Tyrosine kinase-dependent calcium signaling in airway smooth muscle cells

Barbara Tolloczko, Florence C. Tao, Mary E. Zacour, and James G. Martin

Seymour Heisler Laboratory of the Montreal Chest Institute Research Centre and Meakins-Christie Laboratories, Department of Medicine, McGill University, Montreal, Quebec, Canada H2X 2P2


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 gamma  isoform of phospholipase C (PLC-gamma ). 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-beta activity. It is unlikely that PLC-gamma or the mitogen-activated protein kinase pathway is involved in Ca2+ signaling to 5-HT.

cell culture; mitogen-activated protein kinase; phospholipase C


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-beta (PLC-beta ).

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 gamma  isoform of PLC (PLC-gamma ). PLC-gamma causes an increase in [Ca2+]i through the same sequence of events as PLC-beta 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-gamma (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.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha -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 10-5 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-gamma 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-gamma ). 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-gamma 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-gamma antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA), and monoclonal anti-PLC-gamma 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).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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).


View larger version (26K):
[in this window]
[in a new window]
 
Fig. 1.   Effect of genistein on airway smooth muscle cell contraction. Surface area of cells loaded with DiOC6(3) and contracted with 1 µM serotonin (5-HT) or 10 µM ionomycin (iono) in presence and absence of 40 µM genistein was measured using a digitizing tablet and is expressed as a percentage of area of cells before addition of agonist. Base, baseline. Data are presented as means ± SE from 3 experiments (87-108 cells). * P <=  0.001 vs. baseline. # P = 0.01 vs. control 5-HT-stimulated cells by Student's t-test.

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).




View larger version (202019K):
[in this window]
[in a new window]
 
Fig. 2.   Effect of tyrosine kinase inhibition on peak Ca2+ responses to 5-HT. Cells were incubated with 40 µM genistein (gen) or vehicle for 10 min and stimulated with 1 µM 5-HT in Ca2+-containing buffer (A) or Ca2+-free buffer (C) or incubated with 10 µM tyrphostin 23 or tyrphostin A-1 (tyr) for 10 min and stimulated with 1 µM 5-HT (B). Data are presented as means ± SE from 3 experiments for each treatment; n, no. of samples. * P <=  0.009 vs. control 5-HT-stimulated cells in absence of inhibitor by Student's t-test.

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-gamma , 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-gamma tyrosine phosphorylation caused by 5-HT was also confirmed by the absence of the PLC-gamma band in immunoprecipitates of tyrosine- phosphorylated proteins from 5-HT-stimulated cells, with simultaneous detection of PLC-gamma in the lysates from PDGF-stimulated cells (Fig. 4C).


View larger version (34K):
[in this window]
[in a new window]
 
Fig. 3.   5-HT-induced tyrosine phosphorylation. A: representative blot shows proteins phosphorylated on tyrosine within 10 s of stimulation with 1 µM 5-HT. Similar results were obtained in 3 other experiments. B: representative blot shows effect of 40 µM genistein (G) on 5-HT-induced tyrosine phosphorylation. C, control.


                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Tyrosine phosphorylation evoked by 5-HT in absence and presence of genistein





View larger version (361418K):
[in this window]
[in a new window]
 
Fig. 4.   Effect of 5-HT and platelet-derived growth factor (PDGF) on tyrosine phosphorylation of phospholipase C (PLC)-gamma . Cells were stimulated for 10 min with 1 µM 5-HT or 10 ng/ml of BB isoform of PDGF in presence of sodium orthovanadate, immunoblotted with anti-PLC-gamma antibody (alpha PLCgamma ) and then stripped and reprobed with anti-phosphotyrosine antibody (alpha pY). A: representative blot; left, blot probed with anti-PLC-gamma antibody; right, same blot stripped and reprobed with anti-phosphotyrosine antibody. Arrow, band corresponding to PLC-gamma . B: relative density of tyrosine-phosphorylated bands corresponding to PLC-gamma in control, 5-HT stimulated, and PDGF-stimulated cells. Densities were normalized for PLC-gamma levels by calculating the ratio of densities of bands in blots probed with anti-phosphotyrosine antibody to densities of same bands that were, before stripping, labeled with anti-PLC-gamma antibody. Densities are expressed as multiples of increase over control. Data are means ± SE from 4 experiments. P value vs. control unstimulated cells. C: representative blot of immunoprecipitates of tyrosine-phosphorylated proteins from control, 5-HT-stimulated, and PDGF-stimulated cells probed with anti-PLC-gamma antibody. Similar results were obtained in 3 other experiments.

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).


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 5.   Effect of mitogen-activated protein kinase kinase (MEK) inhibitor on 5-HT-induced Ca2+ responses. Cells were incubated with 30 µM MEK inhibitor PD-98059 or appropriate vehicle for 20 min and stimulated with 1 µM 5-HT. Values are means ± SE of peak Ca2+ responses; n, no. of samples.

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).


View larger version (22K):
[in this window]
[in a new window]
 
Fig. 6.   Effect of tyrosine kinase inhibition on PLC activity. Total inositol phosphate (IP) production was measured as described in MATERIALS AND METHODS in resting cells or in cells stimulated with 5-HT (1 and 10 µM) in presence and absence of 40 µM genistein. * P <=  0.05 vs. 5-HT stimulated in absence of genistein by Student's t-test.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 gamma  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-gamma 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-gamma 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-gamma tyrosine phosphorylation in 5-HT-stimulated rat tracheal smooth muscle cells. It seems more likely, therefore, that interference with activation of PLC-beta 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 alpha - or beta gamma -subunits of this protein is necessary for activation of PLC-beta . 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 alpha -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 alpha -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-gamma isoform. Further studies are required to establish which tyrosine kinase(s) is/are involved and to identify the pertinent tyrosine kinase substrates.


    ACKNOWLEDGEMENTS

This work was supported by the Medical Research Council Grant MT-7852.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Bois, F, Desfougeres A, Boumendjel A, Mariotte AM, Bessard G, Caron F, and Devillier P. Genistein and fluorinated analogs suppress agonist-induced airway smooth muscle contraction. Bioorg Med Chem 7: 1323-1326, 1997.

2.   Chopra, LC, Hucks D, Twort CH, and Ward JP. Effects of protein tyrosine kinase inhibitors on contractility of isolated bronchioles of the rat. Am J Respir Cell Mol Biol 16: 372-378, 1997[Abstract].

3.   Di Salvo, J, Nelson SR, and Kaplan N. Protein tyrosine phosphorylation in smooth muscle: a potential coupling mechanism between receptor activation and intracellular calcium. Proc Soc Exp Biol Med 214: 285-301, 1997[Abstract].

4.   Di Salvo, J, and Nelson SR. Stimulation of G-protein coupled receptors in vascular smooth muscle cells induces tyrosine kinase dependent increases in calcium without tyrosine phosphorylation of phospholipase C gamma. FEBS Lett 422: 85-88, 1998[ISI][Medline].

5.   Eguchi, S, Matsumoto T, Motley ED, Utsunomiya H, and Inagami T. Identification of an essential signaling cascade for mitogen-activated protein kinase activation by angiotensin II in cultured rat vascular smooth muscle cells. Possible requirement of Gq-mediated p21ras activation coupled to a Ca2+/calmodulin-sensitive tyrosine kinase. J Biol Chem 271: 14169-14175, 1996[Abstract/Free Full Text].

6.   Fujitani, Y, and Bertrand C. ET-1 cooperates with EGF to induce mitogenesis via a PTX-sensitive pathway in airway smooth muscle cells. Am J Physiol Cell Physiol 272: C1492-C1498, 1997[Abstract/Free Full Text].

7.   Gould, EM, Rembold CM, and Murphy RA. Genistein, a tyrosine kinase inhibitor, reduces Ca2+ mobilization in swine carotid media. Am J Physiol Cell Physiol 268: C1425-C1429, 1995[Abstract/Free Full Text].

8.   Grynkiewicz, G, Poenie M, and Tsien RY. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260: 3440-3450, 1985[Abstract].

9.   Hollenberg, MD. Tyrosine kinase-mediated signal transduction pathways and the actions of polypeptide growth factors and G-protein-coupled agonists in smooth muscle. Mol Cell Biochem 149-150: 77-85, 1995.

10.   Jayaraman, T, Ondrias K, Ondriasova E, and Marks AR. Regulation of the inositol 1,4,5-trisphosphate receptor by tyrosine phosphorylation. Science 272: 1492-1494, 1996[Abstract].

11.   Jin, N, Siddiqui RA, English D, and Rhoades RA. Communication between tyrosine kinase pathway and myosin light chain kinase pathway in smooth muscle. Am J Physiol Heart Circ Physiol 271: H1348-H1355, 1996[Abstract/Free Full Text].

12.   Kahn, AM, Bishara M, Cragoe EJJ, Allen JC, Seidel CL, Navran SS, O'Neil RG, McCarty NA, and Shelat H. Effects of serotonin on intracellular pH and contraction in vascular smooth muscle. Circ Res 71: 1294-1304, 1992[Abstract].

13.   Kaplan, N, and Di Salvo J. Coupling between [arginine8]-vasopressin-activated increases in protein tyrosine phosphorylation and cellular calcium in A7r5 aortic smooth muscle cells. Arch Biochem Biophys 326: 271-280, 1996[ISI][Medline].

14.   Kelleher, MD, Abe MK, Chao TS, Jain M, Green JM, Solway J, Rosner MR, and Hershenson MB. Role of MAP kinase activation in bovine tracheal smooth muscle mitogenesis. Am J Physiol Lung Cell Mol Physiol 268: L894-L901, 1995[Abstract/Free Full Text].

15.   Linseman, DA, Benjamin CW, and Jones DA. Convergence of angiotensin II and platelet-derived growth factor receptor signaling cascades in vascular smooth muscle cells. J Biol Chem 270: 12563-12568, 1995[Abstract/Free Full Text].

16.   Liu, CY, and Sturek M. Attenuation of endothelin-1-induced calcium response by tyrosine kinase inhibitors in vascular smooth muscle cells. Am J Physiol Cell Physiol 270: C1825-C1833, 1996[Abstract/Free Full Text].

17.   Malarkey, K, McLees A, Paul A, Gould GW, and Plevin R. The role of protein kinase C in activation and termination of mitogen-activated protein kinase activity in angiotensin II-stimulated rat aortic smooth-muscle cells. Cell Signal 8: 123-129, 1996[ISI][Medline].

18.   Marrero, MB, Paxton WG, Duff JL, Berk BC, and Bernstein KE. Angiotensin II stimulates tyrosine phosphorylation of phospholipase C-gamma 1 in vascular smooth muscle cells. J Biol Chem 269: 10935-10939, 1994[Abstract/Free Full Text].

19.   Marrero, MB, Schieffer B, Paxton WG, Schieffer E, and Bernstein KE. Electroporation of pp60c-src antibodies inhibits the angiotensin II activation of phospholipase C-gamma 1 in rat aortic smooth muscle cells. J Biol Chem 270: 15734-15738, 1995[Abstract/Free Full Text].

20.   Molloy, CJ, Taylor DS, and Weber H. Angiotensin II stimulation of rapid protein tyrosine phosphorylation and protein kinase activation in rat aortic smooth muscle cells. J Biol Chem 268: 7338-7345, 1993[Abstract/Free Full Text].

21.   Noh, DY, Shin SH, and Rhee SG. Phosphoinositide-specific phospholipase C and mitogenic signaling. Biochim Biophys Acta 1242: 99-113, 1995[ISI][Medline].

22.   Pavalko, FM, Adam LP, Wu MF, Walker TL, and Gunst SJ. Phosphorylation of dense-plaque proteins talin and paxillin during tracheal smooth muscle contraction. Am J Physiol Cell Physiol 268: C563-C571, 1995[Abstract/Free Full Text].

23.   Semenchuk, LA, and Di Salvo J. Receptor-activated increases in intracellular calcium and protein tyrosine phosphorylation in vascular smooth muscle cells. FEBS Lett 370: 127-130, 1995[ISI][Medline].

24.   Servant, MJ, Giasson E, and Meloche S. Inhibition of growth factor-induced protein synthesis by a selective MEK inhibitor in aortic smooth muscle cells. J Biol Chem 271: 16047-16052, 1996[Abstract/Free Full Text].

25.   Srivastava, AK. Protein tyrosine phosphorylation in cardiovascular system. Mol Cell Biochem 149-150: 87-94, 1995.

26.   Suzuki, A, Shinoda J, Oiso Y, and Kozawa O. Tyrosine kinase is involved in angiotensin II-stimulated phospholipase D activation in aortic smooth muscle cells: function of Ca2+ influx. Atherosclerosis 121: 119-127, 1996[ISI][Medline].

27.   Tang, D, Mehta D, and Gunst SJ. Mechanosensitive tyrosine phosphorylation of paxillin and focal adhesion kinase in tracheal smooth muscle. Am J Physiol Cell Physiol 276: C250-C258, 1999[Abstract/Free Full Text].

28.   Tolloczko, B, Jia YL, and Martin JG. Serotonin-evoked calcium transients in airway smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 269: L234-L240, 1995[Abstract/Free Full Text].

29.   Touyz, RM, Deng LY, and Schiffrin EL. Angiotensin signalling, contraction and growth in smooth muscle cells from human resistance arteries (Abstract). J Vasc Res 35: 27, 1998[ISI][Medline].

30.   Touyz, RM, and Schiffrin EL. Tyrosine kinase signaling pathways modulate angiotensin II-induced calcium ([Ca2+]i) transients in vascular smooth muscle cells. Hypertension 27: 1097-1103, 1996[Abstract/Free Full Text].

31.   Tsuda, T, Kawahara Y, Ishida Y, Koide M, Shii K, and Yokoyama M. Angiotensin II stimulates two myelin basic protein/microtubule-associated protein 2 kinases in cultured vascular smooth muscle cells. Circ Res 71: 620-630, 1992[Abstract].

32.   Umemori, H, Inoue T, Kume S, Sekiyama N, Nagao M, Itoh H, Nakanishi S, Mikoshiba K, and Yamamoto T. Activation of the G protein Gq/11 through tyrosine phosphorylation of the alpha subunit. Science 276: 1878-1881, 1997[Abstract/Free Full Text].

33.   Wang, Z, Pavalko FM, and Gunst SJ. Tyrosine phosphorylation of the dense plaque protein paxillin is regulated during smooth muscle contraction. Am J Physiol Cell Physiol 271: C1594-C1602, 1996[Abstract/Free Full Text].

34.   Watts, SW. Serotonin activates the mitogen-activated protein kinase pathway in vascular smooth muscle: use of the mitogen-activated protein kinase kinase inhibitor PD098059. J Pharmacol Exp Ther 279: 1541-1550, 1996[Abstract].

35.   Watts, SW, Yeum CH, Campbell G, and Webb RC. Serotonin stimulates protein tyrosyl phosphorylation and vascular contraction via tyrosine kinase. J Vasc Res 33: 288-298, 1996[ISI][Medline].

36.   Weiss, RH, and Nuccitelli R. Inhibition of tyrosine phosphorylation prevents thrombin-induced mitogenesis, but not intracellular free calcium release, in vascular smooth muscle cells. J Biol Chem 267: 5608-5613, 1992[Abstract/Free Full Text].


Am J Physiol Lung Cell Mol Physiol 278(6):L1138-L1145
1040-0605/00 $5.00 Copyright © 2000 the American Physiological Society