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Introduction
The relation between diabetes and premature vascular disease has
been well established[1]. One of the defects involves
endothelial dysfunction characterized by impaired endothelium-dependent
relaxation responses. Many metabolic disturbances of diabetes, hyperglycemia
have been suggested to be the main cause of endothelial dysfunction.
High glucose in vitro or in vivo has been reported
to inhibit acetylcholine (ACh)-induced endothelium-dependent relaxation[2]
responses, to impair the biological synthesis pathway of nitric
oxide (NO)[3], and to generate reactive oxygen species[4].
It has been demonstrated that hyperactivity of sodium-hydrogen
exchange subtype 1 (NHE-1) has been implicated in the vascular injury
in diabetes mellitus[5]. Ganz et al[6]
found that the activity and expression of NHE-1 significantly increased
in mesangial cells after exposure to high glucose. Our previous
experiments have demonstrated that the benzoylguanidine compound
cariporide (4-isopropyl-3-methylsulfonyl-benzoylguanidine methanesulfonate),
which is a selective NHE-1 inhibitor, protected against injuries
of endothelial functions induced by high lipid diet in rabbits[7].
However, it is not known whether NHE-1 inhibitor protects against
endothelial function affected by high glucose. The aim of this study
is to explore the effect of cariporide against endothelial dysfunction
of isolated rat aortic rings induced by high glucose and to investigate
its mechanisms.
Materials and methods
Drugs and chemicals Cariporide was obtained from Hoechst
Company (Frankfurt, Germany). SNP, ACh, and phenylephrine (Phe)
were purchased from Sigma Chemical Co (Saint Louis, Mo, USA). The
kits for measurement of nitrite/nitrate (NO), MDA, and SOD activity
were purchased from Nanjing Jiancheng Bioengineering Institute (Jiangsu,
China).
Animal Male Sprague-Dawley rats (No SCXK2003-0003;
Grade II) weighing 180-200 g, supplied by the Animal Center of Xiangya
Medical College (Central South University, China) were used.
Preparation of rat thoracic aorta rings The rats were killed
by exsanguinations after an intraperitoneal anesthesia with pentobarbital
sodium 30 mg/kg and intravenous anticoagulation with heparin sodium
150 U/kg. The descending thoracic aorta was rapidly dissected from
the rats and immersed in Krebs' solution, composed of (mmol/L):
NaCl, 118.3; KCl, 4.7; MgSO4·7H2O, 1.2;
KH2PO4, 1.2; CaCl2, 2.5; NaHCO3,
24.0; glucose, 11; and Na2-EDTA, 0.026; and bubbled with
95% O2+5% CO2 (pH 7.4). After the perivascular
tissue was carefully removed, the aortic rings (which were approximately
4 mm in length), were prepared.
Bioassay of vasoreactivity[8] For isometric force
record-ing, the aortic rings were mounted between two stainless
steel hooks and suspended in a 10 mL organ bath containing above
Krebs' solution at 37 ºC bubbled with 95% O2+5%
CO2 gas mixture (pH 7.4). An initial load of 2.0 g was
applied, and the tension of the aortic ring was monitored by a force
transducer and recorded on a polygraph (Model YL-1, Chengdu Instruments,
China). After a 90-min equilibration period, the ring was precontracted
by Phe 1 µmol/L. When the development tension attained its
peak value, the ring was relaxed by ACh or SNP, respectively. Accumulative
concentration-response curves to ACh (0.003, 0.01, 0.03, 0.1, 0.3,
1, and 3 µmol/L) or SNP (0.001-1 µmol/L) were recorded.
Determination of MDA concentration After a 6-h incubation
of aortic segments, the aortic segments were blotted dry and weighed,
then made into 5% tissue homogenate in ice-cold 0.9% NaCl solution.
A supernatant was obtained from tissue homogenate by centrifugalization
(1000×g, 4 ºC, 10 min). The MDA concentration (thiobarbituric
acid reactive substances, TBARS) in the supernatant was measured.
Briefly, 1.0 mL of 20% trichloroacetic acid and 1.0 mL of 1% TBARS
reagent were added to 100 µL supernatant, then mixed and incubated
at 100 ºC for 80 min. After cooling on ice, samples were centrifuged
at 1000×g for 20 min and the absorbance of the supernatant
was read at 532 nm. TBARS results were expressed as MDA equivalents
using tetraethoxy-propane as standard.
Assay of SOD activity in aortic rings The supernatant of
tissue homogenate of the aortic rings were obtained as described
earlier. A competitive inhibition assay was performed by using xanthine/xanthine
oxidase reaction-generated superoxide radicals to reduce nitro blue
tetrazolium (NBT) quantitatively to blue formazan. Conversion of
superoxide radicals to hydrogen peroxide by superoxide
dismutase inhibited dye formation and served as a measure
of superoxide dismutase activity. Briefly, the supernatant of 0.5
mL with xanthine 50 µmol/L and xanthine oxidase 2.5 µmol/L
in potassium phosphate buffer 50 mmol/L (pH 7.8, 37 ºC) were
incubated for 40 min and NBT was added. Blue formazan was then monitored
spectrophotometrically at 550 nm. The amount of protein that inhibited
NBT reduction to 50% maximum was defined as 1 nitrite unit (NU)
of SOD activity.
Assay of NO concentration of incubation medium The incubation
medium of the aortic artery was centrifugated (1000×g,
15 min, 4 ºC) and the supernatant was used for NO measurement.
NO was assayed by the Griess method. Because NO is a compound with
a short half life and is rapidly converted to the stable end products
nitrate (NO3-) and nitrite (NO2-),
the principle of the assay is the conversion of nitrate into nitrite
by cadmium and followed by color development with Griess reagent
(sulfanilamide and N-naphthyl ethylenediamine) in acidic
medium. The total nitrite was measured by Griess reaction. The absorbance
was determined at 540 nm with a spectrophotometer.
Protocol of experiment The first series of experiments were
designed in order to evaluate the protective effects of cariporide
against ACh-induced endothelium-dependent and SNP-induced endothelium-independent
relaxing response of isolating rat aortic rings affected by high
glucose. The experiment was divided into 7 groups with 8 aortic
rings from 8 rats in each group. First, a normal control bioassay
of vasoreactivity was formed in normal Krebs' solution. The rings,
of which a percentage of relaxation induced by ACh 3 µmol/L
to the contraction elicited by Phe 1 µmol/L is more than 80%,
were considered as intact endothelium and used in the study. The
aortic rings of each group were then continually incubated for 6
h in the following medium: (1) control group: glucose 11 mmol/L
in Krebs' solution[9]; (2) high glucose group: glucose
44 mmol/L in Krebs' solution[9]; (3)-(5) cariporide-treated
groups: cariporide 0.01, 0.1, and 1 µmol/L in Krebs' solution
with glucose 44 mmol/L, respectively; (6) cariporide alone group:
cariporide 1 µmol/L in Krebs' solution; (7) mannitol group:
mannitol 44 mmol/L in Krebs' solution. The incubation mediums were
changed every 30 min and cariporide was present throughout the incubation.
After a 6-h incubation of aortic rings, the perfusion solution was
changed to Krebs' solution, and bioassay of vasore-activity was
performed.
The second series of experiments were designed to assay the effects
of high glucose on SOD activity, MDA concentration, NO and the effects
of cariporide on the biochemical parameters in rat aortic rings.
The experiment was divided into 5 groups with 8 aortic segments
of 2 cm from 8 rats in each group; control group, high glucose group,
mannitol group, cariporide-treated group, and cariporide-alone group.
The components of incubation medium were the same as described earlier
except that cariporide only had a dose of 1 µmol/L. The incubations
media were changed every 30 min. Cariporide was added throughout
incubation. After a 6-h incubation, the segments were transfered
to 1 mL normal Kreb's solution which contained ACh 1
µmol/L for 30 min. The aortic segments and the medium were
then collected and frozen at -70 ºC until analyzed.
Data analysis The ACh (3 µmol/L) or SNP (1 µmol/L)
-induced maximal relaxation (Emax) in aortic rings
was calculated as a percentage of the contraction to Phe (1 µmol/L).
The half maximum effective concentration (EC50) was defined
as a concentration of the ACh that induced 50% of maximum
relaxation response to contraction elicited by Phe (1 µmol/L)
and calculated from the concentration-response curve
generated by linear regression analysis. All data were expressed
as mean±SD. Statistical comparisons were made using one-way
ANOVA followed by Newman-Keuls test. P<0.05 was statistically
significant.
Results
Effects of high glucose on EDR and endothelium-independent relaxation
in aortic rings There were no significant differences in ACh-induced
relaxation responses of rat isolated aortic rings before the 6-h
incubation among various groups (data not shown). After a 6-h incubation
of aortic rings in control glucose (11 mmol/L) buffer, ACh (0.003-3
µmol/L) still evoked a normal concentration-dependent relaxation
(Figure 1), the Emax of aortic rings reached 88.4%±12.3%,
and the EC50 value was 94.5±10.8 nmol/L. After the
6-h incubation of the aortic rings and exposure to high glucose
(44 mmol/L), the Emax fell to 43.7%±16.1%
and the EC50 value increased to 154.8±22.9 nmol/L
(P<0.01 vs control group, n=8). However,
no significant changes of Emax and EC50
were shown in the rings incubated in the cariporide (1 µmol/L)-alone
group or mannitol (44 mmol/L) group, compared with the control group
(Figure 1).
The endothelium-independent relaxation induced by SNP (0.001-1
µmol/L) was not significantly different between the different
treated groups (data not shown).
Effects of cariporide on the impairment of EDR induced by high
glucose Treatment with cariporide in different concentration
(0.01, 0.1, and 1 µmol/L, n=8, respectively) significantly
prevented inhibition of EDR induced by high glucose (Figure 2).
The Emax were 61.7%±10.5%, 76.0%±10.5%,
and 83.4%±10.1%, and the EC50 value was 131.5±15.9
nmol/L, 117.1±13.7 nmol/L, and 109.6±10.5 nmol/L, respectively.
There was a significant difference (P<0.05 or P<0.01),
compared with those in the high glucose group (Figure 2).
Effects of cariporide on biochemical index in aortic segments
A 6-h incubation of isolated aortic segments in high glucose
resulted in an elevation of MDA content, decrease of SOD activity
in aortic tissue, and reduction of NO releasing from aortic segments.
Treatment with cariporide (1 µmol/L) in the high glucose (44
mmol/L) group significantly prevented the increase of MDA content,
and protected the activity of SOD and release of NO in aortic segments
(Table 1). Mannitol (44 mmol/L) or cariporide (1 µmol/L) alone
had no effects on MDA, SOD, and NO, compared with the control group
(Table 1).
Discussion
Diabetes mellitus is characterized by chronic hyperglycemia and
associated with significant morbidity as a result of long-term complications,
including diabetic nephropathy, atherosclerosis, and hypertension.
High glucose had a lot of toxicity effects in endothelial cells,
such as impairment of endothelial dependent relaxation[10],
decrease of NO release, generation of free radicals, and increase
in apoptosis[11]. The endothelial dysfunction has been
thought to be the major cause of vascular disease due to hyperglycemia
condition. It was reported that hyperglycemia resulted in an increase
of NHE-1 activity in diabetes[12,13]. In addition, high
glucose may induce the activation of NHE-1 and increase NHE-1 mRNA
expression in vitro smooth muscle cells or myocytes [14,15].
However, there are no reports as to whether NHE-1 inhibitor is able
to protect against the impairment of endothelial functions caused
by high glucose. The present study explores the effects of cariporide
against high glucose-induced endothelial dysfunction of rat isolated
aorta and discusses its mechanisms.
In the present study, we employed a mimic pronounced hyperglycemia
model in which the rat isolated aortic rings were exposed to high
glucose for 6 h. High glucose significantly inhibited EDR and the
release of NO in rat isolated aortic rings, but did not affect vasodilatation
induced by SNP, a NO donor. It has been demonstrated that endothelium
dysfunction induced by high glucose related to the release
of NO from endothelial cells. The same concentration of mannitol
had no effect on EDR of aortic rings, which showed that the damage
of EDR of aortic rings induced by high glucose was not due to a
hyperosmotic effect. These results in the present study were consistent
with a previous study[2]. We also found that cariporide
in dose-dependent manners prevented the inhibition of EDR induced
by high glucose in rat isolated thoracic aorta (Figure 1), and simultaneously
maintained SOD activity and NO release and decreased MDA concentration
caused by high glucose in rat aortic segments (Table 1). It was
reported that cariporide significantly inhibited the injuries of
mitochondrial and pulmonary endothelial cells, and protected cardiac
ischaemia and reperfusion in vitro by reducing intracellular
pH and inhibiting NHE-1 activation, which simultaneously attenuated
oxidant production[16-18]. The Na+/H+
exchanger inhibitor, amiloride, significantly reduced oxidant production
of hepatic stellate cells including intracellular hydroperoxides
and MDA induced by oxidant[19]. Accordingly, it is believed
that the protective effects of cariporide against endothelial dysfunctions
induced by high glucose may partly be due to anti-oxidation and
protective activity of anti-oxidative enzymes.
In the resting state, NHE-1 is relatively quiescent. When intracellular
pH falls, the NHE-1 is activated, and the rate of H+
efflux mediated by NHE-1 increases and causes intracellular alkalinization
and Na+ overload, which leads to intracellular Ca2+
overload through a mechanism of reverse Na+-Ca2+
exchange. It is presumed that during high glucose condition, there
is a metabolic mismatch between glycolysis and glucose oxidation
that results in the accumulation of hydrogen ions, which, in turn,
activates NHE-1, leading to intracellular alkalinization and Ca2+
overload. Therefore, we hypothesized that, in the present study,
the mechanisms of cariporide against high glucose-induced endothelial
dysfunction of rat isolated aortas might be also related to decreasing
intracellular alkalinization and Ca2+ overload. Although
intracellular pH and intracellular Ca2+ concentration
were not assayed in the study, it has been reported that cariporide
could protect from releasing NO in vascular endothelium through
inhibiting activity of NHE-1, decreasing intracellular alkalinization
and Ca2+ overload[17,20].
In conclusion, this study demonstrates that cariporide significantly
prevents endothelial dysfunction, decreases NO release, elevates
MDA concentration and reduces SOD activity induced by high glucose
in rat isolated thoracic aorta. The mechanisms of protective effects
of cariporide may be related to the inhibition of NHE-1 and the
decrease of oxidative stress injury. These results provide a potential
new target for intervention in the prevention of diabetic
complica-tions.
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