Seymour Heisler Laboratory of 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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We tested the hypothesis that, in airway smooth muscle cells, stimulation of G-protein-coupled receptors by contractile agonists activates Src kinase and that this kinase modulates cell contractility and Ca2+ signaling by affecting the levels of the phospholipase C substrate phosphatidylinositol 4,5-bisphosphate (PIP2). Stimulation of cultured rat tracheal smooth muscle cells with serotonin (5-HT) induced an increase in Src activity, Ca2+ mobilization, and contraction (decrease in cell area). 5-HT-evoked cell contraction was reduced by a specific inhibitor of Src family kinases, 4-amino-5(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1). Peak Ca2+ responses to 5-HT were attenuated by PP1 and an anti-Src-blocking antibody and augmented by expression of constitutively activated Y529F Src. Sustained phases of Ca2+ responses to 5-HT and Ca2+ influx resulting from emptying of Ca2+ stores in the endoplasmic reticulum by thapsigargin were also decreased after PP1 treatment. PP1 significantly reduced the turnover of inositol phosphates produced on 5-HT stimulation and the amount of PIP2 in the Triton X-100-insoluble lipid fraction. Overall, these data demonstrate that, in rat tracheal smooth muscle cells, Src kinase modulates 5-HT-evoked cell contractility and Ca2+ signaling by regulating PIP2 levels and Ca2+ influx.
phospholipase C; smooth muscle contraction; phosphoinositides
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INTRODUCTION |
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IT IS WELL ESTABLISHED that tyrosine kinases activated by ligation of G-protein-coupled receptors (GPCRs) take part in the "cross-talk" with pathways involving mitogen-activated protein kinases. There are also data indicating that tyrosine kinases activated by the stimulation of GPCRs may be involved in Ca2+ signaling (10, 16, 24). Therefore, in smooth muscle, in which Ca2+ is the main determinant of contraction and in which the majority of contractile agonists act through GPCRs, tyrosine kinases may modulate contractility and potentially contribute to the altered smooth muscle responsiveness observed in such diseases as asthma or hypertension. Indeed, it has been reported that contraction of vascular and airway smooth muscle (ASM) depends, at least partially, on tyrosine kinase activity (3, 4, 28). However, it is not clear how much of this effect is the result of the modulation of Ca2+ signaling or which tyrosine kinases are involved.
The reputed effects of tyrosine kinase inhibition on Ca2+
responses to a variety of agonists are contradictory, showing a lack of
effect on peak Ca2+ response (29) or its
inhibition (10, 16). The mechanisms of these phenomena are
not known, with the exception of the events after stimulation of
vascular smooth muscle AT1 receptors, where pp60 Src kinase
induces tyrosine phosphorylation and activation of the -isoform of
phospholipase C (PLC-
; see Ref. 11). However, the
majority of GPCRs are known to be linked to the
-isoform of PLC
(PLC-
), and activation of 5-HT receptors in rat tracheal smooth
muscle cells, as well as of several types of GPCRs in A10 vascular
smooth muscle cells, does not appear to result in PLC-
tyrosine
phosphorylation despite the observed reductions in Ca2+
signals after tyrosine kinase inhibition (6, 24).
Regardless of how and which PLC isoform is activated, the availability of its substrate phosphatidylinositol 4,5-bisphosphate (PIP2) is an important determinant of inositol phosphate production (5, 18), and it has been reported that PIP2 levels may be controlled by tyrosine kinase activity (15). However, the kinase involved in this process was not identified, and the effects of lowering PIP2 levels by tyrosine kinase inhibitors on Ca2+ signaling were not studied.
The effect of tyrosine kinases on the sustained phase of the Ca2+ response to contractile agonists in smooth muscle is equally unclear. Potentiation (25) or inhibition (10) of this phase of the response by tyrosine kinase inhibitors has been described, and tyrosine kinases have been implicated in regulation of voltage-dependent L-type Ca2+ channels (30), nonspecific cation channels (1), and store-operated Ca2+ channels (2).
In the present study, we employed several strategies to clarify the role of Src kinases in GPCR-mediated ASM contraction and Ca2+ signaling, focusing mainly on the effects of Src family kinases on Ca2+ release from the endoplasmic reticulum (ER). First, we confirmed that Src was activated by serotonin (5-HT). Next, we measured 5-HT-evoked Ca2+ transients in cells in which Src was inhibited by a selective inhibitor {4-amino-5(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d] pyrimidine (PP1)} or a neutralizing antibody as well as in cells expressing constitutively activated Src. Subsequently, we tested the effects of Src inhibition on inositol phosphate turnover to establish whether Src acted proximally or distally to inositol trisphosphate (IP3) synthesis. Finally, we investigated the effect of Src inhibition on the amount of PIP2 to establish whether Src played a role in maintaining PLC substrate levels.
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MATERIALS AND METHODS |
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Cell culture.
Tracheal smooth muscle cells from 7- to 9-wk-old male Fisher rats
(Harlan Sprague-Dawley, Walkersville, MD) were isolated and cultured
according to previously described methods (24). Briefly,
the cells were enzymatically dissociated with 0.05% elastase type IV
and 0.2% collagenase type IV and cultured in 1:1 DMEM-Ham's F-12
medium supplemented with 10% FBS, 0.224% NaHCO3, and 1%
penicillin/streptomycin in the presence of 5% CO2. Cell
culture reagents were purchased from GIBCO Canada (Mississauga, ON).
First- or second-passage cells were rendered quiescent by incubation in
medium containing 0.5% FBS for 2-4 days before experiments.
Confirmation of a smooth muscle phenotype was based on typical
morphology, positive smooth muscle-specific -actin staining,
negative keratin staining, and contractile responses to agonists.
Src kinase activity assay.
Confluent and growth-arrested cells unstimulated or stimulated for 10 min with 1 µM 5-HT were lysed and clarified, and Src kinase was
immunoprecipitated with monoclonal anti c-Src antibody (Upstate
Biotechnology, Lake Placid, NY) followed by protein G-agarose. Immunoprecipitates from lysates containing 1 mg of protein were washed
and resuspended in Src kinase reaction buffer [100 mM
Tris · HCl (pH 7.2), 125 mM MgCl2, 25 mM
MnCl2, 2 mM EGTA, 0.25 mM sodium orthovanadate, and 2 mM
dithiothreitol] and incubated with the Src-specific substrate peptide
(Upstate Biotechnology) and [-32P]ATP for 10 min at
30°C. Purified Src (15 units; Upstate Biotechnology) was used as a
positive control. The reaction was stopped by adding 40% TCA for 5 min. Aliquots (25 µl) were spotted on P81 paper, which was allowed to
dry. Assay squares were washed with 0.75% phosphoric acid followed by
acetone, and radioactivity was measured in the scintillation counter.
Activity was expressed as a ratio of counts per minute in the sample
from the cells stimulated with 5-HT over control, unstimulated sample.
Measurement of cell contraction. Cells used for contraction measurements were plated on microscopic coverslips with homologous cell substrate (9) and rendered quiescent by serum deprivation at ~70% confluence. The cells were incubated for 20 min with 10 µM PP1 or with the appropriate vehicle. Images of cells stimulated with 1 µM 5-HT or Hanks' buffer were acquired at the rate of one image per minute by using a CCD camera (TM 9701; Pulnix America, Sunnyvale, CA) mounted on a microscope equipped with Nomarski optics (Olympus Optical, Lake Success, NY). The images were digitized and analyzed with Scion software (National Institutes of Health). The surface area of the cells was measured before and 10 min after stimulation. Three experiments were performed with data from 29-36 cells recorded in each experiment.
Cell permeabilization. Confluent and growth-arrested cells on microscope coverslips were temporarily permeabilized by using a previously described method (17). Briefly, the cells were rinsed with serum-free culture medium and incubated on ice with 1.2 M glycerol in PBS for 10 min followed by an 8-min incubation with lysophosphatidylcholine (Sigma, St. Louis, MO) at a final concentration of 40 µg/ml or corresponding volume of vehicle (distilled water). Polyclonal anti-Src-blocking antibody (Santa Cruz Biotechnology, Santa Cruz, CA) or rabbit IgG (1:200) was added for 10 min at 37°C. The cells were transferred to fresh culture medium and allowed to recover for 60 min. Ca2+ responses to 1 µM 5-HT were measured by imaging as described below. To confirm that permeabilization and antibody treatments were effective, parallel cell samples were fixed for 10 min in a 1:1 mixture of acetone and methanol, air-dried, and incubated with anti-rabbit secondary antibody conjugated to fluorescent dye Alexa 488 (Molecular Probes, Eugene, OR) and examined with an Olympus microscope with a confocal attachment (Insight Plus, Meridian, Okemos, MI).
Retroviral infections.
Cell populations that express constitutively activated Y529F murine Src
were generated by retroviral transduction of smooth muscle cells and
selection in the aminoglycoside antibiotic G418. Briefly, the cDNA
encoding Y529F murine Src (kindly provided by Dr. D. Shalloway, Cornell
University) was transferred to a derivative of the pLXSN retroviral
vector (12) to generate pLXSN/Y529FSrc. Recombinant
retrovirus encoding Y529F Src was produced by transfecting human
embryonic kidney 293T cells with pLXSN/Y529FSrc and a second plasmid
encoding the retroviral coat proteins, pSV--E-MLV. To generate
control "empty" virus, 293T cells were transfected with pLXSN
only. Later (3 days), cell supernatants containing the retrovirus were
collected and stored in aliquots at
80°C. To generate ASM cells
that expressed Y529F Src, first-passage rat tracheal smooth muscle
cells (3 × 105) were plated in 60-mm cell culture
dishes. Later (24 h), cells were incubated with recombinant
retroviruses in the presence of 4 µg/ml polybrene. The next morning,
cells were washed two times with PBS and placed in DMEM-F-12 media
containing 10% FBS and antibiotics. Later (6 h), the cells were
trypsinized and replated on coverslips in six-well dishes. Cells that
contained the retroviral DNA were selected by maintaining the cultures
in G418 for 7-10 days, at which time multiple G418-resistant
colonies were present. Src protein levels and Ca2+
responses to 5-HT in cells, both control and those expressing Y529F
Src, were recorded as described below.
SDS-PAGE and Western blotting. Electrophoresis reagents were obtained from Bio-Rad (Mississauga, ON). Populations of control cells infected with pLXSN or those expressing Y529F Src were serum starved overnight, rinsed with ice-cold PBS, lysed with 1 ml of lysis buffer (50 mM HEPES, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM EGTA, 1.5 mM MgCl2, 1 mM phenylmethylsulfonyl fluoride, 20 µM leupeptin, 15 U aprotinin/ml, and 1 mM sodium orthovanadate), and clarified by centrifugation. Protein concentration was determined, and equal amounts (20 µg) were resolved by 10% SDS-PAGE, transferred to 0.22-µm pore nitrocellulose filters, and probed with monoclonal antibodies that recognized either activated Src (Biosource International) or total Src (Upstate Biotechnology). Proteins were visualized by using a secondary antibody conjugated to horseradish peroxidase (Amersham) and enhanced chemiluminescence (ECL; Amersham Canada, Oakville, ON) on a FluorChem 8000 imaging system (Alpha Innotech, San Leandro, CA).
Ca2+ measurements: imaging. This method was used to measure intracellular free Ca2+ concentration ([Ca2+]i) in permeabilized cells, retrovirally transduced cells, and in cells treated with thapsigargin. Cells were loaded with the Ca2+-sensitive dye fura 2-AM (Molecular Probes) according to previously described methods (21) and imaged by using an intensified camera (Videoscope or IC200) and PTI (Photon Technology International, Princeton, NJ) software at a single emission wavelength (510 nm) with a double excitatory wavelength (345 and 380 nm). The fluorescence ratio (345/380) was measured in cells stimulated with 5-HT after treatment with anti-Src-blocking antibody or rabbit IgG (in permeabilized cells), in retrovirally transduced cells in the presence or absence of PP1, and in thapsigargin-treated cells in the presence or absence of PP1.
To assess the effect of Src inhibition on Ca2+ influx, fura 2-AM-loaded cells were treated with 10 µM PP1 or vehicle for 10 min. Ca2+-containing Hanks' buffer was then replaced by Ca2+-free Hanks' buffer, 20 µM endoplasmic ATPase inhibitor thapsigargin (Sigma) was added, and the fluorescence ratio was monitored. When the ratio stabilized after the initial increase resulting from the depletion of Ca2+ from the sarcoplasmic reticulum, Ca2+ levels in the buffer were restored, and the fluorescence ratio was recorded. [Ca2+]i was calculated according to the formula of Grynkiewicz et al. (7). The dissociation constant was assumed to be 224 nM, mean maximal fluorescence ratio (Rmax; 345/380 fluorescence ratio of Ca2+-saturated cells, measured in cells exposed to 10Ca2+ measurements: photometry. The effects of pharmacological inhibition of Src kinases on Ca2+ transients were measured by using microscope-based photometry. The cells were exposed to PP1 or an equivalent volume of vehicle for 10 min before the administration of 1 µM 5-HT. Fluorescence of a small sample (3-5) of cells was recorded at the rate of 20 points/s by using a D401 microphotometer and PTI software. Background fluorescence was automatically subtracted. Rmax measured with this method was 7.6, Rmin was 0.55, and B was 8.89. Three experiments were conducted, with three samples for each treatment per experiment.
Measurement of inositol phosphates. Confluent cells plated on 60-mm-diameter dishes were growth arrested and radiolabeled for 48 h with inositol-free DMEM containing 0.5% FBS and 1 µCi/ml myo-[3H]inositol (Amersham Canada). Cells were washed free of unincorporated myo-[3H]inositol with Krebs-Henseleit buffer (KHB; 117 mM NaCl, 4.7 mM KCl, 1.1 mM MgSO4, 1.2 mM KH2PO4, 20 mM NaHCO3, 2.4 mM CaCl2, 1 mM glucose, and 20 mM HEPES) and preincubated with 10 mM LiCl in 1 ml PBS at 37°C for 10 min with or without 10 µM PP1. 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 ice-cold 3 M TCA. Cells were chilled on ice for 30 min and then scraped off the dishes. After sonication, cellular debris was removed by centrifugation at 4,000 g for 5 min at 4°C. The inositol polyphosphates were extracted with 10 mM EDTA-trichlorotrifluoroethane-tri-n-octylamine (1:2:2), and the aqueous and organic fractions were separated by centrifugation. The aqueous layer was neutralized with 60 mM NaHCO3 and assayed for total myo-[3H]inositol phosphates by anion exchange chromatography. Samples were loaded on columns containing 1 ml of the formate form of Dowex 1-X8 resin (Bio-Rad Laboratories), and myo-[3H]inositol and 3H-glycerophosphoinositides were removed by washing with 60 mM ammonium formate and 5 mM sodium tetraborate. myo-[3H]inositol phosphates were eluted with 1 M ammonium formate and 100 mM formic acid. Five experiments were conducted, each with duplicate samples. Data are expressed as a percentage of control.
Phosphoinositide analysis. Confluent cells cultured on 150-mm dishes were labeled with 2.5 µCi/ml myo-[3H]inositol in inositol-free DMEM containing 0.5% FBS for 48 h. Cells were washed with KHB, incubated with 10 µM PP1 or the vehicle for 10 min, washed two times with ice-cold PBS, and scraped from the plates in 1 ml PBS. To extract lipids, cells were centrifuged at 10,000 g for 5 min, and the cell pellet was resuspended in 400 µl Triton extraction buffer [25 mM HEPES (pH 7.2), 250 mM NaCl, 2 mM MgCl2, 2 mM MnCl2, 1 mM CaCl2, and 0.5% Triton X-100] and incubated for 20 min at 4°C with mixing as described previously (18). Lysates were centrifuged at 15,000 g for 10 min, and the supernatant was collected as the Triton-soluble fraction to which 400 µl of stop solution (methanol-concentrated HCl, 10:1 vol/vol) was added. The Triton-insoluble pellet was washed two times with PBS, and 200 µl of stop solution and 200 µl of PBS were added. Lipids from Triton-soluble and Triton-insoluble fractions were extracted by using chloroform-methanol (1:1 vol/vol). Samples were centrifuged at 15,000 g for 2 min at 4°C, and the lower phase containing phosphoinositides was collected. After reducing the solvent volume of the sample to ~100 µl under vacuum, the samples were applied with a glass capillary on potassium oxalate-impregnated silica gel thin-layer chromatography plates. Potassium oxalate plates were prepared by placing silica gel plates (Analtech, Newark, DE) in a 1% potassium oxalate solution in methanol-water (60:40 vol/vol) for ~30 min. After being dried in the fume hood for ~1 h, the plates were dried in a 110°C oven for 15-20 min and stored in a low-humidity cabinet. The plates were developed in chloroform-acetone-methanol-acetic acid-water (40:15:13:12:8 vol/vol/vol/vol/vol). Chromatographed lipids were localized by iodine staining and identified by migration with the following authentic standards: phosphatidylinositol, phosphatidylinositol 4-phosphate, and PIP2. Lipids were scraped from the plates, and 3H radioactivity was measured by using a scintillation counter. Four independent experiments were conducted, and the amount of PIP2 was expressed as a percentage of the total counts.
Statistical analysis.
Statistical analysis was performed by using unpaired Student's
t-test, and P values of 0.05 were considered as
statistically significant.
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RESULTS |
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Src activity regulates 5-HT-induced ASM cell contraction.
Src kinase activity assays revealed that Src kinase activity was
approximately threefold greater in immunoprecipitates from the cells
stimulated with 1 µM 5-HT for 10 min compared with immunoprecipitates from unstimulated cells (Fig. 1). The
same stimulation procedure resulted in protein tyrosine phosphorylation
that was reduced by incubation with 10 µM PP1, the selective Src
inhibitor (data not shown).
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Src regulates 5-HT-induced Ca2+
signaling.
Ca2+ responses triggered by 1 µM 5-HT were measured in
cells in which Src activity was inhibited either with PP1 or with an anti-pp60 Src-blocking antibody. PP1 caused a concentration-dependent decrease in peak and plateau [Ca2+]i after
5-HT stimulation, reaching statistical significance with 10 µM PP1
(Fig. 3). Resting
[Ca2+]i was 76.2 ± 8.5 nM in the
control cells and 55.9 ± 4.2 nM (P < 0.05) in
the cells incubated for 10 min with 10 µM PP1. The 5-HT-triggered
peak [Ca2+]i was 312.7 ± 50.2 nM in
controls and 103.5 ± 14.9 nM in PP1-treated cells
(P < 0.05). [Ca2+]i at the
sustained phase of the response, reflecting Ca2+ influx and
measured 5 min after the administration of 5-HT, was 106 ± 15.6 and 31.3 ± 2.9 nM in the control cells and cells treated with 10 µM PP1, respectively (P < 0.05; Fig. 3).
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Src regulates production of inositol phosphate production and
PIP2.
To establish which step of Ca2+ release from the ER was
affected by Src kinase, the production of inositol phosphates triggered by 5-HT was measured in the presence and absence of 10 µM PP1. 5-HT
induced an almost threefold increase in inositol phosphate turnover
(286% of basal level). PP1 had no effect on the basal level of
inositol phosphates (106% of control) but significantly reduced the
quantity of inositol phosphates produced on 5-HT stimulation (150% of
basal level; Fig. 6). Inositol phosphate
production reflects PLC activity, which in turn depends on the
availability of the substrate PIP2 (5).
Because tyrosine kinase inhibition may significantly reduce levels of
PIP2 (15), amounts of PIP2 in control and PP1 (10 µM)-treated cells were measured. No effect of PP1
was detected when the whole pool of phosphoinositides was assayed (data
not shown). Therefore, PIP2 levels in Triton-soluble and
Triton-insoluble fractions, representing primarily solubilized plasma
membrane and caveolae, respectively, were assessed. Approximately 40%
of PIP2 was localized to the Triton-insoluble fraction
(PIP2 accounted for 9.7% of total counts in the
Triton-soluble and 6.1% of total counts in the Triton-insoluble
fraction). A significant decrease in PIP2 levels in
PP1-treated samples in the Triton-insoluble fraction was observed (from
6.1 ± 1% total counts to 3.4 ± 0.3%), with no significant
change in the Triton-soluble fraction (9.7 ± 1 and 12 ± 1.6%; Fig. 7). Thus the results suggest
that inhibition of Src family kinases reduces inositol phosphate
production levels by decreasing PIP2 levels in the
Triton-insoluble fraction.
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DISCUSSION |
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The role and identity of tyrosine kinases in inositol phosphate/Ca2+ signaling initiated by GPCRs remain poorly defined. It is generally recognized that binding of a contractile agent to a GPCR is followed by the activation of PLC, leading to the production of the following two second messengers: IP3 and diacylglycerol. IP3 interacts with its receptors on the ER, causing a release of Ca2+ from intracellular stores followed by Ca2+ influx from the extracellular milieu. An increase in [Ca2+]i is necessary for the initiation of many processes and is of particular importance in smooth muscle, where Ca2+ plays a key role in contraction. Enhanced Ca2+ mobilization and contraction of ASM may contribute to airway hyperresponsiveness (21). Several studies have shown that inhibition of tyrosine kinases impairs the ability of gastric, vascular, and ASM rings or strips to contract on stimulation with agonists acting on GPCRs (3, 4, 28). Using cultured ASM cells to avoid the possible compounding effects of other cells present in these preparations, we have shown that tyrosine kinases may have a direct effect on ASM contraction and Ca2+ signaling evoked by 5-HT (24). Our data from the present study, also conducted on cultured cells, have identified the kinase as a member of the Src family, based on the observations that PP1, a specific inhibitor of several members of the Src kinase family, caused a decrease in both cell contraction and Ca2+ mobilization induced by 5-HT. Furthermore, our data implicate pp60 Src directly in these phenomena because anti-pp60 Src blocking antibody had a similar effect on peak Ca2+ responses to 5-HT as did PP1, although maximal responses in the former experiment were lower overall than in the latter. This difference might result either from the lower sensitivity of the imaging system (used in antibody neutralization experiments) than photometry (used to test PP1) or from the permeabilization procedure. The role of Src in Ca2+ mobilization is further supported by our observation that expression of constitutively activated Y529F Src significantly increased peak and sustained responses to 5-HT.
Our data show that both peak and plateau phases of the Ca2+ response to 5-HT were affected by modulating the activity or the expression of Src kinase. We have previously reported that these phases correspond to the release of Ca2+ from ER and entry of Ca2+ from the extracellular milieu and that the sustained phase of the response has characteristics of store-operated Ca2+ influx (23). We conclude, therefore, that Src kinase mediates IP3-induced Ca2+ release from the ER and Ca2+ influx resulting from emptying of intracellular stores. These data are consistent with previous reports showing that tyrosine kinase inhibition led to a decrease of peak and sustained Ca2+ responses to contractile agonists in vascular and ASM (10, 16, 24). Moreover, Ca2+ entry triggered by bradykinin or thapsigargin is dramatically lower in embryonic fibroblasts from Src-deficient mice compared with cells from wild-type animals. Significantly, Ca2+ entry is restored to nearly normal levels by transfecting Src-deficient cells with a c-Src expression plasmid (2). Nevertheless, the mechanism(s) by which tyrosine kinase(s) regulate GPCR-induced Ca2+ entry are poorly defined and probably also depend on the receptor that is activated and in which cell type it is expressed. For example, inhibition of tyrosine kinases by tyrphostin A-23 in rat vascular smooth muscle resulted in a prolonged, sustained [Ca2+]i elevation after ANG II application (25). The identity of the Ca2+ channels involved in tyrosine kinase-sensitive Ca2+ influx is also not well established. It has been reported that agents that increase tyrosine phosphorylation activate a nonselective cation channel (1). Src is implicated in modulation of Ca2+ influx through both store-operated channels (2) and L-type Ca2+ channels (30).
Ca2+ release from the ER critically depends on PLC
activity. It has been reported that, in vascular smooth muscle, pp60
Src kinase induces the activation of PLC- on AT1
receptor stimulation (11). Although Ca2+
responses were not described in this study, it is well established that
activation of PLC-
leads to an increase in
[Ca2+]i by the same sequence of events as
activation of PLC-
, namely by IP3 production and
Ca2+ release from the ER. However, the only functional 5-HT
receptor expressed in rat ASM cells has been identified as 5-HT 2c
(23), and this type of receptor is known to activate
PLC-
. Moreover, we have previously reported (24) that
stimulation of rat ASM with 5-HT does not result in detectable PLC-
phosphorylation, which is usually necessary for its activation.
Similarly, in A10 vascular smooth muscle cells, peak Ca2+
responses triggered by several agonists were significantly attenuated by tyrosine kinase inhibition, whereas stimulation with these agonists
did not result in PLC-
phosphorylation (6).
Nevertheless, our present study revealed that Src inhibition by PP1
resulted in the diminished production of inositol phosphates. This
effect could be explained by the ability of Src to modulate the amount of PLC substrate, PIP2. It has been previously reported
that tyrosine kinase inhibition leads to a rapid decrease of cellular
PIP2 levels in HEK-293 cells (15). The
resynthesis of PIP2 occurs in a sustained fashion in
nonstimulated cells and very rapidly after PLC hydrolysis (26). Therefore, PIP2 resupply is required for
PLC-mediated inositol phosphate production (5). Indeed,
our results show that inhibition of Src by PP1 leads to a decrease in
PIP2 levels in the Triton-insoluble fraction. This
observation is consistent with previous studies showing that, in A431
cells, epidermal growth factor and bradykinin caused a 50% decrease in
PIP2 levels in the caveolin-rich, Triton-insoluble fraction
but no change in the levels of total plasma membrane PIP2
(8, 14). Also consistent with this study are our results
indicating that ~40% of PIP2 recovered from rat ASM
resides in these agonist-sensitive regions.
Although we have observed a significant effect of Src kinase on Ca2+ signaling, it is possible that Src may additionally modulate ASM contractility by other mechanisms such as cytoskeletal rearrangement. It could occur in a PIP2-dependent fashion because PIP2 may bind and thereby regulate a variety of actin-binding proteins (22), or by regulation of tyrosine phosphorylation of proteins in dense plaques (13, 20, 27). Such a possibility is supported by our observation that changes in the cell area triggered by the application of buffer alone were inhibited by PP1, even if the administration of the buffer did not cause an increase in [Ca2+]i (data not shown).
In summary, our data demonstrate that Src kinase modulates ASM contraction and Ca2+ signaling. The mechanism of the effect involves a reduction in inositol phosphate turnover that may be a consequence of alterations in agonist-sensitive pools of the PLC substrate PIP2.
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ACKNOWLEDGEMENTS |
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We thank Jamilah Saeed for technical assistance.
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FOOTNOTES |
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This work was supported by operating Grant MOP/36334 from the Canadian Institutes of Health Research (J. G. Martin) and the Association Pulmonaire du Quebec (E. D. Fixman).
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: james.martin{at}mcgill.ca).
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. Section 1734 solely to indicate this fact.
First published January 4, 2002;10.1152/ajplung.00304.2001
Received 3 August 2001; accepted in final form 28 December 2001.
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