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Cardiovascular Research (2008) 80, 255–262
doi:10.1093/cvr/cvn179
Na1/H1 exchanger is required
for hyperglycaemia-induced endothelial
dysfunction via calcium-dependent calpain
Shuangxi Wang, Qisheng Peng†, Junhua Zhang‡, and Liying Liu*
Department of Pharmacology, Pharmaceutical College, Central South University, 110 Xiang-Ya Road, Changsha, Hunan
410078, China
Received 27 February 2008; revised 24 June 2008; accepted 26 June 2008; online publish-ahead-of-print 30 June 2008
Time for primary review: 34 days
KEYWORDS
Aims Recent studies have reported that the calcium-dependent protease calpain is involved in
hyperglycaemia-induced endothelial dysfunction and that the Naþ/Hþ exchanger (NHE) is responsible
for an increase in the intracellular calcium (Ca2þ
i ) concentration in diabetes. We hypothesized that activation of NHE mediates hyperglycaemia-induced endothelial dysfunction via calcium-dependent calpain.
Methods and results Exposure of human umbilical vein endothelial cells (HUVECs) to high glucose (HG,
30 mM D-glucose) time dependently increased both the Ca2þ
concentration and calpain activity.
i
Chelation of free Ca2þ
with 1,2-bis (2-aminophenoxy) ethane-N, N, N0 ,N0 -tetraacetic acid abolished the
i
HG-increased calpain activity. In addition, HG activated NHE in a time-dependent manner, but
cariporide, an NHE inhibitor, blocked the HG-induced increase in NHE activity. Furthermore, cariporide
or NHE siRNA (small interfering ribonucleic acid) attenuated the HG-induced increases of both Ca2þ
i
concentration and calpain activity. All of these HG-induced effects in HUVECs, including decreased
endothelial nitric oxide synthase (eNOS) activity and NO (nitric oxide) production and increased
dissociation of heat shock protein (hsp90) from eNOS, were NHE or calpain reversible. In vivo
experiments showed that cariporide treatment via inhibition of NHE activity signiﬁcantly attenuated
the hyperglycaemia-induced impairment of acetylcholine-induced endothelium-dependent relaxation
in streptozotocin-injected diabetic rats.
Conclusion Activation of NHE via calcium-dependent calpain contributes to hyperglycaemia-induced
endothelial dysfunction through dissociation of hsp90 from eNOS.
1. Introduction
Diabetes is associated with a signiﬁcantly increased risk of
cardiovascular disease, including atherosclerosis,1 coronary
artery disease,2 and microvascular complications.3 Hyperglycaemia is thought to play a key pathogenic role in the development of diabetic cardiovascular disease. High blood glucose
concentration results in endothelial dysfunction that is associated with a loss of endothelium-derived nitric oxide (NO),
increased vascular permeability, increased endothelial adhesiveness, and thickening of the basement membrane of blood
vessels.4 However, the exact mechanism responsible for
†
Present address. College of Veterinary Medicine, Jilin University, Changchun 130062, China.
‡
Present address. Department of Anesthesiology, Peking Union Medical
College Hospital, Chinese Academy of Medical Sciences, Beijing 100730,
China.
* Corresponding author. Tel: þ86 731 471 6249.
E-mail address: liyingliu2004@yahoo.com.cn
endothelial dysfunction in diabetes remains largely unknown,
limiting effective therapeutic interventions.
In endothelial cells, NO is produced from L-arginine in the
catalysis of endothelial nitric oxide synthase (eNOS). Previous studies5,6 showed that the association of heat shock
protein 90 (hsp90) with eNOS plays an important role in
the generation of NO in endothelial cells. This process is
controlled by calpain because hsp90 is a natural substrate
for calpain.7,8 For example, exposure of pulmonary artery
endothelial cells to hypoxia triggers calpain-mediated loss
of hsp90 from the eNOS complex, resulting in decreased
eNOS activity and NO release.9 Recent study by Stalker
et al.10,11 has also reported that acute experimental hyperglycaemia up-regulated the endothelial-expressed m-calpain
isoform in the microcirculation and induced endothelial
dysfunction, however, the mechanism of calpain activation
in hyperglycaemia is not fully understood.
The Naþ/Hþ exchanger (NHE) is expressed ubiquitously in
the plasma membrane of mammalian cells and exchanges
Published on behalf of the European Society of Cardiology. All rights reserved. & The Author 2008.
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Naþ/Hþ exchanger;
Diabetes;
Endothelial function;
Calpain;
eNOS
256
intracellular Hþ for extracellular Naþ to regulate intracellular
pH (pHi) value and the concentration of intracellular Naþ
12
(Naþ
The activation of NHE increases Naþ
i ).
i that leads to
Ca2þ overload through the Naþ/Ca2þ exchanger, which is
assumed to be the crucial factor in diabetic injuries.13 Inhibition of NHE has been shown to have protective effects
against diabetic nephropathy.14 Our previous study has
demonstrated that cariporide, an NHE inhibitor, inhibited
the high glucose (HG)-induced adhesion of monocytes to
endothelial cells.15 These ﬁndings support the hypothesis
that HG or hyperglycaemia-induced dissociation of hsp90
from eNOS via Ca2þ-dependent calpain is mediated by
NHE. Accordingly, the aim of the current study is to investigate if hyperglycaemia-induced endothelial dysfunction is
mediated by NHE and Ca2þ/calpain. Our results suggest
that hyperglycaemia-induced endothelial dysfunction is
due to the activation of NHE via dissociation of hsp90 from
eNOS by Ca2þ-dependent calpain.
2. Methods
2.1 Animals
2.2 Materials
The NHE inhibitor, cariporide, was kindly provided by Hoechst (Frankfurt, Germany). Calpain inhibitor, ZLLal (benzyloxycarbonyl-leucylleucinal) was from Biomol Research Laboratories, PA, USA. Calcium
chelator [1,2-bis (2-aminophenoxy) ethane-N, N, N0 ,N0 -tetraacetic
acid (BAPTA)] was obtained from Invitrogen Corporation, CA, USA.
Fluorescent indicators, diaminoﬂuorescein (DAF) and 2-carboxyethyl5(6)-carboxyﬂuorescein were purchased from Calbiochem (CA,
USA). Both [3H]L-arginine and [32P]ATP were obtained from NEN
(Boston, MA, USA). Streptozotocin (STZ), phenylephrine, acetylcholine
(ACh), sodium nitroprusside (SNP), hydroxyethyl piperazine ethanesulphonic acid (HEPES), and other chemicals were purchased from Sigma
Chemical Co, MO, USA. Primary antibodies (NHE, hsp90, eNOS, and
b-actin), human NHE siRNA (small interfering ribonucleic acid), and
control siRNA were obtained from Santa Cruz Biotechnology (CA,
USA). All chemicals were of reagent grade.
2.3 Cell culture
Human umbilical vein endothelial cells (HUVECs) purchased from
the American Type Culture Collection were grown in endothelial
basal medium (EBM) (Clonetics Inc., Walkersville, MD, USA) supplemented with 2% fetal bovine serum, 12.5 mg/mL ECGF, 1 mg/
mL hydrocortisone, 100 m/mL penicillin and 100 mg/mL streptomycin. The cells were cultured at 378C in a humidiﬁed atmosphere of
5% CO2 and 95% air. Culture medium was replaced twice a week,
and cells were subcultured when 80% conﬂuent. Cells at passage 4
were used for all experiments.
2.4 High-glucose treatment of human umbilical
vein endothelial cells
After reaching 80% conﬂuence, HUVECs were exposed to normal
glucose (NG; 5 mM D-glucose), HG (30 mM D-glucose), or hyperosmotic
control (HO, 5 mM D-glucose plus 25 mM L-glucose) for different
lengths of time with a daily change of culture media.
2.5 Transfection of small interfering ribonucleic
acid into human umbilical vein endothelial cells
Transient infection of siRNA into cells was carried out according to
Santa Cruz’s protocol.16 Brieﬂy, 100 mL transfection medium
containing 6 mL siRNA (10 mM) stock solution was added to 100 mL
transfection medium containing 6 mL transfection reagent (Lipofectamine 2000, Invitrogen, CA, USA) and mixed gently. After 30 min
incubation at room temperature, 200 mL siRNA–lipid complex solution was added to each well (6-well plate) in 1.0 mL transfection
medium. After incubation for 6 h at 378C, the medium was replaced
with normal medium and cultured for 24–48 h.
2.6 Western blot
After treatment, HUVECs were lysated in cell-lysis buffer (Cell Signaling Company, MA, USA). The protein content was assayed by bicinchoninic acid protein assay reagent (Pierce, IL, USA). Twenty microgram
of protein was loaded to sodium dodecyl sulphate polyacrylamide gel
electrophoresis (SDS–PAGE) and then transferred to membrane.
Membrane was incubated with a 1:1000 dilution of primary antibody,
followed by a 1:2000 dilution of horseradish peroxidase-conjugated
secondary antibody. Protein bands were visualized by enhanced
chemiluminescence (GE Healthcare, Chicago, IL, USA). The intensity
(area density) of the individual bands on western blots was measured
by densitometry (model GS-700, Imaging Densitometer; Bio-Rad, CA,
USA). The background was subtracted from the calculated area.
2.7 Measurement of intracellular calcium
concentration
The intracellular calcium (Ca2þ
i ) concentration was measured by
using a Fluo-4 NW kit from Invitrogen following kit protocol.
Brieﬂy, HUVECs were treated as indicated, the cell culture
medium was aspirated, washed with HEPES buffer (pH 7.4) once,
and 1 mL of HEPES buffer with ﬂuorescent dye was added to cultured cells. After 30 min incubation, ﬂuorescence strength was
measured in wavelength of excitation/emission of 485/520 nm.
2.8 Calpain activity
The calpain activity was measured by using the ﬂuorogenic peptide
Suc-Leu-Leu-Val-Tyr-AMC as a substrate following the procedure
described previously with slight modiﬁcation.17 Shortly, cells were
cultured in 24-well plates in EBM with different treatments. After
being washed twice with phosphate buffered saline (PBS), ﬂuorogenic peptide was added to a ﬁnal concentration of 80 mM in PBS.
Immediately after addition of ﬂuorogenic peptide, ﬂuorescence
was recorded at 2 min intervals for 20 min at excitation 360 nm
and emission 460 nm using a Synergy HT Multi-Detection Microplate
Reader (BIO-TEK Instruments Inc., VT, USA). The initial rate of
peptidyl-AMC hydrolysis was used as the velocity of enzyme activity.
2.9 Determination of Na1/H1 exchanger activity
To evaluate NHE activity in HUVECs or rat aortas, the pHi, rate of
recovery from an induced acidiﬁcation, cellular buffer capacity of
the HUVECs or rat aortas, and the calibration of the ﬂuorescence
to pH values were determined as described previously.14,15
2.10 Endothelial nitric oxide synthase activity
assay
eNOS activity was monitored by L-[3H]citrulline production from
L-[3H]arginine as described previously.18 Brieﬂy, protein samples
were incubated in reaction buffer [1 mM L-arginine/100 mM
NADPH/1 mM tetrahydrobiopterin/0.2 mCi of L-[3H] arginine
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Male Sprague–Dawley rats, 180 + 20 g, 6–8 weeks of age, were
obtained from the Animal Department of Central South University
(Changsha, China). Rats were housed in temperature-controlled
cages with a 12 h light–dark cycle and given free access to water
and normal feed. The animal protocol was reviewed and approved
by the Central South University Institute Animal Care and Use Committee. The current investigation conforms to the Guide for the
Care and Use of laboratory Animals published by the US National
Institutes of Health (NIH Publication No. 85-23, revised 1996).
S. Wang et al.
257
Hyperglycaemia activates NHE
(.66 Ci/mmol) per reaction] for 15 min at 378C, separated by
Dowex-50W ion-exchange chromatography in 20 mM HEPES (pH
5.5), 2 mM ethylene-diamine-tetra-acetic acid, and 2 mM ethylene
glycol tetra-acetic acid, and the ﬂow-through was used for liquid
scintillation counting.
and STZ-injected rats were given cariporide (1 mg/kg body weight
daily) in their drinking water for 28 days.
2.14 Organ chamber
Immunoprecipitations were performed to study the association of
eNOS and hsp90 in endothelial cells extracts.19 Cell lysates
(500 mg protein) were incubated with anti-eNOS monoclonal antibody for 1 h followed by incubation with pre-washed Protein
G-agarose for 2 h. The resulting pellet was washed three times,
boiled in SDS sample buffer, and resolved by SDS–PAGE with immunoblot analysis by using primary antibodies against eNOS or hsp90.
Organ chamber experiments were performed as described
previously.22 Brieﬂy, rings (3–5 mm in length) from rat aortas, free
of fat and connective tissue, were mounted in organ bath in 5 mL
Kreb’s solution at 378C, gassed with 95%O2 þ 5%CO2, under a
tension of 2 g, for 1 h equilibration period. After the equilibration,
rings were contracted with 60 mM KCl. After washing and another
30 min equilibration, contractile response was evoked by phenylephrine (1 mM) to elicit reproducible responses. At the plateau of
contraction, accumulative ACh (0.003–3 mM) or SNP (0.001 to
1 mM) was added into the organ bath to induce the endotheliumdependent/independent relaxation.
2.12 Detection of nitric oxide
2.15 Apoptosis assay
NO production in cultured cells was detected using the ﬂuorescent
probe DAF as described previously.20 In brief, before the end of
treatment, 10 mM DAF was added to the medium and incubated
for 30 min at 378C, then washed with PBS. The DAF ﬂuorescent
intensity was recorded by ﬂuorescence spectrometry at the wavelength of excitation (485 nm) and emission (545 nm).
After treatment, HUVECs were ﬁxed with 4% paraformaldehyde in
PBS. Apoptosis was assessed by terminal deoxynucleotidyl
transferase-mediated dUTP nick end labelling (TUNEL) staining
(TMR red) using a kit from Roche Applied Science and following
the provided instruction manual. The percentage of apoptosis was
calculated from the number of TUNEL positive cells divided by the
total number of cells counted.
2.11 Association of endothelial nitric oxide
synthase with heat shock protein 90
2.13 Streptozotocin-induced diabetes
2.16 Statistical analysis
All values are expressed as means + SEM. Data were analysed using
a one-way or two-way ANOVA followed by Newman-Student’s t-test.
P , 0.05 was considered signiﬁcant.
Figure 1 High glucose (HG) increases intracellular calcium (Ca2þ
i ) and calpain activity in human umbilical vein endothelial cells (HUVECs). Cultured HUVECs
concentration was detected by Fluo-4 ﬂuorescence. (B) Calpain activity was
were incubated with HG (30 mM D-glucose) for 3, 6, 12, 24, and 48 h. (A) Ca2þ
i
assayed by ﬂuorogenic peptide in situ. (C) HUVECs were incubated with normal glucose (NG, 5 mM D-glucose), HG, or hyperosmotic control (HO, 5 mM
2þ
0
0
D-glucose plus 25 mM L-glucose) for 24 h in presence or absence of BAPTA [1,2-bis (2-aminophenoxy) ethane-N, N, N ,N -tetraacetic acid] (0.5 mM), a Cai
chelator, and then calpain activity was detected. Data are expressed by mean + SEM (n ¼ 5). *P , 0.05 vs. Control, #P , 0.05 vs. HG.
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For induction of diabetes, rats were anaesthetized with ketamine/
xylocaine and injected with a single dose of STZ (60 mg/kg bodyweight in 5 mM pH 4.5 citrate buffer, I.P.).21 Hyperglycaemia is
deﬁned as random blood glucose levels of .300 mg/dL. Control
258
3. Results
3.1 High glucose increases intracellular
calcium and calpain activity in human umbilical
vein endothelial cells
Previous studies have demonstrated that calpain is activated
by hyperglycaemia via calcium overload and causes endothelial dysfunction.10,11,23 To investigate whether HG alters
the activity of calpain and Ca2þ
level in HUVECs, both
i
concentration and calpain activity were determined
Ca2þ
i
in HUVECs incubated with HG (30 mM) at different times.
As shown in Figure 1A and B, HG signiﬁcantly increased
both Ca2þ
concentration and calpain activity in a timei
dependent manner.
3.2 High glucose-induced increase in calpain
activity is calcium dependent
3.3 Inhibition of Na1/H1 exchanger abolishes high
glucose-induced intracellular calcium and calpain
activity
Activation of NHE by some pathologic factors, such as
ischaemia25 and hypoxia,26 leads to an increased Ca2þ
level
i
and causes cellular damage in the vascular system. Next we
determined if the HG-increased Ca2þ-dependent calpain
activity is mediated by NHE. HUVECs were treated with HG
in absence or presence of cariporide (10 mM) for 24 h. As
shown in Figure 2A and B, in both NG and HO groups,
inhibition of NHE by cariporide did not change basal calpain
activity and Ca2þ
level. However, cariporide blocked the
i
increase in calpain activity and Ca2þ
level induced by HG.
i
In order to further investigate whether the inhibitory effect
of cariporide on HG-increased calpain activity is speciﬁc to
NHE, we used siRNA to silence NHE protein expression. In
Figure 2C, speciﬁc siRNA of NHE reduced NHE protein
expression to 20%, but control siRNA did not change NHE
protein expression. In Figure 2D, control siRNA did not block
the increase in calpain activity induced by HG, however,
NHE siRNA inhibited the HG-increased calpain activity.
3.4 High glucose induces Na1/H1 exchanger
activation
Since inhibition of NHE by pharmacologic inhibitor or siRNA
abolished HG-induced increase in calpain activity, we next
Figure 2 Inhibition of Naþ/Hþ exchanger (NHE) by pharmacologic inhibitor or small interfering ribonucleic acid (siRNA) abolishes high glucose (HG)-induced
increase in intracellular calcium (Ca2þ
i ) level and calpain activity. Human umbilical vein endothelial cells (HUVECs) were incubated with NG (normal glucose),
HG or HO (hyperosmotic control) for 24 h in presence or absence of cariporide (10 mM). Cells were subjected to detect (A) calpain activity and
(B) concentration of Ca2þ
i . Conﬂuent HUVECs were transfected with control siRNA or NHE siRNA and then incubated with HG for 24 h. (C ) NHE siRNA silenced
NHE protein expression by western blot. The blot is a representative of ﬁve blots obtained from ﬁve independent experiments. (D) NHE siRNA abolished
HG-induced increase in calpain activity. Data are expressed by mean + SEM (n ¼ 5). *P , 0.05 vs. Control, #P , 0.05 vs. HG.
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Calpains are a family of Ca2þ-dependent cysteine proteases
found in mammals and many lower organisms. In the preconcentration, calpain is actisence of an elevated Ca2þ
i
vated in endothelial cells.24 We next detected whether the
HG-increased calpain activity is dependent on Ca2þ
in
i
HUVECs. As shown in Figure 1C, in NG and HO groups,
BAPTA, a Ca2þ
remover, did not change basal calpain
i
activity. However, BAPTA inhibited the enhanced calpain
activity induced by HG.
S. Wang et al.
Hyperglycaemia activates NHE
investigated the effects of HG on NHE activity in HUVECs. In
Figure 3A, HG increased NHE activity in a time-dependent
manner. However, cariporide, a new NHE inhibitor, dosedependently inhibited NHE activity when HUVECs were
treated with HG for 24 h (Figure 3B).
3.5 High glucose decreases endothelial nitric oxide
synthase activity, association of endothelial nitric
oxide synthase with heat shock protein 90 and
nitric oxide production
One of the main functions of endothelium is to produce NO,
which is catalysed by eNOS. We next studied whether HG
259
affected NO production and eNOS activity in HUVECs. In
Figure 4A, HG decreased NO release from endothelial cells
in a time-dependent manner, associated with a decreased
eNOS activity (Figure 4B).
There is evidence that hsp90 plays an important role in
positively regulating eNOS activity.16,27 We further investigated whether HG decreased eNOS activity by decreasing
its association with hsp90. The interaction of hsp90 and
eNOS was assayed using immunoprecipitation of eNOS or
hsp90 with the speciﬁc antibodies. In Figure 4C, exposure
of HUVECs to HG up to 30 mM for 24 h did not alter the
expression of hsp90 and eNOS. Compared with NG,
however, decreased amounts of eNOS were detected when
Figure 4 High glucose (HG) decreases nitric oxide (NO) production, endothelial nitric oxide synthase (eNOS) activity, and association of eNOS with heat shock
protein (hsp90). (A, B) Human umbilical vein endothelial cells (HUVECs) were incubated with HG (30 mM) for 3, 6, 12, 24, and 48 h. Cell lysates were subjected to
detect NO production by DAF (diaminoﬂuorescein) ﬂuorescence and eNOS activity by L-[3H]citrulline production from L-[3H]arginine. Data are expressed by
mean + SEM (n ¼ 5). *P , 0.05 vs. Control [or normal glucose (NG)]. (C ) HUVECs were incubated with NG, HO (hyperosmotic control) or HG for 24 h. After treatment, hsp90 or eNOS were ﬁrst immunoprecipitated from the cell lysates and then detected in western blots with the speciﬁc antibody. The blot is a representative of ﬁve blots obtained from ﬁve independent experiments.
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Figure 3 High glucose (HG) increases cariporide-sensitive Naþ/Hþ exchanger (NHE) activity. (A) Human umbilical vein endothelial cells (HUVECs) were incubated with HG (30 mM) for 3, 6, 12, 24, and 48 h. (B) HUVECs were incubated with NG (normal glucose), and HG with or without cariporide (1, 10, 100 mM),
a selective NHE inhibitor. Cells were subjected to detect NHE activity by NH4Cl pulse method. Data are expressed by mean + SEM (n ¼ 5). *P , 0.05 vs.
Control, #P , 0.05 vs. HG.
260
S. Wang et al.
hsp90 was immunoprecipitated from HG-treated HUVECs.
These results were further corroborated by decreased
detection of hsp90 when eNOS was immunoprecipitated
from HG-treated cells.
3.6 Inhibition of Na1/H1 exchanger or calpain
abolishes high glucose-induced reduction of
endothelial nitric oxide synthase activity,
association of endothelial nitric oxide synthase with
heat shock protein 90, and nitric oxide production
Activation of calpain caused hsp90 degradation from eNOS–
hsp90 complex, leading to inactivation of eNOS.9 Thus, we
further investigated whether NHE or calpain was involved
in HG-reduced eNOS association with hsp90. As shown in
Figure 5A, inhibition of either NHE with cariporide or
calpain with ZLLa1 reversed HG-decreased eNOS activity.
Although inhibition of either NHE with cariporide or
calpain with ZLLa1 alone did not alter NO production in
basal condition, it signiﬁcantly increased the NO release
from HG-treated HUVECs (Figure 5B). In addition, inhibition
of either NHE with cariporide or calpain with ZLLa1 restored
the association of eNOS with hsp90 (Figure 5C).
3.7 Inhibition of Na1/H1 exchanger with cariporide
reverses streptozotocin-induced endothelial
dysfunction via apoptosis-independent pathway
We next investigated whether cariporide reversed diabetes
mellitus-induced endothelial dysfunction in vivo. As shown in
Figure 6A, cariporide alone did not change ACh-induced
endothelium-dependent relaxation in control rats, however,
STZ-induced hyperglycaemia impaired ACh-induced endothelium-dependent relaxation. Furthermore, administration
of cariporide in diabetic rats abolished hyperglycaemiaimpaired endothelium-dependent relaxation but had no
effects on NO donor-triggered endothelium-independent
relaxation (Figure 6B).
In order to conﬁrm whether HG activates NHE in rat aorta
endothelium, the activity of NHE was assayed in rat aortas.
As shown in Figure 6C, STZ-induced hyperglycaemia
activates NHE in rat aortas. Administration of cariporide
attenuated the hyperglycaemia-increased NHE, but cariporide did not change the NHE activity in control rat aortas.
It has been reported that HG-induced apoptosis contributes
to endothelial dysfunction in diabetes. In order to study
whether apoptosis is involved in the NHE-mediated endothelial dysfunction in diabetes, we detected the apoptosis
of endothelial cells treated with HG. We used tumour necrosis
factor, TNFa (20 ng/mL), a well-known positive control of
apoptosis inducer, and HG to treat HUVECs for 24 h. As
shown in Figure 6D, we did not see the increased apoptosis
in HG-treated HUVECs. However, TNFa caused HUVECs apoptosis after 24 h incubation without altering NHE activity and
the association of eNOS with hsp90 (data not shown).
4. Discussion
The current study demonstrates that HG (or hyperglycaemia) via NHE induces vascular endothelial dysfunction by
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Figure 5 High glucose (HG)-induced reduction of endothelial nitric oxide synthase (eNOS) activity, association of eNOS with heat shock protein (hsp90) or nitric
oxide (NO) production is both NHE (Naþ/Hþ exchanger) and calpain dependent. HUVECs (human umbilical vein endothelial cells) were incubated with NG (normal
glucose) or HG for 24 h in presence or absence of cariporide (10 mM) or calpain inhibitor [ZLLa1 (benzyloxycarbonyl-leucyl-leucinal), 50 mM]. Cells were used to
detect (A) eNOS activity by L-[3H]citrulline production from L-[3H]arginine and (B) NO production by DAF (diaminoﬂuorescein) ﬂuoresence. Data are expressed by
mean + SEM (n ¼ 5). *P , 0.05 vs. HG. (C ) After treatment, hsp90 or eNOS were ﬁrst immunoprecipitated from the cell lysates and detected in western blots
with the speciﬁc antibody. The blot is a representative of ﬁve blots obtained from ﬁve independent experiments.
Hyperglycaemia activates NHE
261
activating calpain-dependent dissociation of eNOS from
hsp90. Not only did inhibition of NHE by a pharmacologic
inhibitor, cariporide, reverse HG-induced increase in Ca2þ
i concentration and calpain activity, but its actions were
also mimicked by silencing of NHE with siRNA. In addition,
inhibition of either NHE by cariporide or calpain by ZLLa1
blocked the decrease in eNOS activity and eNOS association
with hsp90 caused by HG. Treatment of diabetic mice
with cariporide restored endothelial function in vivo.
These results strongly suggest that NHE is required for
hyperglycaemia-induced endothelial dysfunction via
calcium-dependent protease calpain.
Recent studies have reported that calpain inhibition
exerts anti-inﬂammatory effects in diabetes. Ruetten
et al.28 showed that calpain inhibitors signiﬁcantly
improve leukocyte-endothelium interactions cardiovascular
outcome and ameliorate multiple organ dysfunction during
endotoxic shock. Similarly, Scalia R et al.29 reported that
hyperglycaemia is a major determinant of albumin permeability in diabetic microcirculation via calpain. These
studies imply that calpain plays a role in diabetes-induced
endothelial dysfunction. Our data clearly indicated that
inhibition of NHE by cariporide or siRNA blocked the increase
in either calpain activity or Ca2þ
concentration caused by
i
HG and improved endothelial function in diabetic rats.
It strongly suggested to us that NHE is essential for
hyperglycaemia-induced endothelial dysfunction. To our
knowledge, this is the ﬁrst study to report that inhibition
of NHE prevents endothelial dysfunction in diabetes via
Ca2þ/calpain-dependent pathway.
Our data also clearly indicate that inhibition of NHE abolished HG-induced increase in calpain activity via Ca2þ. The
calpains are a family of calcium-dependent proteases that
cleave a number of cellular substrates, including kinases,
phosphatases, transcription factors, and cytoskeletal proteins.30 In this study, we found that HG increased calpain
activity in vascular endothelial cells as well as increased
Ca2þ
i . In addition, chelation of intracellular free-calcium
by BAPTA inhibited the activation of calpain induced by
HG. So we speculated that HG-induced calpain activation
is calcium dependent because it has been reported that
calpain is activated in response to large calcium ﬂuxes.24
To determine whether or not the elevated glucose produced
a cariporide-sensitive activation of NHE in endothelial cells,
we measured NHE activity in vitro and in vivo. These results
showed that HG produced a cariporide-sensitive activation
of NHE in endothelial cells. A potential mechanism of
glucose-induced increase in NHE activity is a phosphorylationdependent increase in the activity of existing exchangers or
the activation of dormant membrane-associated exchangers.
Indeed, Sardet et al.31 had demonstrated that the NHE is
rapidly phosphorylated in response to various mitogens and
concluded that this phosphorylation of the NHE is temporally
correlated with its activation. Additional experiments32
support the hypothesis that protein kinase C is one of the
kinases responsible for this phosphorylation.
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Figure 6 Hyperglycaemia-induced endothelial dysfunction via calpain-dependent but apoptosis-independent pathway. Hyperglycaemia was induced by streptozotocin (STZ) in male rats. Cariporide (1 mg/kg body weight daily) was given to rats in their drinking water. Twenty-eight days later, endothelium-dependent/
independent relaxation was detected by organ chamber. (A) Ach (acetylcholine)-induced endothelium-dependent relaxation. (B) SNP (sodium
nitroprusside)-induced endothelium-independent relaxation. (C ) Naþ/Hþ exchanger (NHE) activity in rat aortas. (D) HUVECs (human umbilical vein endothelial
cells) were incubated with high glucose (HG), and tumour necrosis factor, TNFa (20 ng/mL), for 24 h. Cell apoptosis was detected by TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling) in situ. Data are expressed by mean + SEM (n ¼ 7). *P , 0.05 vs. Control or NG (normal glucose),
#
P , 0.05 vs. STZ. (E) Proposed scheme of HG-induced endothelial dysfunction mediated by NHE via calpain.
262
In summary, we have demonstrated that diabetic
hyperglycaemia activates NHE and results in the increased
Ca2þ
and calpain activity, which degraded hsp90, a positive
i
regulator of eNOS activity, and subsequently impaired
endothelium-dependent vessel relaxation (Figure 6E).
These results are particularly relevant to hyperglycaemic
conditions and endothelial dysfunction, both of which are
prevalent in type 1 diabetes mellitus.
Acknowledgement
The authors acknowledge Mrs Vivian for reading proof.
Conﬂict of interest: none declared.
Funding
This work was supported by National Natural Science Foundation of
China (No. 30600248).
References
14. Jandeleit-Dahm K, Hannan KM, Farrelly CA, Allen TJ, Rumble JR,
Gilbert RE et al. Diabetes-induced vascular hypertrophy is accompanied
by activation of Na(þ)-H(þ) exchange and prevented by Na(þ)-H(þ)
exchange inhibition. Circ Res 2000;87:1133–1140.
15. Wang SX, Sun XY, Zhang XH, Chen SX, Liu YH, Liu LY. Cariporide inhibits
high glucose-mediated adhesion of monocyte-endothelial cell and
expression of intercellular adhesion molecule-1. Life Sci 2006;79:
1399–1404.
16. Cheng KK, Lam KS, Wang Y, Huang Y, Carling D, Wu D et al. Adiponectininduced endothelial nitric oxide synthase activation and nitric oxide production are mediated by APPL1 in endothelial cells. Diabetes 2007;56:
1387–1394.
17. Dong Y, Tan J, Cui MZ, Zhao G, Mao G, Singh N et al. Calpain inhibitor
MDL28170 modulates Abeta formation by inhibiting the formation of
intermediate Abeta46 and protecting Abeta from degradation. FASEB J
2006;20:331–333.
18. Davis BJ, Xie Z, Viollet B, Zou MH. Activation of the AMP-activated kinase
by antidiabetes drug metformin stimulates nitric oxide synthesis in vivo
by promoting the association of heat shock protein 90 and endothelial
nitric oxide synthase. Diabetes 2006;55:496–505.
19. Xu H, Shi Y, Wang J, Jones D, Weilrauch D, Ying R et al. A heat shock
protein 90 binding domain in endothelial nitric-oxide synthase inﬂuences
enzyme function. J Biol Chem 2007;282:37567–37574.
20. Thomas S, Kotamraju S, Zielonka J, Harder DR, Kalyanaraman B. Hydrogen peroxide induces nitric oxide and proteosome activity in endothelial
cells: a bell-shaped signaling response. Free Radic Biol Med 2007;42:
1049–1061.
21. Hurdag C, Uyaner I, Gurel E, Utkusavas A, Atukeren P, Demirci C. The
effect of alpha-lipoic acid on NOS dispersion in the lung of streptozotocin-induced diabetic rats. J Diabetes Complications 2008;22:56–61.
22. Wang SX, Xiong XM, Song T, Liu LY. Protective effects of cariporide on
endothelial dysfunction induced by high glucose. Acta Pharmacol Sin
2005;26:329–333.
23. Scalia R, Gong Y, Berzins B, Zhao LJ, Sharma K. Hyperglycemia is a major
determinant of albumin permeability in diabetic microcirculation: the
role of mu-calpain. Diabetes 2007;56:1842–1849.
24. Goll DE, Thompson VF, Li H, Wei W, Cong J. The calpain system. Physiol
Rev 2003;83:731–801.
25. Toda T, Kadono T, Hoshiai M, Eguchi Y, Nakazawa S, Nakazawa H et al.
Naþ/Hþ exchanger inhibitor cariporide attenuates the mitochondrial
Ca2þ overload and PTP opening. Am J Physiol Heart Circ Physiol 2007;
293:H3517–H3523.
26. Peters SC, Piper HM. Reoxygenation-induced Ca2þ rise is mediated via
Ca2þ inﬂux and Ca2þ release from the endoplasmic reticulum in cardiac
endothelial cells. Cardiovasc Res 2007;73:164–171.
27. Miao RQ, Fontana J, Fulton D, Lin MI, Harrison KD, Sessa WC. Dominantnegative Hsp90 reduces VEGF-stimulated nitric oxide release and
migration in endothelial cells. Arterioscler Thromb Vasc Biol 2008;28:
105–111.
28. Ruetten H, Thiemermann C. Effect of calpain inhibitor I, an inhibitor of
the proteolysis of I kappa B, on the circulatory failure and multiple
organ dysfunction caused by endotoxin in the rat. Br J Pharmacol 1997;
121:695–704.
29. McDonald MC, Mota-Filipe H, Paul A, Cuzzocrea S, Abdelrahman M,
Harwood S et al. Calpain inhibitor I reduces the activation of nuclear
factor-kappa B and organ injury/dysfunction in hemorrhagic shock.
FASEB J 2001;15:171–186.
30. Sorimachi H, Ishiura S, Suzuki K. Structure and physiological function of
calpains. Biochem J 1997;328:721–732.
31. Sardet C, Counillon L, Franchi A, Pouyssegur J. Growth factors induce
phosphorylation of the Naþ/Hþ antiporter, glycoprotein of 110 kD.
Science 1990;247:723–726.
32. Williams B, Howard RL. Glucose-induced changes in Naþ/Hþ antiport
activity and gene expression in cultured vascular smooth muscle cells.
Role of protein kinase C. J Clin Invest 1994;93:2623–2631.
Downloaded from by guest on December 3, 2014
1. Colette C, Monnier L. Acute glucose ﬂuctuations and chronic sustained
hyperglycemia as risk factors for cardiovascular diseases in patients
with type 2 diabetes. Horm Metab Res 2007;39:683–686.
2. Bax JJ, Young LH, Frye RL, Bonow RO, Steinberg HO, Barrett EJ.
Screening for coronary artery disease in patients with diabetes. Diabetes
Care 2007;30:2729–2736.
3. Beckman JA, Creager MA, Libby P. Diabetes and atherosclerosis: epidemiology, pathophysiology, and management. JAMA 2002;287:2570–2581.
4. Stehouwer CD, Lambert J, Donker AJ, van Hinsbergh VW. Endothelial dysfunction and pathogenesis of diabetic angiopathy. Cardiovasc Res 1997;
34:55–68.
5. Garcia-Cardena G, Fan R, Shah V, Sorrentino R, Cirino G,
Papapetropoulos A et al. Dynamic activation of endothelial nitric oxide
synthase by Hsp90. Nature 1998;392:821–824.
6. Shah V, Wiest R, Garcia-Cardena G, Cadelina G, Groszmann RJ, Sessa WC.
Hsp90 regulation of endothelial nitric oxide synthase contributes to vascular control in portal hypertension. Am J Physiol 1999;277:G463–G468.
7. Minami Y, Kimura Y, Kawasaki H, Suzuki K, Yahara I. The carboxy-terminal
region of mammalian HSP90 is required for its dimerization and function
in vivo. Mol Cell Biol 1994;14:1459–1464.
8. Bellocq A, Doublier S, Suberville S, Perez J, Escoubet B, Fouqueray B
et al. Somatostatin increases glucocorticoid binding and signaling in
macrophages by blocking the calpain-speciﬁc cleavage of Hsp 90. J Biol
Chem 1999;274:36891–36896.
9. Su Y, Block ER. Role of calpain in hypoxic inhibition of nitric oxide
synthase activity in pulmonary endothelial cells. Am J Physiol Lung Cell
Mol Physiol 2000;278:L1204–L1212.
10. Stalker TJ, Gong Y, Scalia R. The calcium-dependent protease calpain
causes endothelial dysfunction in type 2 diabetes. Diabetes 2005;54:
1132–1140.
11. Stalker TJ, Skvarka CB, Scalia R. A novel role for calpains in the endothelial dysfunction of hyperglycemia. FASEB J 2003;17:1511–1513.
12. Slepkov ER, Rainey JK, Sykes BD, Fliegel L. Structural and functional
analysis of the Naþ/Hþ exchanger. Biochem J 2007;401:623–633.
13. Vial G, Dubouchaud H, Couturier K, Lanson M, Leverve X, Demaison L.
Naþ/Hþ exchange inhibition with cariporide prevents alterations of coronary endothelial function in streptozotocin-induced diabetes. Mol Cell
Biochem 2008;310:93–102.
S. Wang et al.