. Cryovial test tubes (2 ml) were
used as the center wells, each containing 0.5 ml 1% zinc ace-
tate as trapping solution. Reaction was performed in a 25 ml
Erlenmeyer flask. The flasks containing the reaction mixture
and center wells were flushed with N
2
gas before being sealed
with a double layer of parafilm. Reaction was initiated by
transferring the flasks from ice to a 378C shaking water bath.
After incubating at 378C for 90 min, 0.5 ml of 50% tri-
chloroacetic acid was added to stop the reaction. The flasks
were sealed again and incubated at 378C for another 60 min to
ensure a complete trapping of released H
2
S gas from the
mixture. Contents of the center wells were then transferred to
test tubes, each containing 0.5 ml of water. Subsequently,
0.5 ml of 20 mMN,N-dimethyl-p-phenylenediamine sulfate in
7.2 NHCl was added immediately followed by addition of
0.5 ml 30 mMFeCl
3
in 1.2 NHCl. The mixture was kept at
room temperature, protected from light, for 20 min followed
by recording the absorbance at 670 nm with a spectropho-
tometer. H
2
S concentration was calculated using a calibration
curve of standard NaHS solutions.
RNA isolation and real-time quantitative PCR
Total RNA was isolated using RNeasy Mini Kit (QIAGEN,
Mississauga, Ontario, Canada) according to the manufactur-
er’s instructions. First strand cDNA was prepared from total
RNA (5 mg) by reverse transcription using M-MLV reverse
transcriptase (Invitrogen, Burlington, Ontario, Canada) and
oligo(dT) primer. Real-time quantitative PCR was performed
on iCycler iQ Real-time PCR Detection System (Bio-Rad,
Hercules, CA). The primers of rat CSE were as following:
forward 50-GGACAAGAGCCGGAGCAATGGAGT-30, reverse
50- CCCCGAGGCGAAGGTCAAACAGT-30.Theprimersfor
rat b-actin were: forward 50-CGTTGACATCCGTAAAGAC-30
and reverse 50-TAGGAGCCAGGGCAGTA-30. The PCR con-
ditions were as following: denaturation at 958C for 3 min,
followed by 40 cycles of denaturation at 958C for 30 s, an-
nealing at 558C for 1 min, and extension at 728C for 30 s.
Specificity of the amplification was determined by melting
curve analysis. Data were expressed as a ratio of the quantity
of CSE mRNA to the quantity of b-actin mRNA.
Western blot analysis of CSE expression
Total proteins were extracted from harvested cells with
300 ml of cell lysis buffer as described above. Proteins (40 mg)
were subject to Western blot analysis according to the pro-
cedure reported (4). The primary antibody dilutions were
1:500 for antibodies against CSE (Abnova, Taipei, Taiwan)
and 1:5000 for b-actin. Western blots were digitized with
Chemi Genius
2
Bio Imaging System (SynGene, Frederick,
MD), quantified using software of GeneTools from SynGene
and normalized against the quantity of loaded b-actin.
Measurement of ROS production
The formation of oxidized DCF was determined by a DCFH
assay as described previously with minor modification
(2). Briefly, starved cells were loaded with a membrane-
1094 CHANG ET AL.
permeable and nonfluorescent probe DCFH-DA for 2 h at
378C in phenol red-free DMEM, protected from light. There-
after, the cells were washed three times with phenol red-free
DMEM to remove the excess probe, followed by the treatment
with or without MG or MG plus NaHS at desired con-
centrations for different time periods in phenol red-free
DMEM. Once inside cells, DCFH-DA becomes the membrane-
impermeable DCFH
2
in the presence of cytosolic esterases and
further oxidized by H
2
O
2
or ONOO
to form oxidized DCF
with detectable fluorescence. Oxidized DCF was quantified
by monitoring DCF fluorescence intensity with excitation at
485 nm and emission at 527 nm with a Fluoroskan Ascent
plate reader (Thermo Labsystem, Beverly, MA) using Ascent
software and expressed in arbitrary units.
Measurement of GSH, L-cysteine,
and homocysteine levels
Levels of reduced glutathione (GSH), L-cysteine, and
homocysteine in the supernatant of cell lysis were determined
by derivation with 5, 5’-dithiobis (2-nitrobenzoic acid)
(DTNB) and reverse-phase HPLC using ultraviolet detection,
as described in our previous study (20). Briefly, the reaction
mixture for the analysis of free reduced sulfhydryl groups
contained 250 ml 500 mMTris–HCl buffer (pH 8.9), 65 ml
sample or standard, 10 ml internal standard (400 mMD(-)-
penicillamine in cold 5% sulfosalicylic acid containing 0.1 mM
EDTA), and 175 ml10mMDTNB made up in 0.5 mMK
2
HPO
4
(pH 7.2). After 5 min of derivatization, the mixture was
acidified with 21.5 ml7MH
3
PO
4
, and 50 ml of the mixture was
injected into the HPLC system. Chromatography was ac-
complished using isocratic elution on a Supelcosil LC-18-T
column (1504.6 mm, 3 mm) incubated at 378C. The mobile
phase consisted of 12.5% methanol (v=v) and 100 mM
KH
2
PO
4
(pH 3.85) at a flow rate of 0.9 ml=min. Sulfhydryl–
DTNB derivatives were detected by ultraviolet absorbance at
345 nm. After 10 min of isocratic elution, the methanol con-
centration was increased to 40% and pumped for 8 min to
elute excess DTNB reagent from the column. The methanol
concentration was then decreased to 12.5% and pumped for
7 min before the next sample injection. For analyte quantifi-
cation, standard curves were constructed by spiking the su-
pernatant with various known amounts of GSH, L-cysteine,
and homocysteine (Sigma, Oakville, Ontario, Canada). Sam-
ples were run in duplicate. Data were collected digitally with
D-7000 HPLC System Manager software, Hitachi, Ltd., (Mis-
sissauga, Ontario, Canada) and peak areas were quantified.
Direct reaction of H
2
S with MG
MG (10 mM) was mixed with H
2
S at different concentra-
tions (10, 50, and 100 mM) in cell-free PBS buffer and incubated
at 378C for 1*24 h. H
2
S stock solution was prepared by
bubbling H
2
S in distilled H
2
O for 30 min (30). After incuba-
tion, free MG was measured with HPLC as described above.
Chemicals and data analysis
MG and NaHS were obtained from Sigma-Aldrich (Oak-
ville, Canada). The data are expressed as mean SEM from at
least three independent experiments. Statistical analyses were
performed using Student’s ttest or ANOVA. Statistical sig-
nificance was considered at p<0.05.
Results
MG treatment decreased H
2
S level
in VSMCs and vice versa
Cultured VSMCs were treated with MG at different con-
centrations for 24 h. After MG treatment, H
2
S levels in cell
lysates were significantly decreased in a MG concentration-
dependent fashion (Fig. 1). In another group of experiments,
the effect of H
2
S treatment on MG level was studied. Cultured
VSMCs were treated with H
2
S donor, NaHS, at different con-
centrations for 24 h. After NaHS treatment, MG levels in cell
lysates were significantly decreased in a NaHS concentration-
dependent manner (Fig. 2).
MG-induced downregulation of CSE
protein expression
In VSMCs, CSE is the major enzyme responsible for H
2
S
production. Thus, the CSE expression level in MG-treated
cells was investigated. Quantitative-PCR results indicated
that MG treatment did not significantly affect CSE mRNA
levels (Fig. 3A). Western blot results showed that CSE protein
level was significantly lower in 30 and 50 mMMG-treated
cells, but not with 10 mMMG treatment (Fig. 3B and C). As
shown in Fig. 3D, CSE activity in 30 and 50 mMMG-treated
cells was significantly decreased by 14% and 29% in com-
parison to the untreated control. CSE activity in 10 mMMG-
treated group was lower than the untreated control although
the difference was not significant.
Effect of H
2
S on MG-induced ROS production
ROS formation in VSMCs was significantly increased by MG
(10, 30, and 50 mM) in both time- and concentration-dependent
manners (Fig. 4A and B). Interestingly, 30 mMNaHS signifi-
cantly decreased 10 mMMG-induced ROS production, but not
30 or 50 mMMG (Fig. 4A). The antioxidant effect of H
2
S was
observed within 8 h of application and continued thereafter
(Fig. 4B). NaHS at 60 and 90 mMdecreased 10 mMMG-
induced ROS production (Fig. 4C). This effect of NaHS was
more potent when the cells were treated with 30 mMMG
FIG. 1. H
2
S level in MG-treated VSMCs. VSMCs were
treated with MG at 10, 30, and 50 mMfor 24 h, respectively.
H
2
S level in cell lysis was expressed in nmol=mg protein.
n¼4*6; *p<0.05 vs. control;
#
p<0.05 vs. MG (10 mM).
INTERACTION OF MG WITH H
2
S 1095
(Fig. 4D). However, NaHS no longer offered the antioxidant
effect against MG-induced ROS production once its concen-
tration reached 120 mM(Fig. 4C and D).
GSH, cysteine, and homocysteine levels
in MG- or NaHS-treated VSMCs
GSH is an important antioxidant agent that protects the cells
from oxidative stress (31). After VSMCs were treated with MG
for 24 h, cellular GSH level was significantly increased cor-
respondingly to the concentrations of MG (10–50 mM) (Fig.
5A).Homocysteine is a precursor of cysteine synthesis, while
cysteine is a precursor of GSH. MG treatment at 10 mM, but
not 30 or 50 mM, significantly increased cysteine and homo-
cysteine levels in cultured VSMCs (Fig. 5B and C).
The effects of H
2
S on GSH, cysteine, and homocysteine
levels were also investigated. Cellular GSH levels were sig-
nificantly increased when cells were exposed to NaHS treat-
ment as compared with control cells (Fig. 6A). However, GSH
levels in 60 and 90 mMNaHS-treated cells were significantly
lower than that in 30 mMNaHS-treated cells. L-cysteine levels
were also significantly increased by NaHS treatments (30, 60,
and 90 mM) (Fig. 6B).
FIG. 2. MG level in NaHS-treated VSMCs. VSMCs were
treated with H
2
S donor NaHS at 30, 60, and 90 mMfor 24 h,
respectively. MG level in cell lysis was measured using HPLC
as described in Materials and Methods; n¼3, *p<0.05 or
**p<0.01 vs. control;
#
p<0.05 vs. NaHS (30 or 60 mM).
FIG. 3. Effects of MG treatment on CSE expression and activity. VSMCs were treated with MG at 10, 30, and 50 mMfor
24 h, respectively. Treated cells were collected for RNA or protein extraction. (A) Real-time PCR results of CSE mRNA level in
MG-treated cells; n¼6 for each group. (B) and (C) CSE protein level in MG-treated cells; n¼4 for each group, *p<0.05 vs.
control. (D) CSE activity in MG-treated cells; n¼6*9, *p<0.05 or **p<0.01 vs. control;
#
p<0.05 vs. MG (30 mM).
1096 CHANG ET AL.
Direct reaction of MG with H
2
S
To understand the mechanism of H
2
S and MG interaction,
we tested whether MG directly reacts with H
2
S molecule. For
this purpose, MG (10 mM) was mixed with H
2
S at different
concentrations in cell-free PBS buffer and incubated at 378C
for 24 h. After incubation, free MG was measured with HPLC.
H
2
S at 50 and 100 mM, but not at 10 mM, significantly de-
creased MG levels (Fig. 7A). When the H
2
S incubation time
was <4 h, no change in MG level was observed. However,
significant decreases in MG levels were detected after 8 h of
incubation with the lowest level detected after 18 h incubation
with H
2
S (Fig. 7B).
Discussion
Under physiological condition, MG level is generally
higher in vascular tissue than in other types of tissues (20).
CSE is responsible for H
2
S production in vascular tissue,
endothelium, pancreas, and liver, while CBS produces H
2
S
mainly in brain and kidney (19, 25, 29). Obviously, MG and
H
2
S are co-produced in VSMCs. For instance, we found that
the interaction of MG and H
2
S lowers their respective cellular
levels. We also found that CSE protein level was down-
regulated by MG (30 and 50 mM) although no change of CSE
mRNA level in MG-treated cells was observed. The above
results indicate that MG treatment may decrease the transla-
tion of CSE mRNA or the stability of CSE proteins. However,
the underlying mechanisms of MG treatment on CSE mRNA
translation and the protein stability are not yet clear and will
need further investigation. Consistent with decreased CSE
protein levels after MG treatments, CSE enzyme activity was
also decreased as indicated by a lower production rate of H
2
S.
Therefore, MG-induced decrease in CSE protein level could at
least in part explain the decrease in H
2
S level.
Whereas endogenous cellular H
2
S level was significantly
decreased by 10 mMMG treatment, CSE protein level or CSE
activity was not significantly different from the untreated
control. This phenomenon could be explained by a direct re-
action between MG and H
2
S, considering H
2
S as a reducing
agent and MG as a reactive dicarbonyl molecule. We found
FIG. 4. Effect of NaHS on MG-induced ROS production in VSMCs. (A) Oxidized DCF level in cells treated with MG at
different concentrations in the presence of NaHS (30 mM). (B) Time-dependent effect of NaHS (30 mM) on MG (10 mM)-
induced ROS production. (C) and (D) Concentration-dependent effect of NaHS on MG (10 and 30 mM)-induced ROS pro-
duction, n¼8 for each group, *p<0.05 or **p<0.01 vs. untreated control;
#
p<0.05 vs. MG (10 mM)orvs. MG (30 mM)þNaHS
(30 mM);
þ
p<0.05 vs. MG (10 mM)þNaHS (30 mM).
INTERACTION OF MG WITH H
2
S 1097
that MG level in the cell-free MG=H
2
S mixture was decreased
8 h after the incubation was started. This chemical reaction
between MG with H
2
S occurred in both time- and concen-
tration-dependent manners. These data suggest that a direct
reaction of MG with H
2
S may be responsible for lower MG
(10 mM)-induced decrease in H
2
S level, while MG at 30–50 mM
caused both a direct molecule-to-molecule reaction, as well as
the downregulation of CSE protein expression. Consistently,
the direct reaction of MG with H
2
S may have caused the de-
creased MG level in H
2
S donor-treated VSMCs.
In our previous study, we showed that MG increased ROS
production in VSMCs by increasing ONOO
,H
2
O
2
, and O
2
levels (2, 3). We also showed that H
2
S protected VSMCs
against homocysteine-induced oxidative stress (26). It was of
interest to study the effect of H
2
S on MG-induced ROS pro-
duction. Our results support the notion that H
2
S acts as an
antioxidant (26). At the concentrations lower than 90 mM,H
2
S
decreased 10 and 30 mMMG-induced ROS production in a
concentration-dependent manner, but had no effect on 50 mM
MG-induced ROS generation. This could be due to the fact
that MG-induced ROS formation at high concentrations
overwhelms the antioxidant ability of H
2
S. It is also important
to note that H
2
S at concentration higher than physiological
related concentration, for example, 120 mMfails to inhibit
low concentrations of MG (10 and 30 mM)-induced ROS pro-
duction. This may be related to the toxicity and pro-apoptosis
effect of H
2
S at high concentrations (6, 27, 28).
One of the most important and abundant endogenous an-
tioxidant is GSH, which is found at millimolar range in most
FIG. 5. GSH, cysteine, and homocysteine levels in MG
treated VSMCs. Cells treated with MG at different concen-
trations were harvested after 24 h to determine cellular GSH
(A), cysteine (B), and homocysteine (C) levels using HPLC
method as described in Materials and Methods; n¼4*7 for
each group; *p<0.05 or **p<0.01 vs. untreated control.
FIG. 6. GSH and cysteine levels in NaHS-treated VSMCs.
Cells treated with NaHS at different concentrations were
harvested after 24 h to determine cellular GSH (A) and cys-
teine (B) levels using HPLC method as described in Materials
and Methods; n¼4 for each group; *p<0.05 or **p<0.01 vs.
untreated control;
#
p<0.05 vs. NaHS (30 mM).
1098 CHANG ET AL.
cells (31). GSH levels are significantly elevated when the cells
are treated with MG (10, 30, and 50 mM) compared to that of
the control group. Cysteine availability is the rate-limiting
step in GSH synthesis, and homocysteine is the precursor to
cysteine. We observed a corresponding increase of cysteine
and homocysteine levels in cells treated with MG at the con-
centration of 10 mM, but not 30 or 50 mM. This may be due to
the huge consumption of cysteine and homocysteine in order
to maintain GSH at a certain level to compensate the higher
ROS levels induced by MG (30 and 50 mM). On the other hand,
H
2
S treatment of VSMCs also increased GSH level, which may
be attributed to H
2
S-enhanced activity of g-glutamylcysteine
synthetase (11). Furthermore, H
2
S may cause a feedback in-
hibition of CSE (12), which could lead to a decreased breakage
of cysteine to produce H
2
S. Consistently, the increased level of
cysteine was observed after H
2
S treatment. The consequent
increased level of cysteine may inhibit the demethylation of
methionine to produce homocysteine (17).
The physiological relevance for the interaction between
MG and H
2
S has not been previously investigated. Elevated
MG level is linked with the development of hypertension and
insulin resistance (3, 8, 23). In vascular tissue, elevated MG
level is expected to lead to a decreased H
2
S level based on the
direct reaction of MG with H
2
S and the downregulation of
CSE expression by MG. One of the consequences of abnor-
mally low H
2
S level would decrease the opening of K
ATP
channels and impair vascular relaxation, causing an increased
peripheral circulation resistance and hypertension develop-
ment or vascular complications of diabetes. In conclusion, MG
can react with H
2
S and cause a downregulation of the ex-
pression of CSE. MG may reduce H
2
S production, whereas
H
2
S may limit the availability of free MG. As mentioned be-
fore, CSE is mainly expressed in vascular tissue, endothelium,
pancreas, and liver, while MG is produced virtually all
mammalian cells. Therefore, the interaction of MG with H
2
S
are expected to occur in VSMCs and possibly other types
of tissues, which may provide one of future directions for
the studies on glucose metabolism and the development of
insulin resistance syndromes.
Acknowledgments
This work was supported by operating grants from the
Canadian Institutes of Health Research (CIHR) and Heart and
Stroke Foundation of Saskatchewan (HSFS) to L Wu.
Author Disclosure Statement
No competing financial interests exist.
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FIG. 7. Reaction of H
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S with MG. MG (10 mM) was mixed
with H
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S at different concentrations (10, 50, and 100 mM)in
PBS buffer and incubated at 378C for 24 h. Free MG in the
mixtures was measured with HPLC as described in Materials
and Methods; n¼3, *p<0.05 or **p<0.01 vs. MG (10 mM);
#
p<0.05 vs. MG (10 mM)þH
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S (50 mM) or MG (10 mM)þH
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S
(50 mM) 8 h after incubation.
INTERACTION OF MG WITH H
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Address correspondence to:
Dr. Lingyun Wu
Department of Pharmacology
College of Medicine
University of Saskatchewan
107 Wiggins Road
Saskatoon, Saskatchewan S7N 5E5
Canada
E-mail: lily.wu@usask.ca
Date of first submission to ARS Central, September 24, 2009;
date of final revised submission, September 27, 2009; date of
acceptance, October 3, 2009.
Abbreviations Used
CBS ¼cystathionine b-synthase
CSE ¼cystathionine g-lyase
GSH ¼reduced glutathione
H2S¼hydrogen sulfide
MG ¼methylglyoxal
NaHS ¼sodium hydrosulfide
ROS ¼reactive oxygen species
VSMCs ¼vascular smooth muscle cells
1100 CHANG ET AL.
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