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Introduction
Bradykinin locally synthesized in the cardiovascular tissue and blood plasma is believed to contribute to the regulation
of cardiovascular homeostasis such as vascular tone control and vascular permeability alteration by stimulating the
endothelial cells to release vasodilators including nitric oxide (NO), prostacyclin
(PGI2) and endothelium-derived hyperpolarizing
factors. In endothelial cells, it is generally considered that the binding of bradykinin to its receptors _ B1 and/or B2 receptors,
which are G-protein coupled receptors, activates phospholipase C to catalyze the hydrolysis of PIP2 to yield
IP3 and DAG. A biphasic elevation of endothelial
[Ca2+]i is observed upon bradykinin stimulation; a transient initial peak followed by a
sustained plateau phase. The initial transient rise is contributed by the
Ca2+ release from the internal stores via the interaction
of IP3 with IP3 receptor in sarcoplasmic/endoplasmic reticulum, whereas the sustained phase is contributed by the
extracellular Ca2+ influx through
Ca2+ permeable channels in the plasma
membrane[1_6].
Flow shear stress is the most important physiological stimulus for the synthesis and release of different potent
vasodilators from endothelial cells to induce vasodilation. Flow may elicit vasodilation either via a
[Ca2+]i-dependent
mechanism[3,5] or a
[Ca2+]i -independent
mechanism[2]. Flow-induced
Ca2+ influx may be attributed to activation of mechanosensitive cation
channels[27], stimulation of
P2X4 purinoceptor [24,25], modulation of cytoskeleton-mediated signal transduction
pathways[2], activation of K channels, causing an increased driving force for extracellular
Ca2+ influx[20], or facilitation of the delivery of
agonists (eg, ATP, ADP and bradykinin) to the unstirred boundary layer at the cell
surface[4].
Until now, the Ca2+-permeable channel(s) involved in bradykinin- and flow-induced
Ca2+ influx in vascular endothelial cells has still not been very clear, but the members of transient receptor potential (TRP) superfamily offer likely
candidates[26]. TRP was first described as a Drosophila mutant that had an impaired visual transduction with transient receptor potential in
response to continuous light. Since the cloning of Drosophila Trp gene in 1989, approximately 30 unique members of TRP
superfamily have been identified on the basis of amino acid sequence and structural similarity. They are classified into TRPC
(C for canonical), TRPV (V for vanilloid), TRPM (M for melastatin), TRPP (P for PKD), TRPML (ML for mucolipin) and TRPN
(N for NOMPC) subfamilies[10]. All TRP channels, except TRPM4 and TRPM5, are cation channels that allow
Ca2+ influx[26]. The TRPC subfamily is composed of seven mammalian members designated to TRPC1 to 7. All these seven TRPC channels
are involved in Ca2+ influx in response to activation of PLC by membrane
receptors[10].
Targeted knockout of TRPC4 has been shown to markedly reduce the agonist (ATP, acetylcholine and thrombin)-induced
Ca2+ influx in mice artery endothelial
cells[7,23]. Endothelium-dependent smooth muscle relaxation in response to vasoactive
agonists ATP and acetylcholine is also impaired in these
mice[7]. These data provide compelling evidence for the key role of
TRPC4 in agonist-induced endothelial
Ca2+ influx and vascular relaxation. Besides TRPC4, several other TRP channels
including TRPC1 and TRPC3 have been suggested to be involved in the agonist-induced
Ca2+ influx in endothelial
cells[12,13]. For TRPC3, it has been shown that bovine pulmonary artery endothelial cells transfected with TRPC3 channels display
increased Ca2+ influx in response to ATP and
bradykinin[12]. In addition to agonist-induced
Ca2+ influx, TRP channels may also participate in flow-induced
Ca2+ influx. For example, shear stress activates TRPV4 in TRPV4-expressing HEK
cells[8] and it also activates the complex of TRPP1 and TRPP2 in renal epithelial
cells[8,19]. Unfortunately, there is still no evidence to show
the role of TRPV4 or TRPP1/TRPP2 complex in flow-induced
Ca2+ influx in vascular endothelial cells. More importantly, the
role of most TRP channels in agonist- or flow-induced dilatation has not been established, except for the case of TRPC4 and
TRPV1, which are known to participate in the vasodilation in response to ATP, acetylcholine and
ananda-mine[7,26].
In the present study, we explored the functional role of TRPC3 channel in flow- and agonist-induced dilatation of isolated
rat small mesenteric arteries using a specially designed antisense oligo against TRPC3. We also examined the role of TRPC3
in bradykinin- and flow-induced Ca2+ influx in the endothelial cells of isolated small mesenteric arteries.
Materials and methods
Animals Male Sprague-Dawley rats were supplied by the Laboratory Animal Service Centre of the Chinese University of
Hong Kong, Hong Kong, China. We followed the Guide for the Care and Use of Laboratory Animals published by the US
Institute for Laboratory Animal Research, National Research Council in 1996.
Preparation of rat small mesenteric arteries
Male Sprague-Dawley rats (~250_280 g) were killed by inhalation of
CO2. Ileum and associated mesentery were removed and immersed in Krebs-Henseleit solution gassed with 95%
O2_5% CO2 mixture. A third- or fourth-order mesenteric artery (~2_3 mm long) was carefully dissected free of surrounding adipose tissue
under a dissecting microscope (Nikon, Japan).
Isobaric diameter experiments-measurement of flow-induced vasodilation
The method for the flow experiments was as described in a previous
study[16]. After dissecting, the mesenteric artery (~3-mm long) was mounted on a pressure myograph
(Danish MyoTechnology, Denmark) with two glass micropipettes and both cannulation pipettes were connected to
independent reservoirs set at the same height and solution level to ensure no flow. Changes in the vessel diameter and pressure were
tracked and measured with MyoView software (version 1.1 P, 2000, Photonics Engineer-ing). After being cannulated onto
micropipettes, the isolated artery was left to stabilize in Tyrode¡¯s solution while the Tyrode¡¯s solution was continuously
superfused around the artery with a peristaltic pump at a rate of 2_3 mL/min and bubbled with pure
O2 for at least 30 min before experiments began. BSA 1% was included in intraluminal solution and ATP (1
mmol/L) was routinely added in both the extraluminal and intraluminal solutions. Temperature was kept at 37 °C
(± 0.5 °C) in flow experiments.
After the arterial viability was assessed by contraction with 3
mmol/L phenylephrine and subsequent dilation to
1 mmol/L acetylcholine, the viable artery was continued to flow procedure after washing and incubation. The flow (shear
stress 3.5_10.7 dynes/cm2) was initiated by a pressure gradient (5_6 mmHg) in phenylephrine-preconstricted artery, by
moving the two reservoirs an equal distance (5 cm) but in opposite directions at the same time. This ensured that the change
in flow did not cause a simultaneous change in the transmural pressure. The flow was then stopped by moving the two
reservoirs back to the same level after observation. The mean intraluminal pressure was maintained at 50 mmHg throughout
the flow protocol.
Isometric tension experiments-measurement of agonist-induced
vasodilation An approximately 2 mm long segment of
mesenteric artery was dissected and transferred to chambers of a Multi Myograph System (Danish MyoTechnology) filled
with 95% O2_5% CO2 mixture bubbled Krebs-Henseleit solution. The artery segments were mounted in
Mulvany-Halpern myograph (model 400A or 610M, Danish Myo-Technology) with gold-plated tungsten wire (25 µm diameter, Goodfellow,
England). Each wire was fixed to mounting jaws of the myograph. Changes in isometric force were continuously recorded by
using Maclab software (version 3.5). The chamber solution was continuously gassed with a 95%
O2_5% CO2 mixture at 37 °C
(pH 7.4). All arteries were set to an optimal resting tension of 1.5 mN, which had been determined by length_tension
relationship experiments.
Before commencement of the experiments, all artery segments were allowed to equilibrate for 60 min during which the
bath solution was replaced every 20 min with pre-warmed and gassed Krebs-Henseleit solution. The resting tension was
readjusted to 1.5 mN when necessary.
After equilibration, endothelial cell
viability was assessed as sustained
maximal relaxation (>95%) to 1 µmol/L
acetylcholine in arteries constricted with submaximal concentrations of phenylephrine (0.5 µmol/L) combined with U46619 (0.05
µmol/L). The healthy segments were then washed with pre-warmed Krebs-Henseleit solution several times (>4 times) until baseline
tone restored. Subsequently, the accumulated concentration-response curves of histamine and bradykinin were then
evaluated in preconstricted arteries with phenylephrine (0.5 µmol/L) plus U46619 (0.05 µmol/L). For ATP and CPA, we examined the
non-accumulated concentration effect, meaning that a single concentration was used. There was a 10_15 min wait before
washing and the next concentration was applied.
Measurement of endothelial
[Ca2+]i-MetaFluor imaging
system In separate experiments, a specifically modified flow
chamber from pressure myograph chamber (Danish MyoTechnology) by the Technical Services Unit, Chinese University of
Hong Kong was used, which ensured the chamber fit to the MetaFluor imaging system. Following equilibration of
pressurized arteries in the flow chamber, 200 µL Tyrode¡¯s solution containing the fluorescent indicator Fluo-4 AM (Molecular
Probes, 20 µmol/L) and 0.02% pluronic F127 was pumped into the artery lumen at a pressure of <10 mmHg and kept in the
lumen for 1 h in the dark at room temperature. The pressure was kept low during the dye loading to ensure the dye could be
selectively loaded into endothelial cells. After the dye loading, the pressure was raised to 50 mmHg and the artery was
allowed to stabilize for another 10 min. The chamber was placed on an inverted microscope (IX70, Olympus, NY, USA),
equipped with a 20X Olympus water immersion objective (0.50W, UMPLANFL, Olympus). Fluorescence intensity was
measured with a MetaFluor imaging system (Universal Imaging, West Chester, PA, USA). The Fluo-4 AM loaded artery was
excited at 490 nm and the images of the respective 510 nm emissions were collected at every 2-s interval using MetaFluor
v4.6 software (Universal Imaging). The emitted light was transmitted to the collecting device and then to a cooled charge
coupled device (CCD) camera (Photometrics Quantix, Roper Scientific, Trenton, NJ, USA). Video frames containing
fluorescence images were digitized at 512×480 pixel resolution and then analyzed with MetaFluor.
Antisense oligo treatment The second generation of antisense oligo modified by phosphothioates (s) at both ends of
the sequence for three nucleotides was used in the present study. An antisense oligo (antisense-TRPC3) specially targeting
the region surrounding the AUG starting codon for rat TRPC3 mRNA was designed by us and synthesized by Tech Dragon,
Hong Kong, China. Its sequence was as follows: 5¡¯-gsCsAsTCTAgTTAAsAsgsC-3¡¯. A reversed
sequence of the antisense oligo was used as a control: 5¡¯-CsgsAsAATTgATCTsAsCsg-3¡¯.
Antisense oligos or control oligos at 100 mg were directly delivered into the rat blood stream by tail vein injection. After
72-h treatment, the rats were killed and their mesenteric arteries were collected. Then flow- and agonist-induced
vasodilations were separately evaluated using a pressure myograph or a multi myograph system. In addition, a few arteries were
prepared for immunohistochemical staining for assessment of the expression level of TRPC3.
Immunohistochemical staining of TRPC channels
After excision, the small mesenteric arteries were fixed in 10% neutral
buffered formalin solution (Sigma, St Louis, USA) overnight, and then embedded with paraffin following a series of dehydration.
Tissue sections (4-µm thick) were used in this study. After deparaffination and rehydration, tissue sections were stained with
appropriate anti-TRPC polyclonal antibody using a standard SABC method. Tissue sections were permeabilized with PBST
and incubated with primary antibody overnight at 4 °C. Secondary antibody [biotinyl-ated-goat anti-rabbit IgG (H+L)] was
applied for 1 h at room temperature. After washing with PBS 3 times, HRP-streptavidin conjugate was added onto tissue
slides and 1-h incubation was allowed at room temperature. The color was then developed using
DAB-H2O2 solution (0.3 g/mL DAB in
PBS with 0.003% H2O2) and the cell nuclei were subsequently counter-stained with Mayer¡¯s haematoxylin. Slides were
viewed using a Leica microscope (Wetzlar, Germany).
Two negative controls were set for each tissue section. One was the competitive control that was used to test the
specificity of the antibody and the other was the blank control that was applied to monitor the whole staining process. The
competitive control was prepared by incubating the antibody with its respective antigen (provided by Alomone with antibody)
in 1:1 weight ratio for 2.5 h at room temperature and the supernatant was collected after centrifugation at 13 200 rpm for 5 min,
proceeding to the same procedure in parallel with primary antibody staining. The blank control was designed by replacing
the primary antibody with PBS while staining.
SDS/PAGE and immunoblots Immunoblots were performed as described in a previous
study[14]. Rat aortas were homogenized and the lysates were
extracted with protein extraction buffer, which contained 50 mmol/L Tris-HCl, 150 mmol/L NaCl,
1% Nonidet P-40, 0.1% SDS, 50 mmol/L NaF, 2 mmol/L EDTA, 0.5% sodium deoxycholate, pH 7.5, with the addition of
complete protease inhibitor cocktail tablets. Protein concentrations were determined by Bradford assay (Bio-Rad). Proteins
100 µg were loaded onto each lane and separated on an 8%
SDS/PAGE gel after being boiled in SDS loading buffer. After
electrophoresis, proteins were transferred to a
PVDF membrane, and the membrane was then immersed in a blocking solution
containing 5% non-fat milk and 0.1% Tween 20 in PBS buffer for 1 h at room temperature with constant shaking. The
incubation with the primary anti-TRPC3 antibodies (1:200 dilution) was carried out overnight in PBS buffer containing 5%
non-fat milk and 0.1% Tween 20. Immunodetection was accomplished with horseradish peroxidase (HRP)-conjugated
secondary antibody, followed by ECL Plus Western Blotting Detection system. The intensity of the bands was analyzed by the
FluorChem 8000 imaging system.
Chemicals and solutions Anti-TRPC antibodies were purchased from Alomone Laboratories
(Jerusalem, Israel). Phenylephrine hydrochloride was purchased from RBI. U46619 was obtained from Tocris (Mo, USA). Bradykinin was from Calbiochem
(San Diego, USA). Fluo-4 AM (acetoxymethyl ester) and Pluronic F127 were from Molecular Probes . Other chemicals were
from Sigma.
Krebs-Henseleit solution contained: NaCl 119 mmol/L,
NaHCO3 25 mmol/L, MgCl2 1 mmol/L, KCl 4.7 mmol/L,
CaCl2 2.5 mmol/L,
KH2PO4 1.2 mmol/L, and
D-glucose 11 mmol/L. Tyrode¡¯s solution contained: NaCl 117 mmol/L,
MgCl2 1 mmol/L, KCl 4.7 mmol/L,
KH2PO4 1.2 mmol/L,
CaCl2 1.6 mmol/L, HEPES 10 mmol/L,
D-mannitol 30 mmol/L, and D-glucose 11 mmol/L,
pH 7.4. PBS contained: NaCl 140 mmol/L,
Na2HPO4 10 mmol/L, KCl 3 mmol/L, and
KH2PO4 2 mmol/L, pH 7.4. PBST contained 0.01%
Tween-20 in PBS, pH 7.4.
Data analysis Vasodilation to flow was calculated as a percentage using the following equation:
% vasodilation=100×(Df_D
phe) /
(Di_Dphe),
where D represents vessel external diameter;
Df is the maximum vessel diameter during flow;
Dphe is the diameter after phenylephrine constriction and before flow;
Di is the initial diameter before phenylephrine constriction without any treatments.
For measurement of agonist-induced relaxation,
EC50 values were calculated as the agonist concentration that caused
50% maximum relaxation. The effects of agonists were expressed as percentage relaxation from phenylephrine plus
U46619-induced contraction. Concentration-relaxation relationship was analyzed with a non-linear regression curve fitting (GraphPad
Prism, version 3.0, San Diego, CA).
For Ca2+ imaging, fluorescence intensity before the initiation of flow or the application of bradykinin was normalized to 1
(F0). The responses to flow or bradykinin were displayed as the ratio of fluorescence relative to the intensity before flow
(F1/F0).
Statistical evaluation of the effect of antisense oligos was made using a two-tailed Mann-Whitney test by comparing the
maximal % of vasodilation to flow, EC50 for agonist-induced relaxation, and
F1/F0 for endothelial
[Ca2+]i measurement. All data
were shown as means±SEM of n experiments on the vessel segments prepared from different rats. Significance was assumed
at P<0.05.
Results
Effect of antisense oligo on TRPC3 expression In order to study the possible involvement of TRPC3 in flow- and
agonist-induced vasodilation, TRPC3 antisense oligo (antisense-TRPC3) or its control oligo was injected into the rat tail vein.
After 72-h treatment, the inhibitory efficiency of antisense-TRPC3 was evaluated using immunohistochemical staining.
Immunohistochemical staining showed that TRPC3 proteins were expressed in both endothelial cells and smooth muscle
cells in small mesenteric arteries of rats that were treated with control oligos (100
mg) for 72 h (Figure 1A). In contrast, in rats treated with the antisense-TRPC3, no positive signal was observed in endothelial cells and only weak signals were found in
smooth muscle cells (Figure 1D). Two additional control experiments were performed to verify the specificity of
immunohistochemical staining, one with antigen preabsorption (Figure 1B,1E) and the other in the absence of primary anti-TRPC3
antibody (Figure 1C,1F). No signals were observed in both sets of controls (Figure 1B,1C,1E,1F). These results suggested
that the injected antisense-TRPC3 was effective in suppressing TRPC3 protein expression in endothelial cells as well as
smooth muscle cells.
Cellular localization of other TRPC homologs in vascular tissues was explored with immunohistochemical staining using
antibodies against other TRPC homologs in small mesenteric arteries. Strong positive signals in brown color could be also
observed in vascular endothelial cells as well as in vascular smooth muscle layer for TRPC1, 4_6 homologs (Figure
2A,2C,2E,2G). No signal was observed in competitive controls (Figure 2B,2D,2F,2H) and blank controls (data not shown). The
differences in all TRPC staining between positives and competitive controls were very large and obvious, indicating that the
immunohistochemical stainings were TRPC-specific. Note that the blue color in experiments and in controls was due to
hematoxylin counterstaining, which stained the cell nucleus.
Immunoblots were performed to verify the suppressing effect of antisense-TRPC3 on the expression level of TRPC3
proteins. Because of the difficulty in isolating enough proteins from small mesenteric arteries, proteins from rat aortas were
used for the purpose. Immunoblots detected a single band with the molecular size of approximately 150 kDa, which may
represent a glycosylated form of TRPC3. Several previous publications have also reported TRPC3 proteins of similar
molecular sizes[15,22]. Figure 1G shows that antisense-TRPC3 markedly suppressed the expression of TRPC3 proteins in rat
aorta with protein levels decreased by 20%. Note that the aorta
contains many layers of vascular smooth muscle cells, which
are not in direct contact with circulating blood, therefore the suppressing effect of antisense TRPC3 on the expression of
TRPC3 in the aorta is expected to be smaller than that in small mesenteric arteries.
Effect of antisense oligo on flow-induced vasodilation
Flow-induced vasodilation was measured using a pressure
myograph system. Small mesenteric arteries treated with antisense-TRPC3 or control oligos pressurized to 50 mmHg had an
external diameter of 350_400 µm (n=45). After preconstriction with phenylephrine (0.5_1.5 µmol/L) to a similar level
(65%_75% of its initial diameter), the artery was exposed to an intraluminal flow initiated by a pressure gradient (5_6 mmHg) to evoke
dilation. Flow dilation consisted of an initial transient peak followed by a sustained plateau phase (>10 min), which was
flow-dependent. The vessels rapidly contracted again as soon as the flow stopped (Figure 3A). In this study, the peak dilation
in response to flow was compared between the control group and the antisense-TRPC3 group. Analysis revealed that,
compared to the control oligo treatment, antisense-TRPC3 treatment significantly inhibited the magnitude of flow-induced
vasodilation in rat small
mesenteric arteries. The maximal percentage of dilation was reduced from the control value of 85.8%±4.5% to 73.1%±
2.7%, a reduction of approximately 13% (Figure 3B). These results suggested an involvement of TRPC3 in flow-induced
vasodilation.
Effect of antisense oligo on agonist-induced relaxation
The relaxation to common vasodilators including bradykinin,
histamine, ATP and CPA was studied using a multi-myograph system. All these agonists elicited a concentration-dependent
relaxation in rat small mesenteric arteries that were preconstricted with phenylephrine (0.5 µmol/L) plus U46619 (0.05 µmol/L).
Two representative concentration-response curves for bradykinin in control oligo or antisense-TRPC3 treated arteries are
shown in Figure 4A and 4B. While antisense-TRPC3 treatment did not show any effect on histamine-, ATP- and CPA-induced
relaxation (Figure 5), it significantly suppressed the relaxation to bradykinin. In the antisense-TRPC3 group, the
EC50 value of bradykinin was raised to 92.6±0.1 nmol/L from the control value of 32.6±0.1 nmol/L in the control group (Figure
4C). These data suggest that TRPC3 might participate in bradykinin-induced relaxation.
Effect of antisense oligo on flow-induced endothelial
Ca2+ influx Flow-induced endothelial
[Ca2+]i changes were compared between the arteries pretreated with antisense-TRPC3 or control oligos. In both types of arteries, flow initiated a
transient rise in endothelial
[Ca2+]i, which reached its peak in 20_30 s (Figure 6A). Treatment of the arteries with
antisense-TRPC3 oligos significantly attenuated the magnitude of endothelial
[Ca2+]i rise in response to flow (Figure 6B). The peak
F1/F0 value was significantly decreased from 1.75±0.17 to 1.27±0.1.
Effect of antisense oligo on 30 nmol/L bradykinin-induced endothelial
Ca2+ influx Because antisense-TRPC3
treatment significantly suppressed the relaxation to 30
nmol/L bradykinin, 30 nmol/L bradykinin-induced endothelial
[Ca2+]i changes were compared between the arteries treated with antisense-TRPC3 and control oligos. Similar to flow responses, 30 nmol/L
bradykinin induced a transient rise in endothelial
[Ca2+]i in all tested arteries (Figure 6C). Treatment of the arteries with
antisense-TRPC3 oligos significantly reduced the magnitude of endothelial
[Ca2+]i rise in response to 30 nmol/L bradykinin
(Figure 6D). The peak
F1/F0 value was significantly decreased from 1.41±0.07 to 1.14±0.03. We also tested the effect of
Gd3+ and nifedipine on bradykinin-induced
Ca2+ influx. Gd3+ (10 µmol/L), a putative TRPC channel inhibitor, reduced the
bradykinin-induced Ca2+ rise by 89%±2%
(n=4), whereas an L-type Ca2+ channel blocker, nifedipine (1 µmol/L), had no effect
(n=3).
Discussion
In the present study, we provided evidence that TRPC3 channel is involved in bradykinin- and flow-induced vasodilation
in isolated rat small mesenteric arteries. In vessels isolated from antisense TRPC3 oligo-treated rats, the peak magnitude of
flow-induced vasodilation was decreased by approximately 13% (Figure 3B). Antisense oligo treatment also reduced the
sensitivity of vasodilation to bradykinin by increasing its
EC50 value from the control value 32.6±0.1 nmol/L
to 92.6±0.1 nmol/L (Figure 4), though the treatment does not appear to affect the maximal amplitude of relaxation to bradykinin. We also
demonstrated that bradykinin- and flow-induced
Ca2+ influx in the endothelial cells of isolated small mesenteric arteries was
partly mediated by TRPC3. Further-more, antisense oligo treatment reduced flow- and bradykinin-induced
Ca2+ influx in the endothelial cells of isolated small mesenteric arteries (Figure 6). Therefore, it is likely that bradykinin- and flow-induced
vasodilation is partly attributed to the
Ca2+ influx through TRPC3.
Evidence suggests that TRPC3 may participate in agonist-induced
Ca2+ influx in endothelial cells in culture. In human
vascular endothelial cells, expression of an N-terminal fragment of TRPC3, which exerts a dominant negative effect on the
TRPC3 channel function, eliminated store-operated currents induced by thapsigargin or
IP3, suggesting that TRPC3 channel plays a significant role in store-operated cation
conductance[9]. Furthermore, bovine pulmonary artery endothelial cells
transfected with TRPC3 channels display an increased
Ca2+ influx in response to ATP and
bradykinin[12]. These results suggest that TRPC3 forms agonist-activated
Ca2+ permeable ion channels in endothelial cells in culture. Until now, however,
there is still no evidence indicating the role of TRPC3 in endothelial
Ca2+ influx and vasodilation in intact vessels. The results
from the present study clearly indicated an involvement of TRPC3 in bradykinin- and flow-induced
Ca2+ influx as well as
vasodilation in isolated rat small mesenteric arteries. However, the effect of "knocking down" the expression of TRPC3 on
bradykinin- and flow-induced vasodilation is relatively small. This could be because multiple TRPC isoforms may participate
in endothelium-dependent dilation in rat mesenteric arteries. Previous data has demonstrated that TRPC4 is involved in
acetylcholine-induced vasodilation in mice
aorta[7]. Using immunohistochemical staining, we have found the expression of
all TRPC proteins except TRPC7 (no commercial antibody available) in endothelial cells and smooth muscle cells of rat
mesenteric arteries (Figure 2). In addition, it is possible that the expression of other TRPC channels may be upregulated for
functional substitution when TRPC3 expression is suppressed.
Liu et al recently showed that TRPC3 channel expression was increased and this was responsible for an increased
Ca2+ influx into monocytes in primary hypertension. More importantly, after specific TRPC3-knockdown using siRNA, the
reduced TRPC3 expression in cells was accompanied by a significantly reduced
Ca2+ influx[17]. In the present study, we have
not only found an association between TRPC3 and bradykinin-induced
Ca2+ influx, but also established a linkage between
TRPC3 and bradykinin- and flow-induced vascular dilation. Therefore, it appears that TRPC3 is an important channel in the
maintenance of normal vascular function, and that the overexpression of the channels could lead to the development of
cardiovascular diseases.
CPA has been reported to be a specific inhibitor of
Ca2+-dependent ATPase in the endoplasmic reticulum, which depletes
the IP3-sensitive intracellular
Ca2+ stores by blocking the refilling of
Ca2+ stores. In the rat aorta, it may promote
Ca2+ influx into endothelial cells to induce NO release from endothelial cells and then relax the
aorta[18]. A different mechanism underlies
the CPA-induced relaxation in mesenteric artery. Here CPA relaxes the mesenteric artery through the production of cAMP but
not cGMP[11]. In the present study, we found that TRPC3 was not involved in the relaxation to CPA, ATP or histamine. It is
unclear why TRPC3 is involved in dilation to bradykinin but not to CPA, ATP or histamine. One possibility is that different
agonists may be coupled to different
Ca2+ influx channels.
In the present study, immunohistochemical research clearly showed that the TRPC3 protein level was significantly
reduced by the treatment of specific antisense oligo in endothelial cells as well as in smooth muscle cells (Figure 1). The
suppression of TRPC3 expression in smooth muscle cells may change the basal vascular tone and/or alter the contractile
response of the smooth muscle cells to the agonists that bind to smooth muscle cells, because TRPC is also a molecular
component of Ca2+-permeable cation channels in smooth
muscle cells. It has been demonstrated that the suppression of
arterial TRPC3 expression in smooth muscle cells with antisense oligos significantly reduces the depolarization and
constriction of intact cerebral arteries in response to UTP, not to increased intravascular pressure, that is, myogenic
responses[21]. Nevertheless, because both bradykinin and flow first act on vascular endothelial cells to initiate vascular dilation, the
suppression of smooth muscle TRPC3 is not expected to significantly alter the bradykinin- and flow-
induced vasodilation.
In conclusion, with the use of antisense oligo, the present study directly demonstrated that TRPC3, as a
Ca2+ influx channel, was involved in flow-induced vasodilation as well as in bradykinin-induced relaxation in rat small mesenteric
arteries.
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