Seymour Heisler Laboratory of the Montreal Chest Institute Research Centre and Meakins-Christie Laboratories, Department of Medicine, McGill University, Montreal, Quebec, Canada H2X 2P2
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ABSTRACT |
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Contractile agonists may stimulate mitogenic responses in
airway smooth muscle by mechanisms that involve tyrosine kinases. The
role of contractile agonist-evoked activation of tyrosine kinases in
contractile signaling is not clear. We addressed this issue using
cultured rat airway smooth muscle cells. In these cells, serotonin
(5-HT, 1 µM) caused contraction (quantitated by a decrease in cell
area), which was blocked by the tyrosine kinase inhibitor genistein (40 µM). Genistein and tyrphostin 23 (40 and 10 µM, respectively)
significantly decreased 5-HT-evoked peak Ca2+ responses,
and the effect of genistein could be observed in the absence of
extracellular Ca2+. The specific inhibitor of
mitogen-activated protein kinase kinase PD-98059 (30 µM) had no
significant effect on peak Ca2+ levels. Western analysis of
cell extracts revealed that 5-HT caused a significant increase in
tyrosine phosphorylation of proteins with molecular masses of ~70 kDa
within 10 s of stimulation but no measurable tyrosine phosphorylation
of the isoform of phospholipase C (PLC-
). Tyrosine
phosphorylation was inhibited by genistein. Furthermore, genistein (40 µM) significantly attenuated 5-HT-induced inositol phosphate
production. We conclude that in airway smooth muscle contractile
agonists acting on G protein-coupled receptors may activate tyrosine
kinase(s), which in turn modulate calcium signaling by affecting,
directly or indirectly, PLC-
activity. It is unlikely that PLC-
or the mitogen-activated protein kinase pathway is involved in
Ca2+ signaling to 5-HT.
cell culture; mitogen-activated protein kinase; phospholipase C
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INTRODUCTION |
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ALTHOUGH CONTRACTILE AGONISTS and growth factors act through distinct categories of receptors, G protein coupled and tyrosine kinases, the effects of these two classes of stimuli may be similar. It is well established that contractile agonists may have mitogenic properties, whereas growth factors can have contractile effects. A growing number of studies conducted on several tissues, including vascular smooth muscle, have confirmed that indeed there is considerable "cross talk" between signaling pathways mediating contraction and those mediating growth (7, 11, 15, 17).
Smooth muscle contraction is critically dependent on the increase in
intracellular Ca2+ concentration
([Ca2+]i). For most contractile
agonists, this increase is caused by release of Ca2+ from
intracellular stores, resulting from the binding of inositol 1,4,5-trisphosphate (IP3) to the receptors on sarcoplasmic
reticulum followed by Ca2+ influx from the extracellular
milieu. IP3, in turn, is produced by a rapid hydrolysis of
phosphatidylinositol 4,5-bisphosphate regulated by the interaction of
heterotrimeric G proteins with phospholipase C- (PLC-
).
The response of cells to classical growth factors and other mitogens is
much more complex, with several possible pathways being activated
depending on the stimulus. In most of these pathways, the signal is
transmitted via sequential activation of cytoplasmic protein kinases,
leading to the activation of nuclear transcription factors.
Mitogen-activated protein kinase kinase (MEK) and its substrates,
mitogen-activated protein kinases (MAPKs) p44 [extracellular signal-regulated kinase (ERK) I] and p42 (ERKII), are recognized as
central regulators of mitogenic signaling pathways in smooth muscle
(14, 17, 24). These kinases also have been identified as
potential sites of cross talk between contractile and mitogenic pathways because their activation has been correlated with mitogenic effects of contractile agonists acting on G protein-coupled
receptors (6, 15, 17). The contractile effects of growth
factors, on the other hand, are mediated by the isoform of PLC
(PLC-
). PLC-
causes an increase in
[Ca2+]i through the same sequence
of events as PLC-
on its association with tyrosine-phosphorylated
domains of growth factor receptors (21).
Recent studies suggest that tyrosine kinases may also have a role in
contractile responses evoked by agonists acting through G
protein-coupled receptors (1, 2, 29, 35). Few reports, however, have
addressed the issue of mechanisms involved in this process. Studies of
vascular smooth muscle indicate a possible role for MAPK (29, 34) and
PLC- (18). The aim of our study was to investigate whether tyrosine
kinase inhibition affects contraction of airway smooth muscle cells,
and if so, whether modulation of Ca2+ signaling has a role
in this effect. We also wished to examine if contractile agonist-evoked
changes in Ca2+ signaling involved mechanisms similar to
those required for mitogenic effects of contractile agonists, as well
as establish which step of the contractile signal transduction pathway
was affected.
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MATERIALS AND METHODS |
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Cell culture. Tracheal smooth muscle cells from 7- to 9-wk-old
male Fischer rats (Harlan Sprague Dawley; Frederick, MD) were cultured
according to previously described methods (28). Briefly, the cells were
enzymatically dissociated with 0.05% elastase type IV and 0.2%
collagenase type IV and cultured in 1:1 Dulbecco's modified Eagle's
medium (DMEM)-Ham's F-12 medium supplemented with 10% fetal bovine
serum (FBS), 0.224% NaHCO3, and 1%
penicillin-streptomycin in the presence of 5% CO2. On
reaching confluence (~7 days after the culture was established) the
cells were trypsinized and subcultured at a density of 5,000 cells/ml.
Cells from a first or second passage were rendered quiescent by
incubation in medium with 1% FBS 36-48 h before experiments.
Based on our previous studies of rat airway smooth muscle
proliferation, the estimated number of divisions was ~14-21 for
first- and second-passage cells, respectively. Confirmation of a smooth
muscle phenotype was based on typical morphology, positive -actin
staining, negative keratin staining, and contractile responses to agonists.
Measurement of cell contraction. Cells used for contraction measurements were plated on microscopy coverslips coated with rat tail collagen (12) and rendered quiescent by serum deprivation at ~50% confluence. The cells were labeled with a lipophilic fluorescent dye DiOC6(3) and incubated for 10 min with 40 µM genistein or with the appropriate vehicle. Cells were stimulated with 1 µM serotonin (5-HT) and 5 min later with 10 µM ionomycin, and images were acquired at the rate of 1 image/min using an intensified charge-coupled device camera (VideoScope) mounted on a Nikon microscope and commercial software [Photon Technology International (PTI); Princeton, NJ]. The excitation light wavelength was 490 nm and emission wavelength was 530 nm. Digitized images of unstimulated cells and maximally contracted cells were printed, and the surface area of individual cells was measured with a digitizing tablet. Three experiments were performed, with data from 29 to 36 cells recorded in each experiment.
Ca2+ measurements.
Cells grown on microscopy coverslips were loaded with the
Ca2+-sensitive dye fura 2-acetoxymethyl ester (AM)
according to previously described methods (28). The cells were exposed
to inhibitors (20-40 µM genistein, 10 µM tyrphostin, and 30 µM PD-98059) or an equivalent volume of an appropriate vehicle or
control substance for 10 min (for genistein and tyrphostin 23) or 20 min (for PD-98059) before the addition of 1 µM 5-HT. Fluorescence of
a small sample of cells (3-5) was recorded before and after
stimulation at a single emission wavelength (510 nm) with a double
excitatory wavelength (345 and 380 nm) using a PTI D401 microphotometer
and PTI software. Background fluorescence measured in the presence of
the inhibitors in cells not loaded with fura 2 was subtracted. The
[Ca2+]i was calculated according to
the formula of Grynkiewicz et al. (8). The dissociation constant was
assumed to be 224 nM, mean Rmax (345- to 380-nm
fluorescence ratio of calcium-saturated cells measured in cells exposed
to 105 M ionomycin-Hanks' buffer) was
7.6, Rmin (345- to 380-nm fluorescence ratio of
Ca2+-free cells measured in cells exposed to
10
5 M ionomycin and
10
6 M EGTA in Ca2+-free
buffer) was 0.55, and B (the ratio of fluorescence at 380-nm excitation
wavelength in Ca2+-free and Ca2+-saturated
cells) was 8.89.
SDS-PAGE and Western blotting. Confluent cells grown on 150 × 25-mm tissue culture dishes (Becton Dickinson) were growth
arrested with 1% FBS for 36-48 h and washed with Hanks' buffer
consisting of (in mmol/l) 137 NaCl, 4.2 NaHCO3, 10 glucose,
3 Na2HPO4, 5.4 KCl, 0.4 KH2PO4, 1.3 CaCl2, 0.5 MgCl2, and 0.8 MgSO4. For the time-course
experiments, the cells were stimulated with 5-HT for 10, 20, and 40 s.
To study the effects of tyrosine kinase inhibition the cells were
incubated for 10 min with 40 µM genistein or appropriate vehicle
before stimulation with 1 µM 5-HT in the presence of the phosphatase
inhibitor sodium orthovanadate (1 mM) for 10 min. To stimulate PLC-
phosphorylation, cells were incubated with the BB isoform of
platelet-derived growth factor (PDGF) at a concentration of 10 ng/ml
for 30 min in the presence of sodium orthovanadate. Cells were rinsed
with ice-cold PBS with sodium orthovanadate, 1 ml of lysis buffer
[1% Nonidet P-40 detergent, 20 mM Tris (pH 8.0), 137 mM NaCl,
10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 0.15 U aprotinin/ml,
and 1 mM sodium orthovanadate] was added to each plate, and cells
were scraped and left on ice for ~30 min. Lysates were centrifuged at
4°C for 10 min at 14,000 rpm (Eppendorf centrifuge 5402), and
supernatants were stored at
80°C.
Protein was quantified by the method of Bradford, and each lane was
loaded with equal amounts of protein (10 or 20 µg). Proteins were
resolved by 7.5% SDS-PAGE (Bio-Rad PROTEAN Mini II apparatus, Bio-Rad;
Mississauga, ON). The separated proteins were electroblotted onto
0.22-µm-pore nitrocellulose filters for 18 h at a constant voltage of
30 V, and the efficacy of the transfer was confirmed by Ponceau
staining as well as posttransfer staining of the gel with Coomassie
blue. The filters were blocked for 1 h at room temperature with 3%
milk powder in Tris-buffered saline with 0.05% Tween 20, incubated for
1 h with a biotinylated monoclonal primary antibody (for phosphorylated
tyrosine), followed by streptavidin-horseradish peroxidase (HRP) or a
primary antibody followed by a polyclonal HRP-conjugated secondary
antibody (for PLC-). Blots were then developed by enhanced
chemiluminescence (ECL, Amersham Canada; Oakville, ON) and presented as
multiples of increase over control. All quantified bands
were of a density that fell within linear range as established by
varying protein loading and exposure times. Molecular weights were
estimated by comparison with biotinylated molecular weight standards
(Bio-Rad). For reprobing with a different antibody, blots were
stripped with 0.1 M glycine, pH 2.9, and the immunoblotting protocol
was repeated.
Immunoprecipitation. One milliliter of cell lysate from each
group (control, 5-HT stimulated, and PDGF stimulated) containing 1 mg
of protein was precleared with agarose-conjugated mouse IgG, and
supernatants were precipitated for 2 h with 20 µl of
agarose-conjugated anti-phosphotyrosine antibody. Immunoprecipitates
were washed with the lysis buffer, resuspended in Laemmli buffer,
subjected to PAGE, and, after transfer to nitrocellulose filters,
probed with anti- PLC- antibody and HRP-conjugated secondary
antibody. The bands were visualized using chemiluminescence and the
FluorChem 8000 chemiluminescence imaging system (Alpha Innotech; San
Leandro, CA).
Measurement of inositol phosphates. Confluent cells plated onto 60-mm-diameter dishes were growth arrested and radiolabeled for 48 h with inositol-free DMEM containing 2% FBS and 1 µCi/ml myo-[3H]inositol (specific activity 17.0 Ci/mmol). Cells were washed free of unincorporated myo-[3H]inositol with PBS (in mmol/l: 137 NaCl, 2.7 KCl, 8.5 Na2HPO4, 1.5 KH2PO4, 0.9 MgCl2, 5.5 glucose, 0.7 CaCl2, and 0.1% BSA) and preincubated with 10 mM LiCl in 1 ml of PBS in the presence and absence of genistein at 37°C for 10 min. The cells were challenged with 5-HT in the presence of LiCl for 10 min at 37°C. Stimulation was terminated by the addition of 100:1 vol/vol methanol-HCl. Chloroform (50 vol/vol to HCl) was added to the supernatant, and the aqueous and organic fractions were separated by centrifugation. The top aqueous layer was assayed for total myo-[3H]inositol phosphate activity by anion-exchange chromatography. Samples were loaded onto columns containing 1 ml of the formate form Dowex 1-X8 resin (Bio-Rad Laboratories), and myo-[3H]inositol and [3H]glycerophosphoinositide were removed by washing with a solution of 60 mM ammonium formate and 5 mM sodium tetraborate. The myo-[3H]inositol phosphates were eluted with a solution containing 1 M ammonium formate and 100 mM formic acid.
Materials. DMEM, FBS, penicillin, and streptomycin were
obtained from GIBCO Canada (Mississauga, ON) and Ham's F-12 from
ICN. DiOC6(3) and fura 2-AM were purchased
from Molecular Probes (Eugene, OR), and genistein, tyrphostins, and
PD-98059 were from Calbiochem (LaJolla, CA). Electrophoresis reagents
were obtained from Bio-Rad, rabbit polyclonal anti-PLC- antibodies
were from Santa Cruz Biotechnology (Santa Cruz, CA), and monoclonal
anti-PLC-
antibodies were from Transduction Laboratories (Lexington,
KY). ECL reagents, film, HRP-conjugated anti-rabbit and anti-mouse
antibodies, and myo-[3H]inositol were
purchased from Amersham Canada. Anti-phosphotyrosine antibodies and all
remaining chemicals were purchased from Sigma (St Louis, MO).
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RESULTS |
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The contractile effects of 5-HT were tested on cultured rat tracheal
smooth muscle cells grown on rat tail collagen. 5-HT (1 µM) caused a
contraction that resulted in a 10 ± 1% decrease in the surface area
of the cells. The subsequent application of the calcium ionophore
ionomycin (10 µM) elicited a greater contraction (18 ± 3% decrease
in area). The tyrosine kinase inhibitor genistein at 40 µM inhibited
5-HT-induced contractions in almost all cells, with the mean surface
area of the stimulated cells at 101 ± 3% of the controls, but had no
significant effect on contraction elicited by ionomycin in the same
cells (26 ± 2% decrease in surface area; Fig.
1).
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Genistein (40 µM) also affected 5-HT-induced Ca2+
transients in a concentration-dependent manner, causing a significant
decrease in peak Ca2+ response (from 441.7 ± 59.49 nM in
controls to 223.6 ± 35.32 nM in genistein-treated cells, P = 0.009; Fig. 2A). The effect of
tyrphostin 23, a tyrosine kinase inhibitor structurally different from
genistein, was similar. At a concentration of 10 µM, it caused a
decrease in 5-HT-induced Ca2+ responses, with a peak
Ca2+ of 294.8 ± 29.10 nM compared with 507.0 ± 68.28 nM
in the cells incubated with the inactive tyrphostin analog tyrphostin
A-1, (Fig. 2B). The effect of genistein was also observed in
the absence of extracellular Ca2+, and the decrease in peak
Ca2+ was even greater than in Ca2+-containing
medium (from 441.73 ± 59.49 nM Ca2+ in the controls to
97.04 ± 26.55 nM Ca2+ in the presence of 40 µM
genistein; Fig. 2C).
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Western analysis demonstrated that 1 µM 5-HT caused an increase in
tyrosine phosphorylation of proteins with approximate molecular masses
of 70 and 115 kDa within 10 s after the application of the agonist
application (Fig. 3A). The increase
in tyrosine phosphorylation was significantly inhibited in the cells
treated with 40 µM genistein (Fig. 3B, Table
1). The identity of the
tyrosine-phosphorylated proteins has not been established. However, we
specifically examined the blots for evidence of tyrosine
phosphorylation of PLC-, which we were not able to detect although
it was apparent in the cells stimulated with PDGF (Fig.
4, A and B). The lack of
PLC-
tyrosine phosphorylation caused by 5-HT was also confirmed by
the absence of the PLC-
band in immunoprecipitates of tyrosine-
phosphorylated proteins from 5-HT-stimulated cells, with simultaneous
detection of PLC-
in the lysates from PDGF-stimulated cells (Fig.
4C).
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To test the possibility that MAPKs were involved in
[Ca2+]i regulation, we used the
specific inhibitor of the upstream kinase MEK, PD-98059. No significant
change in peak Ca2+ responses to 1 µM 5-HT was observed
after 20 min of incubation with 30 µM PD-98059 (Fig.
5).
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To establish whether tyrosine kinase inhibition exerted its effects
downstream or upstream of PLC, we measured 5-HT-evoked inositol
phosphate production in the presence and absence of 40 µM genistein.
As expected, 5-HT induced a dramatic increase in inositol phosphate
production (~600% of the baseline turnover in the cells stimulated
with 1 µM 5-HT and 800% in the cells stimulated with 10 µM 5-HT).
Inositol phosphate production induced by both concentrations of 5-HT
was significantly reduced in the cells incubated with 40 µM genistein
(Fig. 6).
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DISCUSSION |
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Although the existence of cross talk between signaling pathways mediating contractility (PLC and inositol phosphates) and growth (tyrosine kinase pathway) of vascular smooth muscle has been a subject of several studies (6, 11, 15; reviewed in Refs. 9 and 25), little is known about the modulation by tyrosine kinases of the signaling pathways leading to inositol phosphate production after activation of G protein-coupled receptors (7, 13, 18, 23, 35). Indeed, to our knowledge, there are only three reports indicating a possible role for tyrosine kinases in airway smooth muscle contraction (1, 2, 33). Our study has shown that a component of the 5-HT-evoked decrease in the area of cultured rat airway smooth muscle cells is tyrosine kinase dependent. These results imply that the previously described effect of tyrosine kinase inhibition on 5-HT-induced contraction of vascular strips (35) and isolated rat bronchioles (2) reflects altered responses of smooth muscle and not an indirect effect mediated by other cell types present in these preparations. The fact that the effects on cultured smooth muscle cells parallel the effects on muscle strips also suggests that agonist-induced changes in cell area may be a manifestation of the same processes as those taking place during the contraction of the intact muscle. This is further supported by the observation that the 5-HT-induced decrease in cell area can be blocked by PLC inhibitor U-73122 (unpublished data). However, the possibility that in cultured cells signaling pathways may be altered and may be different from those in the intact muscle has not been excluded.
Altered smooth muscle contractility by inhibitors of tyrosine kinase could result from either the modulation of structural or regulatory proteins or alterations of Ca2+ signaling. For example, acetylcholine- and 5-HT-induced tyrosine phosphorylation of paxillin and focal adhesion kinase, a protein and tyrosine kinase, respectively, present in dense plaques, have been described in canine tracheal smooth muscle (22, 27, 33), suggesting that interference with cytoskeletal rearrangement might be a potential mechanism for the effects. However, our data have shown that tyrosine phosphorylation may also have an important role in the coupling of G protein-linked receptor activation to increases in [Ca2+]i. The observation that genistein did not affect Ca2+ ionophore-induced contraction suggests that in rat airway smooth muscle cells the main effect of tyrosine kinase was exerted on signaling involved in Ca2+ release and not on sensitivity of the contractile apparatus to Ca2+. This conclusion is consistent with a study showing that the genistein-dependent decrease in [Ca2+]i quantitatively accounted for the decrease in myosin light chain phosphorylation in swine carotid arteries (7) and with the observation that tyrosine kinase inhibitors did not alter increases in isometric force evoked by direct activation of the contractile apparatus by Ca2+ in skinned vascular smooth muscle fibers (3). However, we observed a complete genistein-induced inhibition of cell contraction, although there was only partial inhibition of Ca2+ increase and inositol phosphate production. Although this observation may be construed as indicating that tyrosine kinase may also modulate smooth muscle contractility in a Ca2+-independent fashion, there are alternate explanations. We postulate that the modest contractions of the smooth muscle cells in our experimental conditions result from limitations imposed by their attachments to substratum or from phenotypical changes occurring in culture. Perhaps under these circumstances, the partial decrease in [Ca2+]i may have been sufficient to result in an apparent complete inhibition of contraction.
There are only a few studies of the effects of tyrosine kinase inhibitors on Ca2+ signaling in vascular smooth muscle, and the results are contradictory, showing a decrease of peak responses (16, 23) or no effect on peak responses (36), a decrease of Ca2+ influx (26) and of the sustained levels of the Ca2+ response (16), or an increase of the sustained phase of the Ca2+ response (30). Our observation of the inhibitory effects of genistein on Ca2+ responses in Ca2+-free medium indicates that tyrosine kinases are involved in the modulation of Ca2+ release from sarcoplasmic reticulum. This effect could be explained either by the modulation of IP3 binding to its receptors, as has been postulated for T cells where tyrosine phosphorylation of IP3 receptors has been found (10), or by modulation of PLC activity and therefore IP3 production. The latter explanation is supported by our observation of the genistein-induced decrease of inositol phosphate production stimulated by 5-HT. This result is also consistent with the observation in vascular smooth muscle where the increase in [Ca2+]i induced by the direct application of IP3 was not inhibited by genistein (16).
The mechanism of the modulation of PLC activity by tyrosine kinases is
well established only for the isoform of PLC, which is activated by
phosphorylation on tyrosine on binding to tyrosine-phosphorylated residues of growth factor receptors. It also has been found that in rat
vascular smooth muscle PLC-
1 is associated with the G protein-coupled angiotensin AT1 receptor and is responsible
for angiotensin-induced Ca2+ signaling (18). However, it
also has been shown that tyrosine phosphorylation of PLC-
1 is not
required for tyrosine kinase-dependent increases in
[Ca2+]i resulting from stimulation
of diverse G protein-coupled receptors in A10 vascular smooth muscle
cells (4). Likewise, we have not been able to detect PLC-
tyrosine
phosphorylation in 5-HT-stimulated rat tracheal smooth muscle cells. It
seems more likely, therefore, that interference with activation of
PLC-
is responsible for the observed reduction in inositol
phosphates. The 5-HT receptors on these cells have been characterized
as 5-HT2c (28), typical seven-transmembrane-domain receptors coupled to
Gq protein, and the interaction with
- or
-subunits of this protein is necessary for activation of PLC-
.
An alternate mechanism for reduction in inositol phosphates is
suggested by the recent finding that in mouse embryo fibroblasts
expressing the M1 muscarinic acetylcholine receptor,
stimulation with carbachol resulted in tyrosine phosphorylation of the
-subunit of Gq protein and that this phosphorylation was necessary to evoke a Ca2+ signal (32). Perhaps similar
events take place in relation to the airway smooth muscle 5-HT
receptor, and the inhibition of the
-subunit phosphorylation by
genistein leads to the observed decrease of PLC activity and the
consequent diminution of the Ca2+ response. It is not clear
at present which tyrosine kinases are involved in this process, but
soluble tyrosine kinases of the src family are the most likely
candidates because they may be activated by various G protein-coupled
receptors, including the AT1 receptor in vascular smooth
muscle (19).
The ERK MAPK pathway, identified as a site of cross talk between contractile and proliferative pathways, also has been implicated in tyrosine kinase modulation of the contractile response in vascular smooth muscle (34), and it was suggested that it modifies Ca2+ signaling (29). Our experiments demonstrated a lack of effect of a specific inhibitor of MEK kinase on 5-HT-induced Ca2+ transients in airway smooth muscle cells, arguing that any effects that MAPKs may have on smooth muscle contraction are Ca2+ independent. This conclusion is consistent with the studies showing that vascular smooth muscle MAPK activation by agonists acting on G protein-coupled receptors lies downstream of PLC activity (5, 20, 31).
In summary, our study shows that in airway smooth muscle enhanced
protein tyrosine phosphorylation may contribute to the signaling by G
protein-coupled receptors through the modulation of activity of PLC
other than PLC- isoform. Further studies are required to establish
which tyrosine kinase(s) is/are involved and to identify the pertinent
tyrosine kinase substrates.
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ACKNOWLEDGEMENTS |
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This work was supported by the Medical Research Council Grant MT-7852.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: J. G. Martin, Meakins-Christie Laboratories, McGill Univ., 3626 St. Urbain St., Montreal, Quebec, Canada H2X 2P2 (E-mail: jmartin{at}meakins.lan.mcgill.ca).
Received 2 March 1999; accepted in final form 10 January 2000.
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