Alteration of Volume-Regulated Chloride Movement in Rat
Cerebrovascular Smooth Muscle Cells During Hypertension
Xiao-Lian Shi, Guan-Lei Wang, Zheng Zhang, Yu-Jie Liu, Jing-Hui Chen, Jia-Guo Zhou,
Qin-Ying Qiu, Yong-Yuan Guan
Downloaded from http://hyper.ahajournals.org/ by guest on June 16, 2017
Abstract—The cerebrovascular remodeling is a prominent feature of hypertension and considered a major risk factor for
stroke. Cerebrovascular smooth muscle cells meet volume challenge during this pathophysiological process. Our
previous studies suggest that volume regulated chloride channels may be critical to the cell cycle of vascular smooth
muscle cells. However, it is unknown whether the volume-regulated chloride movement is altered in hypertension.
Therefore, we directly measured the concentration of intracellular chloride ([Cl⫺]i) in rat basilar arterial smooth muscle
cells isolated from control rats and rats that were made hypertensive for 1 to 12 weeks after partial renal artery
constriction (2-kidney, 2-clip method) using a 6-methoxy-N-ethylquinolinium iodide fluorescence probe. The [Cl⫺]i in
isotonic solution showed no difference in all of the groups. After hypotonic perfusion, the reduction in [Cl⫺]i was more
prominent in hypertensive cerebrovascular smooth muscle cells than in sham control cells. Genistein, a protein tyrosine
kinase inhibitor, inhibited hypotonic-induced reduction in [Cl⫺]i, whereas sodium orthovanadate, a protein–tyrosine
phosphatase inhibitor, enhanced hypotonic-induced reduction in [Cl⫺]i in both groups. The percentage inhibition of
reduction in [Cl⫺]i by genistein on volume-regulated chloride movement has a positive correlation with blood pressure
levels in the 2-kidney, 2-clip hypertensive group, as is the case for the percentage increase of reduction in [Cl⫺]i by
sodium orthovanadate. Antihypertensive therapy with the angiotensin-converting enzyme inhibitor captopril completely
reversed abnormal volume-regulated chloride movement in hypertensive rats. We conclude that volume-regulated
chloride movement is augmented in rat cerebrovascular smooth muscle cells in proportion to the severity of
hypertension. (Hypertension. 2007;49:1371-1377.)
Key Words: hypertension (kidney) 䡲 hypertrophy/remodeling 䡲 chloride channels 䡲 cerebral artery
䡲 vascular smooth muscle
I
n cerebral arteries, hypertension induces vascular remodeling, which was defined as an increase in the crosssectional area of the vessel wall (hypertrophy of the vessel
wall) and a reduction in the external diameter of cerebral
arteries.1,2 These structural changes cause the impairment of
cerebral vasodilation and make stroke outcome worse. In
response to chronic increase in blood pressure (BP) and
vascular hypertrophy, adaptive remodeling of cerebral
smooth muscle cells is closely associated with perturbations
of ionic movements across the cell membrane. The upregulation of L-type Ca2⫹ channels has been demonstrated in
cerebral vessels from several hypertensive animal models.3–5
The increase in high-conductance, Ca2⫹-gated K⫹ channels6
and inward rectifier channels7 and the reduction in voltagegated potassium channels6,8 and ATP-sensitive K⫹ channels9
have been reported to contribute to changes in cerebral
vasodilation during hypertension. However, it is unclear
whether there is a change in anion channels in cerebrovascular smooth muscle (CVSM) cells during hypertension.
The 3D morphometric study has revealed that an increase
in cell volume of vascular smooth muscle cells (VSMCs) is
the primary change responsible for the hypertrophy of mesenteric arterial media in spontaneously hypertensive rats.10
However, it is not known whether CVSM cells undergo a
similar remodeling process in the 2-kidney, 2-clip (2k2c)
renal hypertension model.
Recent growing evidence suggests that volume-regulated
Cl⫺ channels (ICl.vol) play an important role in the control of
cell volume, proliferation, apoptosis, and membrane potential,11 especially in VSMCs.12 Although the molecular nature
of ICl.vol is still under verification, ClC-3, a member of the
voltage-gated ClC Cl⫺ channel family, is currently regarded
as the most potential molecular component involved in the
activation or regulation of ICl.vol in VSMCs.12 The evidence
supporting the idea includes the following. First, ClC-3
antisense inhibited the functional expression of ClC-3 and
endothelin-1–induced proliferation in cultured rat aortic
VSMCs in the same time-dependent manner.13 Second, patch
Received November 15, 2006; first decision December 7, 2006; revision accepted March 22, 2007.
From the Department of Pharmacology, Zhongshan Medical College, Sun Yat-Sen University, Guangzhou, People’s Republic of China.
The first 3 authors contributed equally to this work.
Correspondence to Yong-Yuan Guan, Department of Pharmacology, Zhongshan Medical College, Sun Yat-Sen University, 74 Zhongshan 2 Rd,
Guangzhou, 510089, People’s Republic of China. E-mail [email protected]
© 2007 American Heart Association, Inc.
Hypertension is available at http://www.hypertensionaha.org
DOI: 10.1161/HYPERTENSIONAHA.106.084657
1371
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June 2007
clamp experiments combined with gene-targeting studies
have found that the ClC-3 Cl⫺ channel is responsible for the
volume regulation of endogenous ICl.vol and the concentration
of intracellular chloride ([Cl⫺]i) in A10 aortic VSMCs.14 We,
therefore, hypothesized that volume-regulated Cl⫺ movement
though ICl.vol in CVSM cells might be altered during chronic
hypertension.
A major goal of this study was to determine whether the
volume-regulated Cl⫺ movement is altered during hypertension by directly measuring [Cl⫺]i in rat CVSM cells. In
addition, protein tyrosine kinase (PTK) has been reported to
mediate volume regulation of ICl.vol in VSMCs.14 It is noteworthy that increased PTK activity was involved in the
enhanced contraction of vascular smooth muscle in the
development of hypertension.15 Therefore, we further tested
whether the volume regulation of [Cl⫺]i associated with PTK
activity would change during hypertension.
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Methods
For details regarding animal and cell experiments, please see the
online supplements available at http://hyper.ahajournals.org.
Animal Models
All of the experimental procedures were approved by the Sun
Yat-Sen University Committee for Animal Research and were in
accordance with the National Institutes of Health Guide for the Care
and Use of Laboratory Animals. 2k2c hypertensive rats were
prepared as described previously.16
Tissue Preparation and
Immunofluorescence Analysis
The brain sections containing 3 mm of basilar artery were removed
and fixed in 4% paraformaldehyde. The CVSM cells were visualized
with monoclonal ␣-smooth muscle actin by an immunostaining
method.17 The cross-sectional area of basilar arterial media, the wall
diameter, the lumen diameter, and the wall:lumen ratio (the medial
thickness to the ID) were assessed after immunostaining.1
Cell Isolation
Rat basilar arterial smooth muscle cells were isolated by enzymatic
digestion as described previously.18
Measurement of Cell Membrane Capacitance
The voltage-clamp experiment was performed as described previously.14 The cell membrane capacitance was calculated by integrating the area under an uncompensated capacitive transient elicited by
a 5-mV hyperpolarizing pulse from a holding potential of 0 mV.19
Measurement of [Clⴚ]i
[Cl⫺]i was measured using 6-methoxy-N-ethylquinolinium iodide as
described.14
Statistical Analysis
All of the values are expressed as mean⫾SEM. Comparisons
between 2 groups were analyzed using Student’s t test and among 3
groups by ANOVA followed by a posthoc comparison using the least
significant difference test (SPSS 11.0). Values of P⬍0.05 were
considered statistically significant.
Results
Development of Hypertension and Morphological
Change in Basilar Arterial Smooth Muscle Media
in 2k2c Models
BP in the 2k2c group rose progressively after operation as
described previously.16 At 4, 8, and 12 weeks postopera-
tively, the mean values of BP in the 2k2c group were
significantly higher than those in sham-operated groups.
Development of hypertension in the 2k2c group could be
prevented by chronic captopril treatment (see details in
data supplement).
The cell size of CVSM cells, as assessed by cell membrane
capacitance, was significantly increased in hypertensive
groups at all of the time points, whereas there was no
significant difference between sham controls. Moreover, the
cell membrane capacitance of hypertensive CVSM cells
tended to be larger as time after surgery increased (P⬍0.01;
Table S1). Immunostaining demonstrated a time-dependent
increase in ␣-smooth muscle actin staining in basilar arteries
from the 2k2c group as BP increased. At 4 weeks postoperatively, there was no significant difference in the structure
parameters among all of the groups. However, at the end of
weeks 8 and 12, the mean values of cross-sectional area, wall
diameter, lumen diameter, and wall:lumen ratio in the hypertensive groups were significantly higher than those in the
sham-operated group, which could be reversed by captopril
treatment (Table S2).
Comparison of [Clⴚ]i in Rat CVSM Cells
From the Stern–Volmer equation, the Ksv was 37.1 mmol/L
and the resting [Cl⫺]i of rat CVSM cells in isotonic solution
was 29.0 ⫾ 1.0 mmol/L (n⫽60 cells from 12 rats). The result
shown in Figure 1 summarizes [Cl⫺]i measured from different
groups at different time points after operation; there were no
significant differences in [Cl⫺]i in isotonic solution among all
of the groups (P⬎0.05). Change of the medium from isotonic
solution to hypotonic solution decreased [Cl⫺]i in CVSM
cells, which is consistent with our previous report in A10
VSMCs.14 The mean values of [Cl⫺]i in isotonic solution in
CVSM cells from 1, 4, 8, and 12 weeks sham-operated rats
were 28.7⫾1.0, 29.1⫾1.2, 29.2⫾1.1, and 29.2⫾0.9 mmol/L,
respectively, which were decreased to 24.4⫾1.0, 24.6⫾1.0,
24.5⫾1.1, and 24.7⫾1.0 mmol/L when cells were perfused
with hypotonic solution (n⫽30 cells from 8 rats). Hypotonic
perfusion results in 15.1%⫾2.4%, 15.5%⫾3.0%,
16.1%⫾2.3%, and 15.4%⫾2.0% decrease in [Cl⫺]i as compared with their isotonic controls; there is no significant
difference between sham-operated groups at different time
points.
In the hypertension group, hypotonic solution induced
more of a decrease in [Cl⫺]i than those in age-matched
sham-operated groups. The mean values of [Cl⫺]i from the 1-,
4-, 8-, and 12-week hypertensive group were 28.5⫾0.9,
28.6⫾1.0, 28.3⫾0.9 and 28.6⫾1.0 mmol/L, under isotonic
solution, respectively, which were decreased to 22.7⫾1.7,
20.7⫾1.0, 19.6⫾0.9, and 18.9⫾ 0.8 mmol/L, after exposure
to hypotonic solution, respectively, resulting in
20.4%⫾2.8%, 27.6%⫾4.1%, 30.9%⫾3.7%, and 33.7%⫾
3.8% reduction in [Cl⫺]i, respectively. The percentage decrease of [Cl⫺]i induced by hypotonic solution in CVSM cells
from different hypertension groups showed a greater degree
of reduction (range: 20% to 35%) of [Cl⫺]i as compared with
the degree of reduction (⬇15%) observed in age-matched
sham controls. The hypotonic-induced reduction in [Cl⫺]i, in
Shi et al
Volume-Regulated Chloride Movement and Hypertension
1373
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Figure 1. Comparison of [Cl⫺]i in CVSM cells
under isotonic, hypotonic, and hypertonic
conditions. Representative time course of
changes in [Cl⫺]i were recorded in CVSM
cells from sham-operated (sham, f), hypertensive (Htn, E), and captopril treatment
(Cap, ‚) rats at 1 week (A), 4 weeks (B), 8
weeks (C), and 12 weeks (D) after operation,
respectively. E, Correlation between the
change in hypotonic [Cl⫺]i in rat CVSM cells
and BP.
hypertensive CVSM cells tended to be greater as time after
surgery increased.
We also examined whether the administration of captopril
would reverse hypertension-induced change in volumeregulated [Cl⫺]i in CVSM cells. As shown in Figure 1A
through 1D, no significant differences were observed in
the mean values of [Cl ⫺] i in CVSM cells between
captopril-treated hypertensive rats and age-matched sham
controls. Under isotonic condition, the mean values of
[Cl⫺]i in CVSM cells from the 1-, 4-, 8-, and 12-week
captopril group were 28.8⫾1.0, 28.9⫾1.3, 29.7⫾1.3, and
29.4⫾1.3 mmol/L, respectively. In the presence of captopril, subsequent exposure of CVSM cells to hypotonic
solution caused a decrease in [Cl⫺]i with the mean values
of 24.5⫾0.8, 24.1⫾1.2, 24.6⫾1.1, and 24.7⫾1.1 mmol/L,
respectively (n⫽30 from 8 rats), and the percentage
decrease in [Cl⫺]i induced by hypotonic solution was
15.9%⫾2.4%, 16.3%⫾3.0%, 16.9%⫾2.0%, and
15.7%⫾2.2%, respectively. Captopril treatment prevented
the enhancement of volume-regulated Cl⫺ movement during hypertension. The reduction in [Cl⫺]i induced by
hypotonic solution had a significant positive correlation
with BP levels in the hypertensive group (n⫽30 cells from
8 rats; r⫽0.8501; P⬍0.001; Figure 1E).
Effects of Genistein and Vanadate on [Cl-ⴚ]i in Rat
CVSM Cells
Table 1 summarizes effect of genistein (30 ␮mol/L; a PTK
inhibitor) on osmotic-regulated [Cl⫺]i in CVSM cells from
sham and hypertensive rats. When CVSM cells were exposed
to hypotonic solution containing genistein, inhibition of PTK
caused [Cl⫺]i to rise consistent with our previous reports in
aortic VSMCs.14 The pooled data from all of the groups
were summarized in Figure 2A. The percentage inhibition
of reduction in [Cl⫺]i by genistein (for calculation, see
online supplement) in CVSM cells from sham-operated
rats was ⬇50%, showing no significant difference between
different time points. However, in the hypertensive group,
the percentage inhibition of reduction in [Cl⫺]i by genistein
was higher at every sampling point compared with those of
sham controls. The mean values of percentage inhibition in
CVSM cells from 1-, 4-, 8-, and 12-week hypertensive rats
were 55.8%⫾8.7%, 72.4%⫾5.2%, 80.1%⫾4.5%, and
83.9%⫾4.5% (n⫽30 from 8 rats), respectively, which in-
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June 2007
TABLE 1.
Effect of Genistein on Hypotonicity-Induced 关Clⴚ兴i
关Cl⫺兴i, mmol/L
1 Week
4 Weeks
8 Weeks
12 Weeks
关Cl⫺兴i iso
29.1⫾1.0
29.1⫾1.0
29.1⫾1.0
29.2⫾0.9
⫺
关Cl 兴i.hypo
24.6⫾0.9
24.4⫾1.0
24.6⫾0.9
24.4⫾0.9
关Cl⫺兴i.gen⫹hypo
26.9⫾1.0
26.9⫾0.9
26.9⫾1.0
26.8⫾0.9
关Cl⫺兴i iso
28.7⫾1.4
28.5⫾1.1
28.4⫾1.2
28.4⫾1.3
⫺
关Cl 兴i.hypo
23.6⫾1.1
20.9⫾1.1*
19.8⫾1.1*
18.8⫾1.0*
关Cl⫺兴i.gen⫹hypo
26.4⫾1.3
26.5⫾1.4
26.7⫾1.0
26.8⫾0.8
关Cl⫺兴i iso
28.9⫾0.7
28.9⫾1.0
29.1⫾0.9
29.0⫾0.9
⫺
关Cl 兴i.hypo
24.2⫾0.7
24.5⫾1.0†
24.3⫾0.9†
24.4⫾0.9†
关Cl⫺兴i.gen⫹hypo
26.4⫾0.8
26.8⫾0.9
26.8⫾1.0
26.7⫾1.1
Sham-operated group
2k2c group
2k2c⫹captopril group
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gen indicates genistein; hypo, hypotonic; iso, isotonic.
*P⬍0.01 vs corresponding sham group; †P⬍0.01 vs corresponding 2k2c group (n⫽30 from 8
rats).
creased gradually as time after operation increased. Captopril
treatment completely restored the mean values of percentage
inhibition of reduction in [Cl⫺]i to the level (⬇50%) similar to
those of sham controls. As shown in Figure 2B, there is a
significant positive correlation between the percentage inhibition of reduction in [Cl⫺]i by genistein and BP levels in the
hypertensive group (n⫽30 cells from 8 rats; r⫽0.8653;
P⬍0.001).
To further test whether PTK is indeed involved in the
volume regulation of [Cl⫺]i. in CVSM cells, we examined the
effect of protein–tyrosine phosphatase inhibitor sodium orthovanadate (500 ␮mol/L) on osmotic regulation of [Cl⫺]i. As
shown in Table 2, exposure of the cells to sodium orthovanadate in hypotonic solution caused a further decrease in [Cl⫺]i,
suggesting that the osmotic regulation of [Cl⫺]i in CVSM
cells may be linked to a PTK-mediated phosphorylation and
dephosphorylation activity. As shown in Figure 3A,
hypotonic-induced reduction in [Cl⫺]i in CVSM cells from all
of the sham-operated rats was further decreased by orthovandate by ⬇45%, showing no significant difference when
sham control groups at different time points were compared with each other. However, in the 1-, 4-, 8-, and
12-week hypertension group, the reduction in [Cl⫺]i in
CVSM cells by hypotonic solution was further decreased
much more than those of sham controls, and the mean
percentage increase in hypotonic-induced reduction in
[Cl⫺]i by orthovandate (for calculation, see online supplement) was 44.0%⫾4.2%, 30.6%⫾3.3%, 25.1%⫾3.9%, and
18.8%⫾ 2.6%, respectively. The abnormal response of
hypotonic-induced reduction in [Cl⫺]i to sodium orthovandate was also normalized by captopril treatment. Moreover, correlation analysis showed that the percentage
increase in hypotonic-induced reduction in [Cl⫺]i by sodium orthovandate had a negative correlation with BP
levels in the hypertensive group (n⫽30 cells from 8 rats;
r⫽⫺0.8021; P⬍0.001; Figure 3B).
Discussion
Figure 2. Effects of genistein on [Cl⫺]i induced by hypotonic
solution in rat CVSM cells during hypertension. A, Comparison
of the percentage inhibition of hypotonic-induced [Cl⫺]i by
genistein in CVSM cells from sham-operated, hypertensive, and
captopril treatment groups at different time point during hypertension. *P⬍0.01 vs corresponding sham-operated group;
†P⬍0.01 vs corresponding hypertensive group (n⫽30 from 8
rats). B, Correlation between change in genistein [Cl⫺]i in rat
CVSM cells and BP.
Several new findings are presented in this study. First, in
2k2c hypertensive models, our study is the first to show
basilar arterial media undergoing remodeling, with an increase in CVSM cell size and cross-sectional area of the
media. Second, although the [Cl⫺]i showed no difference
between hypertensive rats and sham controls, hypotonic
perfusion induced much more decrease in [Cl⫺]i in hypertensive rats than those of sham controls, suggesting that the
volume-regulated Cl⫺ movement through ICl.vol could be
Shi et al
TABLE 2.
Volume-Regulated Chloride Movement and Hypertension
1375
Effect of Sodium Orthovanadate on Hypotonicity-Induced 关Clⴚ兴i
关Cl⫺兴i, mmol/L
1 Week
4 Weeks
8 Weeks
12 Weeks
关Cl⫺兴i iso
29.2⫾0.9
28.8⫾1.1
29.3⫾0.9
29.4⫾0.8
⫺
关Cl 兴i.hypo
24.7⫾0.7
24.5⫾0.9
24.7⫾0.9
24.8⫾0.8
关Cl⫺兴i.van⫹hypo
20.4⫾0.8
20.7⫾1.1
20.7⫾0.9
20.7⫾1.0
关Cl⫺兴i iso
28.7⫾1.4
28.7⫾1.0
28.5⫾1.1
28.4⫾1.2
⫺
关Cl 兴i.hypo
23.3⫾0.9
21.0⫾0.8†
19.7⫾1.0†
18.7⫾1.0†
关Cl⫺兴i.van⫹hypo
19.0⫾0.5*
17.6⫾1.0†
16.8⫾0.7†
16.4⫾0.7†
关Cl⫺兴i iso
29.0⫾0.9
29.1⫾0.9
29.2⫾0.9
28.7⫾0.8
⫺
关Cl 兴i.hypo
24.4⫾0.6
24.4⫾0.8§
24.3⫾0.9§
24.2⫾0.7§
关Cl⫺兴i.van⫹hypo
19.8⫾0.9‡
20.5⫾0.9§
20.5⫾0.9§
20.6⫾0.8§
Sham-operated group
2k2c group
2k2c⫹captopril group
enhanced during the development of hypertension. We also
showed a correlation between hypotonic-induced decreases in [Cl⫺]i and an increase in BP in hypertensive
animals. These findings suggest the enhanced ICl.vol activity
in hypertension may be associated with cerebrovascular
structural remodeling. In addition, cell volume regulation
Percentage Increase
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hypo indicates hypotonic; iso, isotonic; van, vanadate.
*P⬍0.05; †P⬍0.01 vs corresponding sham controls; ‡P⬍0.05; §P⬍0.01 vs corresponding 2k2c
group (n⫽30 from 8 rats).
Figure 3. Effects of sodium orthovanadate on hypotonicinduced [Cl⫺]i in rat CVSM cells during hypertension. A, Comparison of the percentage increase of hypotonic-induced
[Cl⫺]i by sodium orthovanadate in CVSM cells from shamoperated, hypertensive, and captopril treatment groups at
different time points during hypertension. *P⬍0.01 vs corresponding sham-operated group; †P⬍0.01 vs corresponding
hypertensive group (n⫽30 from 8 rats). B, Correlation
between change in sodium orthovanadate [Cl⫺]i in rat CVSM
cells and BP.
of Cl⫺ movement in CVSM cells is closely coupled to
phosphorylation/dephosphorylation mediated through
PTK. This regulatory mechanism could also change as
hypertension proceeds.
The present structural remodeling in the basilar artery in
2k2c hypertensive models is in agreement with previous
findings from spontaneously hypertensive rats,1,10,20 suggesting that both genetic and nongenetic hypertension models
share similar morphological alterations in the cerebral basilar
arterial media wall during hypertension. However, it is still
not known whether the increase in the cerebrovascular media
wall in hypertension is because of either hypertrophy and/or
hyperplasia of the VSMCs.
Hypotonic-induced cell swelling has been used to mimic
the increase in cell volume in several cell types in response to
physical stresses.21,22 CVSM cells may not meet osmotic
challenge during hypertension; however, our present study
demonstrated that the cell volume of CVSM cells became
larger as BP increased in the 2k2c renal hypertension model.
Therefore, in response to increased cell volume, it is likely
that ICl.vol would be activated by cell shape change when
CVSM is exposed to increased BP.
The pharmacological and electrophysiological properties of the volume-regulated Cl⫺ current in CVSM cells
were similar to those of ICl.vol reported in other VSMCs and
numerous other cell types.12,23,24 In addition, nonspecific
I C l . v o l blockers, indanyloxyacetic acid, and 4,4⬘diisothiocyanatostilbene-2,2⬘-disulphonic acid have been
demonstrated to hyperpolarize and dilate rat cerebral artery
smooth muscle cells.23 Because the Nernst equilibrium
potential for Cl⫺ (⫺20 to ⫺30 mV) is positive to the
resting membrane potential in VSMCs (⫺70 mV), activation of ICl.vol would theoretically cause depolarization of
CVSM cells. Thus, there is a possible explanation for
enhanced activity of ICl.vol in hypertension: the increasing
BP or vascular hypertrophy may activate ICl.vol, cause Cl⫺
efflux, and provide a depolarizing influence on CVSM
cells. Alteration of ICl.vol activity thereby may contribute to
altered excitation– contraction coupling in hypertensive
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CVSM cells. On the other hand, recent studies have found
that ICl.vol plays dual roles in regulating cell proliferation
and apoptosis,11,12 although it is not clear whether alteration of cell proliferation or apoptosis is involved in
hypertrophy of CVSM; the possible contribution of enhanced activity of ICl.vol to cerebral arteriolar remodeling
may be related to cell cycle regulation.
Our present study could not determine whether the
enhanced ICl.vol is the cause or effect of the proposed
remodeling. However, our results have found that the
enhancement of volume-regulation of ICl.vol and alteration
of its regulatory mechanism occurred before any large
difference in BP existed between hypertensive animals and
sham-operated controls. Because hypertrophic remodeling
is well known as the primary change in the development of
hypertension, we proposed that the activity of ICl.vol may be
enhanced as the volume of CVSM cells increased.
Numerous studies have demonstrated that PTK phosphorylation and dephosphorylation are involved in the volume
regulation of ICl.vol in a variety of cell systems, including
cultured VSMCs,14 atrial myocytes,25 and lymphocytes.26 Our
results in CVSM cells and aortic smooth muscle cells14
indicated that tyrosine phosphorylation favors stimulation of
ICl.vol by exposure to hypotonic solution, and tyrosine dephosphorylation blocks hypotonic-induced ICl.vol activation. These
conclusions were consistent with those reported in human T
lymphocytes26 and rat astrocytes27 but different from those in
human atrial myocytes.25 The possible explanation for the
discrepancy between these studies may be because of
different cell types and species and/or nonspecific effects
of PTK or protein–tyrosine phosphatase inhibitors.25 We
further have found that the hypotonic-induced Cl⫺ movement was more sensitive to these PTK/protein–tyrosine
phosphatase inhibitors as BP levels increased in hypertensive rats. These data suggested that an increase in the
tyrosine kinase activity in hypertension could lead to an
enhanced activity of osmotic-sensitive Cl⫺ movement. The
present data are in line with the previous report demonstrating that an increase in the activity of PTK in hypertensive aorta could lead to an enhanced Ca2⫹-linked aorta
contraction.15 This finding also suggests that PTK is a
shared potential candidate contributing to aortic remodeling in different pathological models.
It is important to know the molecular mechanism responsible for the osmotic-regulated Cl⫺ movement in
CVSM cells. It is, therefore, reasonable to perform additional gene targeting experiments and patch-clamp studies
to answer the above questions. In aortic smooth muscle
cells, our previous studies have provided strong evidence
showing that ClC-3, a member of ClC channel family, may
be the molecular component involved in the activation or
regulation of ICl.vol.14 However, the molecular identity of
ICl.vol remains controversial and needs specific Cl⫺ channel
blockers.12 Therefore, the gene targeting experiments were
not performed in this study.
Perspectives
The present study has shown for the first time that there is an
enhancement of volume-regulated Cl⫺ movement across the
CVSM cell membrane mediating through PTK/protein–
tyrosine phosphatase phosphorylation and dephosphorylation
in the development of hypertension. It is becoming apparent
that ICl.vol may play a key role in more general cell functions,
including cell volume regulation, cell proliferation, and cell
apoptosis.11,12 Our data provide a new insight into our
understanding of the role of ICl.vol in some pathological
processes, such as cerebrovascular remodeling, leading to
stroke in hypertension.
Sources of Funding
This work was supported by the National Natural Science Foundation of China (No. 30472021 and No. 30500616), by Science
Foundation of Ministry of Education in China (No. 20050558072),
and by China Medical Board (No. 00730).
Disclosures
None.
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Alteration of Volume-Regulated Chloride Movement in Rat Cerebrovascular Smooth
Muscle Cells During Hypertension
Xiao-Lian Shi, Guan-Lei Wang, Zheng Zhang, Yu-Jie Liu, Jing-Hui Chen, Jia-Guo Zhou,
Qin-Ying Qiu and Yong-Yuan Guan
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Hypertension. 2007;49:1371-1377; originally published online April 16, 2007;
doi: 10.1161/HYPERTENSIONAHA.106.084657
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