(Circulation. 1999;99:392-399.)
© 1999 American Heart Association, Inc.
Clinical Investigation and Reports |
Role of Extracellular Signal-Regulated Kinases in Angiotensin II–Stimulated Contraction of Smooth Muscle Cells From Human Resistance Arteries
From the Medical Research Council Multidisciplinary Research Group on Hypertension, Clinical Research Institute of Montreal, University of Montreal, Montreal, Quebec, Canada.
Correspondence to Rhian M. Touyz, MD, PhD, Clinical Research Institute of Montreal, 110 Pine Ave W, Montreal, Quebec, Canada H2W 1R7. E-mail touyzr{at}ircm.umontreal.ca
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
Background—We assessed the role of extracellular signal–regulated kinases (ERKs) in Ang II–stimulated contraction and associated signaling pathways in vascular smooth muscle cells (VSMCs) from human small arteries.
Methods and Results—VSMCs derived from resistance arteries (<300 µm in diameter) from subcutaneous gluteal biopsies of healthy subjects (n=8) were used to assess Ang II–stimulated [Ca2+]i, pHi, and contractile responses. [Ca2+]i and pHi were measured with fura 2-AM and BCECF-AM, respectively, and contraction was measured photomicroscopically in cells grown on Matrigel matrix. To determine whether tyrosine kinases and ERKs influence Ang II–stimulated responses, cells were pretreated with 10-5 mol/L tyrphostin A-23 (tyrosine kinase inhibitor) and PD98059 (MEK inhibitor). Ang II–stimulated MEK activity was determined by tyrosine phosphorylation of ERKs. The angiotensin receptor subtypes (AT1 and AT2) were assessed with [Sar1,Ile8]Ang II (a nonselective subtype antagonist), losartan (a selective AT1 antagonist), and PD123319 (a selective AT2 antagonist). Ang II dose-dependently increased [Ca2+]i (pD2=8.4±0.36, Emax=541±55 nmol/L), pHi (pD2=9.4±0.29, Emax=7.19±0.01), and contraction (pD2=9.2±0.21, Emax=36±2.2%). Ang II induced rapid tyrosine phosphorylation of ERKs, which was inhibited by PD98059. Tyrphostin A-23 and PD98059 attenuated (P<0.05) Ang II–stimulated second messengers, and PD98059 reduced Ang II–induced contraction by >50%. [Sar1,Ile8]Ang II and losartan, but not PD123319, blocked Ang II–stimulated responses.
Conclusions—These data demonstrate that in VSMCs from human peripheral resistance arteries, functional Ang II receptors of the AT1 subtype are coupled to signaling cascades involving Ca2+ and pHi pathways that are partially dependent on tyrosine kinases and ERKs. ERKs, the signaling cascades characteristically associated with cell growth, may play an important role in Ang II–stimulated contraction of human VSMCs.
Key Words: arteries • calcium • kinases • signal transduction • angiotensin
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
The major function of vascular smooth muscle cells (VSMCs) is to maintain normal vascular tone via coordinated contraction-relaxation cycling. These cells are also responsible for the structural characteristics of the vessel wall, including growth, development, remodeling, and repair.1 Vascular smooth muscle function is modulated by various growth factors and vasoactive peptides, among which angiotensin II (Ang II) has been identified as an important endogenous regulator. Ang II is a multifunctional peptide that influences both contraction and growth.2 3 It binds at least 2 receptors, AT1 and AT2.4 Most known Ang II–induced effects are mediated via AT1 receptors, whereas the function and signaling pathways of the AT2 subtype remain unclear. The AT1 receptor couples to a wide variety of signal transduction events, including phospholipase C, tyrosine kinases, and mitogen-activated protein kinases (MAPKs) (or extracellular signal–regulated protein kinase [ERK]).5 The ERK cascade consists of a 3-kinase module that includes a MAPK (ERK), which in turn is activated by a MAPK/ERK kinase (MEK), which in turn is activated by a MEK kinase (MEKK).6 7 Multiple mammalian MAPK pathways have been identified, of which the ERK cascade is the best characterized. It consists of Raf isoforms, MEK1/2, and ERK-1 and ERK-2 and is regulated by Ras.7 8
Elucidation of Ang II signaling pathways has been extensively investigated in animal VSMCs.5 9 However, little is known about the intracellular transduction events in human VSMCs. The few human studies performed were done in cells from aortic, coronary, internal mammary, umbilical, and uterine arteries and saphenous veins.10 11 12 13 14 15 16 17 Furthermore, these studies all examined large vessels, which do not contribute significantly to peripheral resistance or blood pressure regulation. Also, in all of these studies, immortalized or passaged cultured cells, which lose their contractile phenotype and do not resemble the cells from which they were originally derived, were investigated. To the best of our knowledge, there are no data in the literature on Ang II signaling events in VSMCs from human peripheral resistance arteries. The aim of the present study was to investigate the receptor subtypes and some of the intracellular transduction pathways through which Ang II mediates its actions in VSMCs from human peripheral resistance arteries. In particular, we examined whether ERK-dependent pathways play a role in Ang II–elicited second messengers and associated contraction in isolated VSMCs. The novelty of this study relates to the facts that humans, and not experimental animals, were studied; that primary cultured cells, which retain their contractile phenotype, were used; and that small arteries, which contribute to blood pressure regulation, were examined. Furthermore, [Ca2+]i and contractile responses were measured simultaneously, which allows for the investigation of the temporal relationship between an intracellular signaling event ([Ca2+]i responses) and a functional effector (contraction).
Our study demonstrates that in VSMCs from human peripheral resistance arteries, Ang II–induced contraction and associated second messengers are mediated via receptors of the AT1 subtype, signaling in part by tyrosine kinases and ERKs. We also show that MEK may influence [Ca2+]i by modulating Ca2+ influx and intracellular Ca2+ mobilization. Our data thus show for the first time that in VSMCs from human small arteries, ERK-dependent pathways, which are characteristically involved in signaling cascades associated with cell growth, may also play an important role in Ang II–mediated contraction. These results have important clinical significance, because small arteries are the vessels that play a critical role in regulating peripheral resistance and blood pressure in humans.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Cell Culture
The study was approved by the Ethics Committee of the Clinical Research Institute of Montreal (IRCM). Written informed consent to participate in the study was obtained from each subject. Healthy men 25 to 50 years old were recruited. They had a normal physical examination, complete blood count, blood biochemistry, and urinalysis. Gluteal biopsies of subcutaneous fat, 1.0x0.5x0.5 cm, were obtained under local anesthesia. Arteries were dissected from gluteal fat under a dissecting microscope immediately after the biopsy had been performed. Small arteries were isolated as previously described.18 Only vessels with lumen diameters <300 µm were used for cell culture. Vessels of this size correspond to resistance arteries, which contribute significantly to the regulation and distribution of blood flow and pressure.19 20 21 VSMCs were isolated and characterized as described in detail previously,22 23 with some modifications. Briefly, cleaned arteries were placed in Ham's F-12 culture medium containing 1% gentamicin, collagenase (type 1), elastase, soybean trypsin inhibitor, and BSA and vortexed in an incubator for 1 hour at 37°C. The digested tissue was further dissociated by repeated aspiration through a syringe (needle gauge, 25). The cell suspension was centrifuged (200g, 5 minutes) and the cell pellet resuspended in Ham's F-12 culture medium containing 10% heat-inactivated FCS. Cells were seeded onto round glass coverslips (25 mm in diameter) that had been coated with Matrigel basement membrane matrix (Becton Dickinson Labware), which is a cell culture preparation optimized for contractile phenotypic states.24 Matrigel was diluted 1:3 and prepared according to the manufacturer's instructions. For the first 48 hours, cells were incubated in Ham's F-12 culture medium containing 10% heat-inactivated FCS. Thereafter, the culture medium was changed to DMEM containing L-glutamine, HEPES, penicillin, streptomycin, and 0.5% FCS as previously described.23 Before experimentation, cells were rendered quiescent by serum deprivation and maintenance in a serum-free medium for 36 hours.
For Western blotting of ERKs, cells (passages 1 to 3) from 3 subjects were used. For these studies, cells were grown in DMEM containing heat-inactivated FCS (10%), L-glutamine, HEPES, penicillin, and streptomycin as previously described.23 VSMCs were maintained at 37°C in a humidified incubator in an atmosphere of 95% air/5% CO2. Before experimentation, confluent cultures of VSMCs were rendered quiescent by serum deprivation and maintenance in a serum-free medium for 36 hours.
Measurement of [Ca2+]i
[Ca2+]i was
measured with fura 2-AM 4 µmol/L according to
previously described methods.23 Cells were investigated
with an inverted microscope (x40 oil-immersion objective) and
Attofluor Digital Fluorescence System (Zeiss) with alternating
excitatory wavelengths of 343 and 380 nm. Video images of
fluorescence at 520-nm emission were obtained with an
intensified CCD camera system with the output digitized to a resolution
of 512x480 pixels.
[Ca2+]i was calculated by
in situ calibration techniques.25
Measurement of Ang II–Induced Contraction
The gel-coated coverslips with attached fura 2–loaded cells
were placed on the stage of the microscope. After a 10-minute
stabilization period, a field of cells was photographed to obtain
baseline images. Ang II (in the absence or presence of
inhibitors) was then added, and serial images were taken of
the same field of cells at 30-second intervals after addition of Ang
II. The images, which were computer-saved, were later scanned with a
Scan Jet 4c/T scanner (Hewlett Packard). The lengths of the longest
axes of cells were measured in the first image, and lengths of the same
cells were measured in the subsequent photographs with Adobe Photoshop
software (version 4.0). The magnitude of cell contraction was expressed
as the percentage reduction in cell length relative to initial baseline
measurements.26 27 28 29 For each cell, the percent contraction
from the baseline was calculated, and these values were averaged for
all cells. To demonstrate that cells did in fact contract in response
to Ang II and that the changes in cell length were not simply due to
volume changes, we assessed the effects of sodium nitroprusside (SNP),
a nitric oxide donor, on Ang II–precontracted cells.
Measurement of pHi
pHi was measured with BCECF-AM 2
µmol/L according to previously described methods.30
Fluorescence was measured with alternating excitatory
wavelengths of 488 and 460 nm and an emission wavelength of 520 nm.
pHi was calculated from a calibration curve
obtained for each experiment by determining the fluorescence
ratios at pHi values of 7.4, 7.2, 7.0, and 6.8.
pHi was set by incubating the coverslip in
K+-rich buffer in the presence of 10
µmol/L nigericin (an exogenous
K+/H+ exchange
ionophore).30
Experimental Protocols
[Ca2+]i,
pHi, and contractile responses were measured in
cells exposed to Ang II 10–12 to
10-5 mol/L in the absence and presence of the
selective tyrosine kinase inhibitor tyrphostin A-23
10-5 mol/L, its inactive analogue tyrphostin A-1
10-5 mol/L, and the MEK inhibitor
PD98059 10-5 mol/L.31 32 For these
experiments, cells were preexposed to the specific
inhibitors for 20 minutes before addition of Ang II. The
effects of SNP were assessed in precontracted cells. In these
experiments, once cells had reached maximal contraction (
3 minutes
after addition of Ang II), cells were exposed to
10-5 mol/L SNP. To determine the receptor
subtype through which Ang II mediates responses, cells were preexposed
to 10-6 mol/L
[Sar1,Ile8]Ang II,
losartan, and PD 123319 for 10 minutes before Ang II
addition.
Western Blotting of ERKs
Quiescent cells, grown on 15 mL-culture plates, were stimulated
with Ang II 10-7 mol/L for 1.5, 3, 5, or 10
minutes. Cells were pretreated with 10-5 mol/L
PD98059 for 30 minutes before Ang II addition. Cells were lysed,
collected, and sonicated for 5 seconds as previously
described.6 Protein concentrations were determined with
the Bio-Rad Assay (Bio-Rad Laboratories). Equal amounts of proteins (15
µg for phosphotyrosine assays and 5 µg for ERK assessment) were
loaded onto a 10% SDS-polyacrylamide gel and transferred to
PVDF membrane (Boehringer Mannheim) for 1 hour at 100 V.
Membranes were blocked in 5% nonfat milk (for ERK) or 5% BSA (for
phosphotyrosine) and incubated with anti-ERK1 diluted 1:5000 or a mouse
antibody anti-phosphotyrosine (PY20) diluted 1:750 for 1 hour at room
temperature. They were then washed, incubated with a goat anti-rabbit
horseradish peroxidase–conjugated antibody (Bio-Rad) (for ERK) or a
goat anti-mouse horseradish peroxidase–conjugated antibody (for
phosphotyrosine) diluted 1:5000 for 1 hour at room temperature, and
washed extensively. Membranes were then incubated with blotting
substrate (POD) (Boehringer Mannheim) according to the
manufacturer's protocol, exposed to film, and developed. The film was
scanned by ScanJet 6100C/T (Hewlett Packard) and computer-saved. Band
intensity was measured by computer analysis with the Image
Quant program.
Statistical Analysis
Data obtained from digital imaging studies, in which multiple
cells (8 to 20 cells) were examined in each experimental field, were
calculated as the mean response per experiment and then as the mean of
multiple experiments. Results are presented as mean±SEM and
compared by Student's t test or by ANOVA where appropriate.
A Tukey-Kramer correction was used to compensate for multiple testing
procedures. Concentration-response curves were fitted by nonlinear
regression, the concentration in moles per liter that gave 50%
response (EC50) was determined, and
pD2 was calculated as -log
EC50. P<0.05 was considered
significant.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Basal and Ang II–Stimulated [Ca2+]iand pHi Responses
Basal [Ca2+]i was 83.5±2.31 nmol/L, and basal pHi was 7.03±0.06. Ang II elicited a biphasic [Ca2+]i wave (Figure 1
). The first phase, which peaked within
a few seconds after addition of Ang II, was acute and transient (Figure 1). The second
[Ca2+]i phase was
sustained at suprabasal levels (Figure 1).
[Ca2+]i approached basal
values 15 minutes after stimulation. The initial
[Ca2+]i peak (first
[Ca2+]i phase) was used
to construct dose-response curves. Ang II increased
[Ca2+]i in a
dose-dependent manner (pD2=8.4±0.36 and
Emax=541±55 nmol/L) (Figure 2). Ang II dose-dependently increased
pHi (pD2=9.4±0.29 and
Emax=7.19±0.01) (Figure 2).
pHi values at maximal alkalinization were used to
construct dose-response curves.
|
|
Effects of Tyrphostin A-23 and PD98059 on Ang II–Stimulated
[Ca2+]i and pHi
Responses
Tyrphostin A-23 alone did not alter
[Ca2+]i or
pHi. Tyrosine kinase inhibition significantly
reduced Ang II–stimulated peak
[Ca2+]i
(P<0.01) (Figures 1
and 3) and prolonged
[Ca2+]i recovery to basal
levels (Figure 1). Tyrphostin A-23 significantly attenuated
(P<0.05) Ang II–stimulated alkalinization (Figure 4). Tyrphostin A-1 had no effect on
agonist-stimulated responses (Figures 1, 3, and 4). These data suggest that Ang II–elicited actions are
partially mediated through tyrosine kinase–dependent pathways.
|
|
PD98059 did not alter basal
[Ca2+]i (85±7 nmol/L) or
basal pHi (7.01±0.03) but significantly
attenuated (P<0.01) Ang II–elicited
[Ca2+]i responses, both
the peak [Ca2+]i
transient and the second
[Ca2+]i phase (Figures 1, 5, and 6). Ang II–induced alkalinization was
significantly reduced by PD98059 (Figure 4).
|
|
To determine whether MEK effects on
[Ca2+]i are mediated via
Ca2+ influx, additional experiments were
performed in Ca2+-free buffer (Hanks buffer
without Ca2+ plus 3 mmol/L EGTA). In the
absence of extracellular Ca2+, basal and Ang
II–elicited [Ca2+]i
responses were reduced (Figures 5 and 6). The peak
[Ca2+]i transient was
slightly but significantly (P<0.05) attenuated, and the
second [Ca2+]i phase was
almost abolished (Figure 5). These results were similar to those
obtained with PD98059 in the presence of extracellular
Ca2+. When cells were pretreated with the MEK
inhibitor in Ca2+-deprived buffer,
the Ang II–induced peak
[Ca2+]i response was
further reduced, whereas the second
[Ca2+]i phase was
unchanged compared with that in Ca2+-free buffer
without PD98059 (Figures 5 and 6). These data suggest
that MEK inhibition decreases Ang II–elicited
Ca2+ influx, which contributes mainly to the
[Ca2+]i plateau phase and
to a lesser extent to the peak
[Ca2+]i transient. The
fact that PD98059 further reduced the peak
[Ca2+]i response in
Ca2+-free buffer suggests that MEK inhibition may
also elicit part of its effects via another mechanism, possibly by
inhibition of intracellular Ca2+
mobilization.
Effects of Ang II on VSMC Contraction
Ang II dose-dependently reduced cell length
(pD2=9.2±0.21,
Emax=36±2.2%) (Figure 7). Maximal contraction occurred within 5
minutes of Ang II stimulation (Figure 8).
Cell contraction was sustained, and 8 minutes after stimulation, cells
were still contracted (Figure 8). MEK inhibition significantly
attenuated Ang II–induced contractile responses and almost abolished
the sustained phase of contraction (Figure 8). Addition of SNP
to Ang II–precontracted cells resulted in a rapid reversion of VSMCs
to their prestimulated length (97±1.2% of initial cell length).
|
|
Characterization of Human VSMC Angiotensin Receptors
[Sar1,Ile8]Ang II (a
nonspecific angiotensin subtype receptor
antagonist) and losartan (a selective
AT1 receptor antagonist) completely
abolished Ang II–mediated responses (Figure 9). PD123319 (a selective
AT2 antagonist) had no effect on Ang
II–stimulated effects (Figure 9). Treatment of VSMCs with any
of the antagonists alone did not influence basal
[Ca2+]i,
pHi, or cell size.
|
Effects of PD98059 on Ang II Effects on ERK Expression and
Tyrosine Phosphorylation
To determine whether Ang II–induced changes in ERK expression and
tyrosine phosphorylation could occur within a time
frame that would be responsible for early signal transduction events
and to assess whether PD98059 could attenuate these effects, cells were
incubated for various periods of time with Ang II, in the absence and
presence of PD98059, before cell lysis. Multiple proteins were
tyrosine-phosphorylated under basal conditions. Ang II
significantly increased tyrosine phosphorylation of 2
proteins with molecular weights of 44 and 42 kDa, corresponding to
ERK-1 and ERK-2. Significant activation was evident within 1.5 minutes
of Ang II stimulation, and maximal activation occurred at 5 minutes
(Figure 10). PD98059 significantly
attenuated Ang II–induced activation of ERKs (Figure 10).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Data from the present study demonstrate that VSMCs from human peripheral resistance arteries possess functional angiotensin receptors of the AT1 subtype that mediate contraction via signaling cascades involving Ca2+ and pHi pathways that are partially dependent on tyrosine kinases and ERKs. Our results suggest that ERKs probably influence [Ca2+]i by modulating Ca2+ influx and intracellular Ca2+ mobilization.
Smooth muscle cells in culture are used routinely in an effort to
elucidate the causes and mechanisms of vascular disease. However, cells
in culture, especially serially passaged cells, undergo many phenotypic
and morphological changes. Accordingly, cultured cells may be very
different from the cells from which they were originally derived. In
the present work, we studied cells that had undergone little
phenotypic change relative to the native cells in blood vessels.
Primary cultured cells were grown on Matrigel basement membrane matrix
in low-serum-containing culture medium. Under these conditions, they
displayed a contractile phenotype, had a low proliferation
rate, expressed the cytoskeletal marker 
-actin as determined by
immunocytochemical analysis, and contracted in response to Ang
II. These features are characteristic of the contractile
phenotype as previously described.25
An important issue in this study is the VSMC contraction in response to Ang II. Cell volume changes could theoretically imitate contraction,27 and in the present study, actin-myosin interaction was not directly evaluated to unambiguously demonstrate that contraction had occurred. The fact that cells shortened in response to Ang II and elongated when exposed to SNP suggests that the cells did carry out excitation-contraction coupling and were probably under tension during the shortening phase. With respect to cell shape changes, we did not notice any ballooning of the cell bodies after exposure to Ang II, indicating that the physical change cells underwent was not simply a change in the shape of the cell. Thus, cell shortening induced by Ang II in our study probably does reflect smooth muscle cell contraction.
[Ca2+]i is a major trigger of actin-myosin interaction, cross-bridge cycling, and VSMC contraction.33 The response elicited by Ang II was typically biphasic, with an acute peak followed by a sustained, suprabasal second phase. The second [Ca2+]i phase was temporally related to maximum contraction, indicating an association between [Ca2+]i and contraction. pHi is also an important second messenger. Intracellular alkalinization stimulates DNA synthesis and cell growth and increases actin-myosin sensitivity to Ca2+, thereby increasing vascular contraction and tone.34 Basal pHi in the present study was similar to that reported in the only other known study of pHi in cultured human VSMCs35 and to the pHi in intact human subcutaneous small arteries.36 Our results, which are the first to show that Ang II induces alkalinization in primary cultured human VSMCs, are in agreement with findings from rat studies.28 37
In addition to stimulation of classic phospholipase C–mediated Ca2+ signaling pathways, Ang II also stimulates protein phosphorylation on tyrosine residues.38 39 Tyrosine kinase pathways are typically involved in cell growth, and it has recently been shown that these pathways also modulate contraction.3 28 To determine whether tyrosine kinases regulate VSMC [Ca2+]i and pHi, we assessed Ang II effects in the presence of the selective tyrosine kinase inhibitor tyrphostin A-23 and its inactive analogue tyrphostin A-1. Tyrphostin A-23, but not tyrphostin A-1, attenuated Ang II–elicited actions, suggesting a contribution of tyrosine kinases in the regulation of agonist-stimulated responses in human VSMCs. The tyrosine kinase effects on Ang II–elicited pHi responses may be mediated via Na+-H+–linked pathways. We have observed in rat VSMCs that activity of the Na+-H+ exchanger is reduced by tyrosine kinase inhibitors. The importance of tyrosine phosphorylation in Ang II signaling has been extensively reviewed recently.5 6 The quoted studies, however, all referred to animal experiments. Our data now demonstrate that tyrosine kinases are also implicated in Ang II–mediated Ca2+ and pHi signaling in human VSMCs.
The specific tyrosine kinase effectors involved in Ang II–stimulated responses are unclear, but ERKs may play a role, because they have been implicated in both contraction and growth in rat VSMCs.3 5 6 40 The immediate ERK activator is MEK, which phosphorylates ERK-1 and ERK-2.41 42 MEK, in turn, is activated by various kinases, which are stimulated by Ang II in cultured VSMCs.43 44 Thus, MEK may be a potential tyrosine kinase candidate activated by Ang II. To assess the role of ERKs in Ang II signaling, the MEK inhibitor PD98059 was used. We demonstrated that PD98059 inhibited Ang II–stimulated ERK activity in VSMCs, indicating that the inhibitor did in fact block agonist-induced ERK activation and that it was effective at a concentration of 10-5 mol/L. In cells pretreated with PD98059, Ang II–elicited alkalinization was reduced, [Ca2+]i peak responses were attenuated, the second [Ca2+]i phase was inhibited, and contraction was significantly decreased. These data suggest that ERKs partially regulate Ang II–stimulated second messengers and associated contraction in human VSMCs.
The putative mechanisms underlying ERK regulation of Ang II–mediated responses are unclear, but ERKs, which are activated by Ca2+,44 may retroactively modulate [Ca2+]i and pHi. We demonstrate here that Ang II–induced tyrosine phosphorylation of ERKs occurs rapidly, certainly within the time frame that would be associated with early signal transduction events. Similar findings have been demonstrated in human saphenous vein cells.10 ERKs might influence [Ca2+]i by modulating inositol 1,4,5-triphosphate, which is the primary mediator of Ca2+ mobilization, and/or by activating Ca2+ channels, which are the major pathway for Ca2+ influx. We demonstrate here that when cells are pretreated with PD98059 in Ca2+-deprived buffer, the Ang II–induced peak [Ca2+]i response was further reduced, whereas the second [Ca2+]i phase was unchanged compared with that in Ca2+-free buffer without the inhibitor. These data suggest that MEK inhibition decreases Ang II–elicited Ca2+ influx, which contributes mainly to the [Ca2+]i plateau phase and to a lesser extent to the peak [Ca2+]i transient. The fact that PD98059 further reduced the peak [Ca2+]i response in Ca2+-free buffer suggests that MEK inhibition may also elicit its effects via another mechanism, possibly by inhibition of Ca2+ mobilization. These data are supported by recent studies that demonstrated that Ang II–induced inositol phosphate generation is mediated through tyrosine kinase pathways in cardiomyocytes45 and that tyrosine kinases activate voltage-dependent Ca2+ channels in vascular smooth muscle.46 It may also be possible that ERKs directly influence regulatory contractile proteins.47 However, the exact cellular pathways by which ERKs influence Ang II–mediated signaling and contraction in VSMCs await further clarification.
Ang receptor subtypes mediating Ang II responses were characterized by use of selective receptor antagonists. [Sar1,Ile8]Ang II and losartan completely blocked the cellular effects of Ang II, whereas PD123319 had no effect. These results indicate that Ang II–elicited actions are exclusively of the AT1 subtype in human VSMCs. Similar results have been reported in cultured cells from human coronary arteries and saphenous vein as well as in cells from rat small arteries.4 10 13 28
In conclusion, this study provides the novel findings that in VSMCs, ERKs modulate Ang II–induced contraction and signaling events that lie upstream from MEK. These effects are mediated via AT1 receptors. Our data have significant physiological implications, because the cells that we studied retained their contractile phenotype so that they resembled, as closely as possible, the cells of origin. Also, cells were derived from peripheral resistance arteries, vessels that contribute to peripheral resistance and blood pressure regulation. The present study thus provides new insights regarding Ang II signaling in VSMCs, which participate in mechanisms involved in the control of blood pressure through changes in peripheral resistance in humans.
|
Acknowledgments |
|---|
This study was supported by grant 14080 and a group grant to the Multidisciplinary Research Group on Hypertension, both from the Medical Research Council of Canada, and by a grant from the Heart and Stroke Foundation of Quebec.
Received July 13, 1998; revision received September 30, 1998; accepted October 9, 1998.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Jackson CL, Schwartz SM. Pharmacology of smooth muscle cell replication. Hypertension. 1992;20:713–736.
2.
Geisterfer AAT, Peach MJ, Owens GK.
Angiotensin II induces hypertrophy, not
hyperplasia, of cultured rat aortic smooth muscle cells. Circ
Res. 1988;62:749–756.
3.
Sauro MD, Sudakow R, Burns S. In vivo effects of
angiotensin II on vascular smooth muscle contraction and
blood pressure are mediated through a protein tyrosine kinase-dependent
mechanism. J Pharmacol Exp Ther. 1996;277:1744–1750.
4. Timmermans PMWM, Benfield P, Chiu AT, Herblin WF, Wong PC, Smith RD. Angiotensin II receptors and functional correlates. Am J Hypertens. 1992;5(pt 2):221S–235S.
5. Griendling KK, Ushio-Fukai M, Lassègue B, Alexander RW. Angiotensin II signaling in vascular smooth muscle: new concepts. Hypertension. 1997;20(pt 2):366–373.
6.
Lucchesi PA, Bell JM, Willis LS, Byron KL, Corson MA,
Berk BC. Ca2+-dependent mitogen-activated
protein kinase activation in spontaneously hypertensive rat vascular
smooth muscle defines a hypertensive signal transduction
phenotype. Circ Res. 1996;78:962–970.
7. Force T, Bonventre JV. Growth factors and mitogen-activated protein kinases. Hypertension. 1998;31(part 2):152–161.
8. Sugden PH, Clerk A. Regulation of the ERK subgroup of MAP kinase cascades through G-protein-coupled receptors. Cell Signal. 1997;9:337–351.[Medline] [Order article via Infotrieve]
9.
Touyz RM, Schiffrin EL. Tyrosine kinase signaling
pathways modulate angiotensin II–induced calcium
transients in vascular smooth muscle cells. Hypertension. 1996;27:1097–1103.
10. Kruse HJ, Bauriedel G, Heimerl J, Hofling B, Weber PC. Role of L-type calcium channels on stimulated calcium influx and on proliferative activity of human coronary smooth muscle cells. J Cardiovasc Pharmacol. 1994;24:328–335.[Medline] [Order article via Infotrieve]
11. Patel MK, Betteridge LJ, Hughes AD, Clunn DG, Schachter M, Shaw RJ, Sever PDS. Effect of angiotensin II on the expression of the early growth response gene c-fos and DNA synthesis in human vascular smooth muscle cells. J Hypertens. 1996;14:341–347.[Medline] [Order article via Infotrieve]
12. Takagishi T, Murahashi N, Azagami S, Morimatsu M, Sasaguri Y. Effect of angiotensin II and thromboxane A2 on the production of matrix metalloproteinase by human aortic smooth muscle cells. Biochem Mol Biol Int. 1995;35:265–273.[Medline] [Order article via Infotrieve]
13. Yazawa H, Iida-Kubota E, Honda K. Human aortic smooth muscle cells containing angiotensin II type I receptors. Jpn J Pharmacol. 1993;62:339–343.[Medline] [Order article via Infotrieve]
14. Cox BE, Word RA, Rosenfeld CR. Angiotensin II receptor characteristics and subtype expression in uterine arteries and myometrium during pregnancy. J Clin Endocrinol Metab. 1996;81:49–58.[Abstract]
15. Hatakeyama H, Miyamori I, Yamagishi S, Takeda Y, Takeda R, Yamamoto H. Angiotensin II upregulates the expression of type A endothelin receptor in human vascular smooth muscle cells. Biochem Mol Biol Int. 1994;34:127–134.[Medline] [Order article via Infotrieve]
16.
Williams B, Baker AQ, Gallacher B, Lodwick D.
Angiotensin II increases vascular permeability factor gene
expression by human vascular smooth muscle cells.
Hypertension. 1995;25:913–917.
17. Svane D, Kahr O, Hansen VB, Holm-Nielsen P, Formar A. Angiotensin-converting enzyme activity and contractile effects of angiotensin I and II in human uteroplacental arteries. Am J Obstet Gynecol. 1995;172:991–997.[Medline] [Order article via Infotrieve]
18. Schiffrin EL, Deng LY, Larochelle P. Morphology of resistance arteries and comparison of effects of vasoconstrictors in mild essential hypertensive patients. Clin Invest Med. 1993;16:177–186.[Medline] [Order article via Infotrieve]
19. Davis MJ, Ferrer PN, Gore RW. Vascular anatomy and hydrostatic pressure profile in the hamster cheek pouch. Am J Physiol. 1986;250(pt 2):H291–H303.
20. Christensen KL, Mulvany MJ. Mesenteric arcade arteries contribute substantially to vascular resistance in conscious rats. J Vasc Res. 1993;30:73–79.[Medline] [Order article via Infotrieve]
21. Joyner WL, Davis MJ. Pressure profile along the microvascular network and its control. Fed Proc. 1987;46:266–269.[Medline] [Order article via Infotrieve]
22. Schiffrin EL, Poissant L, Cantin M, Thibault G. Receptors for atrial natriuretic factor in cultured vascular smooth muscle cells. Life Sci. 1986;38:817–826.[Medline] [Order article via Infotrieve]
23. Touyz RM, Tolloczco B, Schiffrin EL. Mesenteric vascular smooth muscle cells from spontaneously hypertensive rats display increased calcium responses to angiotensin II, but not to endothelin-1. Hypertension. 1994;12:663–673.
24.
Li X, Tsai P, Wieder ED, Kribben A, Putten VV, Schrier
RW, Nemenoff RA. Vascular smooth muscle cells grown on matrigel: a
model of the contractile phenotype with decreased activation of
mitogen-activated protein kinase. J Biol Chem. 1994;269:19653–19658.
25.
Grynkiewicz G, Poenie M, Tsien RY. A new generation of
Ca2+ indicators with greatly improved
fluorescent properties. J Biol Chem. 1985;260:3440–3450.
26.
Kahn AM, Bishara M, Cragoe EJ Jr, Allen JC,
Seidel CL, Navran SS, O'Neil RG, McCarty NA, Shelat H. Effects of
serotonin on intracellular pH and contraction in vascular
smooth muscle. Circ Res. 1992;71:1294–1304.
27.
Touyz RM, Schiffrin EL. Angiotensin II
regulates vascular smooth muscle cell pH, contraction, and growth via
tyrosine-kinase dependent signaling pathways. Hypertension. 1997;30:222–229.
28.
Bodin P, Travo C, Stoclet J-C, Travo P. High
sensitivity of hypertensive aortic myocytes to
norepinephrine and angiotensin. Am J
Physiol. 1993;264:C441–C445.
29.
Bolzon JB, Cheung DW. Isolation and characterization of
single vascular smooth muscle cells from spontaneously hypertensive
rats. Hypertension. 1989;14:137–144.
30. Thomas JH, Buchbaum RN, Zimniak A, Racker E. Intracellular pH measurements in Ehrlich ascites tumor cell utilizing spectroscopic probes generated in situ. Biochemistry. 1979;18:2210–2218. inhibitor of the mitogen-activated protein kinase cascade. Proc Natl Acad Sci U S A. 1995;92:7686–7689.[Medline] [Order article via Infotrieve]
31.
Pang L, Sawada T, Decker SJ, Saltiel AR, Inhibition of
MAP kinase kinase blocks the differentiation of PC-12 cells induced by
nerve growth factor. J Biol Chem.. 1995;270:13585–13588.
32.
Dudley DT, Pang L, Decker SJ, Bridges AJ, Saltiel AR. A
synthetic inhibitor of the mitogen-activated
protein kinase cascade. Proc Natl Acad Sci U S A. 1995;92:7686–7689.
33. Berridge MJ. Spatial and temporal signaling by calcium. Curr Opin Cell Biol. 1994;6:267–274.[Medline] [Order article via Infotrieve]
34.
Wray S. Smooth muscle intracellular pH: measurement,
regulation and function. Am J Physiol. 1988;254:C751–C758.
35.
Neylon CB, Little PJ, Cragoe EJ, Bobik A. Intracellular
pH in human arterial smooth muscle: regulation by
Na+/H+ exchange and a novel
5-(N-ethyl-N-isopropyl) amiloride-sensitive
Na+ and
HCO3--dependent mechanism.
Circ Res. 1990;67:814–825.
36.
Carr P, McKinnon W, Poston L. Mechanisms of
pHi control and relationships between tension and
pHi in human subcutaneous small arteries.
Am J Physiol. 1995;268:C580–C589.
37.
Izzard AS, Cragoe EJ, Heagerty AM. Intracellular pH in
human resistance arteries in essential hypertension.
Hypertension. 1991;17:780–786.
38. Duff JL, Berk BC, Corson MA. Angiotensin II stimulates the PP44 and PP42 mitogen-activated protein kinases in cultured rat aortic smooth muscle cells. Biochem Biophys Res Commun. 1992;188:257–264.[Medline] [Order article via Infotrieve]
39.
Takahashi T, Kawahara Y, Okuda W, Ueno H, Takeshita A,
Yokoyama M. Angiotensin II stimulates
mitogen-activated protein kinases and protein synthesis by a
Ras-independent pathway in vascular smooth muscle cells. J
Biol Chem. 1997;272:16018–16022.
40.
Jin N, Siddiqui RA, English D, Rhoades RA.
Communication between tyrosine kinase pathway and myosin light chain
kinase pathway in smooth muscle. Am J Physiol. 1996;271:H1348–H1355.
41.
Pelech SL, Sanghora JS. MAP kinases: charting the
regulatory pathways. Science. 1992;257:1355–1356.
42. Robinson MJ, Cobb MH. Mitogen-activated protein kinase pathways. Curr Opin Cell Biol. 1997;9:180–186.[Medline] [Order article via Infotrieve]
43.
Liao D-F, Duff JL, Daum G, Pelech SL, Berk BC.
Angiotensin II stimulates MAP kinase activity in vascular
smooth muscle cells: role of Raf. Circ Res. 1996;79:1007–1014.
44.
Berk BC, Corson MA. Angiotensin II signal
transduction in vascular smooth muscle: role of tyrosine kinases.
Circ Res. 1997;80:607–616.
45. Goutsouliak V, Rabkin SW. Angiotensin II-induced inositol phosphate generation is mediated through tyrosine kinase pathways in cardiomyocytes. Cell Signal. 1997;9:505–512.[Medline] [Order article via Infotrieve]
46. Wïjetunge S, Aalkjaer C, Schachter M, Hughes AD. Tyrosine kinase inhibitors block calcium channel currents in vascular smooth muscle cells. Biochem Biophys Res Commun. 1992;189:1620–1623.[Medline] [Order article via Infotrieve]
47.
Adam LP, Franklin MT, Raff GJ, Hathaway DR. Activation
of mitogen-activated protein kinase in porcine carotid
arteries. Circ Res. 1995;76:183–190.
This article has been cited by other articles:
 |
|
 |
|
  C. A. Lemarie, S. M.C. Simeone, A. Nikonova, T. Ebrahimian, M.-E. Deschenes, T. M. Coffman, P. Paradis, and E. L. Schiffrin Aldosterone-Induced Activation of Signaling Pathways Requires Activity of Angiotensin Type 1a Receptors Circ. Res., October 23, 2009; 105(9): 852 - 859. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. K. Stennett, X. Qiao, A. E. Falone, V. V. Koledova, and R. A. Khalil Increased vascular angiotensin type 2 receptor expression and NOS-mediated mechanisms of vascular relaxation in pregnant rats Am J Physiol Heart Circ Physiol, March 1, 2009; 296(3): H745 - H755. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Hus-Citharel, X. Iturrioz, P. Corvol, J. Marchetti, and C. Llorens-Cortes Tyrosine Kinase and Mitogen-Activated Protein Kinase/Extracellularly Regulated Kinase Differentially Regulate Intracellular Calcium Concentration Responses to Angiotensin II/III and Bradykinin in Rat Cortical Thick Ascending Limb Endocrinology, January 1, 2006; 147(1): 451 - 463. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. M Touyz Intracellular mechanisms involved in vascular remodelling of resistance arteries in hypertension: role of angiotensin II Exp Physiol, July 1, 2005; 90(4): 449 - 455. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. L. Schiffrin and R. M. Touyz From bedside to bench to bedside: role of renin-angiotensin-aldosterone system in remodeling of resistance arteries in hypertension Am J Physiol Heart Circ Physiol, August 1, 2004; 287(2): H435 - H446. [Full Text] [PDF]
|
|
|
|
 |
|
 T. A. Khan, C. Bianchi, M. Ruel, P. Voisine, J. Li, J. R. Liddicoat, and F. W. Sellke Mitogen-Activated Protein Kinase Inhibition and Cardioplegia-Cardiopulmonary Bypass Reduce Coronary Myogenic Tone Circulation, September 9, 2003; 108(90101): II-348 - 353. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Zhao, W. Long, L. Zhang, and L. D Longo Extracellular signal-regulated kinases and contractile responses in ovine adult and fetal cerebral arteries J. Physiol., September 1, 2003; 551(2): 691 - 703. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Tao, S. Chaudry, B. Tolloczko, J. G. Martin, and S. M. Kelly Modulation of smooth muscle phenotype in vitro by homologous cell substrate Am J Physiol Cell Physiol, June 1, 2003; 284(6): C1531 - C1541. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. P. Massett, Z. Ungvari, A. Csiszar, G. Kaley, and A. Koller Different roles of PKC and MAP kinases in arteriolar constrictions to pressure and agonists Am J Physiol Heart Circ Physiol, December 1, 2002; 283(6): H2282 - H2287. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. M. Touyz, X. Chen, F. Tabet, G. Yao, G. He, M. T. Quinn, P. J. Pagano, and E. L. Schiffrin Expression of a Functionally Active gp91phox-Containing Neutrophil-Type NAD(P)H Oxidase in Smooth Muscle Cells From Human Resistance Arteries: Regulation by Angiotensin II Circ. Res., June 14, 2002; 90(11): 1205 - 1213. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. M. Touyz, X.-H. Wu, G. He, S. Salomon, and E. L. Schiffrin Increased Angiotensin II-Mediated Src Signaling via Epidermal Growth Factor Receptor Transactivation Is Associated With Decreased C-Terminal Src Kinase Activity in Vascular Smooth Muscle Cells From Spontaneously Hypertensive Rats Hypertension, February 1, 2002; 39(2): 479 - 485. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. E. Cain, D. M. Tanner, and R. A. Khalil Endothelin-1-Induced Enhancement of Coronary Smooth Muscle Contraction via MAPK-Dependent and MAPK-Independent [Ca2+]i Sensitization Pathways Hypertension, February 1, 2002; 39(2): 543 - 549. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. M.A.C. Martens, B. Demeilliers, D. Girardot, C. Daigle, H. H. Dao, D. deBlois, and P. Moreau Vessel-Specific Stimulation of Protein Synthesis by Nitric Oxide Synthase Inhibition: Role of Extracellular Signal-Regulated Kinases 1/2 Hypertension, January 1, 2002; 39(1): 16 - 21. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Berry, R. Touyz, A. F. Dominiczak, R. C. Webb, and D. G. Johns Angiotensin receptors: signaling, vascular pathophysiology, and interactions with ceramide Am J Physiol Heart Circ Physiol, December 1, 2001; 281(6): H2337 - H2365. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Matrougui, L. B. Tanko, L. Loufrani, D. Gorny, B. I. Levy, A. Tedgui, and D. Henrion Involvement of Rho-Kinase and the Actin Filament Network in Angiotensin II-Induced Contraction and Extracellular Signal-Regulated Kinase Activity in Intact Rat Mesenteric Resistance Arteries Arterioscler Thromb Vasc Biol, August 1, 2001; 21(8): 1288 - 1293. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. El Mabrouk, R. M. Touyz, and E. L. Schiffrin Differential ANG II-induced growth activation pathways in mesenteric artery smooth muscle cells from SHR Am J Physiol Heart Circ Physiol, July 1, 2001; 281(1): H30 - H39. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. H. Ratz Regulation of ERK phosphorylation in differentiated arterial muscle of rabbits Am J Physiol Heart Circ Physiol, July 1, 2001; 281(1): H114 - H123. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. M. Touyz, G. He, X.-H. Wu, J. B. Park, M. E. Mabrouk, and E. L. Schiffrin Src Is an Important Mediator of Extracellular Signal-Regulated Kinase 1/2-Dependent Growth Signaling by Angiotensin II in Smooth Muscle Cells From Resistance Arteries of Hypertensive Patients Hypertension, July 1, 2001; 38(1): 56 - 64. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. M. Touyz and E. L. Schiffrin Signal Transduction Mechanisms Mediating the Physiological and Pathophysiological Actions of Angiotensin II in Vascular Smooth Muscle Cells Pharmacol. Rev., December 1, 2000; 52(4): 639 - 672. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. M. Muthalif, N. A. Karzoun, L. Gaber, Z. Khandekar, I. F. Benter, A. E. Saeed, J.-H. Parmentier, A. Estes, and K. U. Malik Angiotensin II-Induced Hypertension : Contribution of Ras GTPase/Mitogen-Activated Protein Kinase and Cytochrome P450 Metabolites Hypertension, October 1, 2000; 36(4): 604 - 609. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Matrougui, Y. E. G. Eskildsen-Helmond, A. Fiebeler, D. Henrion, B. I. Levy, A. Tedgui, and M. J. Mulvany Angiotensin II Stimulates Extracellular Signal-Regulated Kinase Activity in Intact Pressurized Rat Mesenteric Resistance Arteries Hypertension, October 1, 2000; 36(4): 617 - 621. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. C. Martinez, V. Randriamboavonjy, P. Ohlmann, N. Komas, J. Duarte, F. Schneider, J.-C. Stoclet, and R. Andriantsitohaina Involvement of protein kinase C, tyrosine kinases, and Rho kinase in Ca2+ handling of human small arteries Am J Physiol Heart Circ Physiol, September 1, 2000; 279(3): H1228 - H1238. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. D. Intengan and E. L. Schiffrin Structure and Mechanical Properties of Resistance Arteries in Hypertension : Role of Adhesion Molecules and Extracellular Matrix Determinants Hypertension, September 1, 2000; 36(3): 312 - 318. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. D Hughes AT 1-signalling in vascular smooth muscle Journal of Renin-Angiotensin-Aldosterone System, June 1, 2000; 1(2): 125 - 130. [PDF]
|
|
|
|
 |
|
 K. Aoki, A. Y. Zubkov, A. D. Parent, J. H. Zhang, and R. L. Macdonald Mechanism of ATP-Induced [Ca2+]i Mobilization in Rat Basilar Smooth Muscle Cells Editorial Comment Stroke, June 1, 2000; 31(6): 1377 - 1385. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. L. Schiffrin, J. B. Park, H. D. Intengan, and R. M. Touyz Correction of Arterial Structure and Endothelial Dysfunction in Human Essential Hypertension by the Angiotensin Receptor Antagonist Losartan Circulation, April 11, 2000; 101(14): 1653 - 1659. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Samain, H. Bouillier, S. Miserey, C. Perret, J.-F. Renaud, M. Safar, and G. Dagher Extracellular Signal-Regulated Kinase Pathway Is Involved in Basic Fibroblast Growth Factor Effect on Angiotensin II-Induced Ca2+ Transient in Vascular Smooth Muscle Cell From Wistar-Kyoto and Spontaneously Hypertensive Rats Hypertension, January 1, 2000; 35(1): 61 - 67. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||