Abstract
Our experiments were conducted to evaluate, in rat myometrium, the potential contribution of a protein tyrosine kinase (PTK) pathway in the hydrolysis of phosphatidylinositol-4,5-bisphosphate mediated by bombesin, endothelin-1 (ET-1), and carbachol. The production of inositol phosphates (InsP) by agonists and AlF4 − was partly inhibited (35–40%) by genistein and tyrphostins, two PTK inhibitors. Genistein attenuated uterine contractions elicited by the stimulation of muscarinic and bombesin receptors, whereas pervanadate, a protein tyrosine phosphatase inhibitor, potentiated receptor-mediated contraction. Tyrosine-phosphorylated proteins were detected in detergent extracts from agonist- and pervanadate-stimulated myometrium. The amount of InsP produced in response to pervanadate was related to the tyrosine phosphorylation status of phospholipase C-γ1. In contrast, with ET-1 and bombesin, phosphorylated phospholipase C-γ1 made a minor contribution. Additional findings were rather consistent with a role for Ca2+. In fura-2-loaded cells, genistein partly decreased both the transient and sustained intracellular Ca2+ concentration phases induced by bombesin. The removal of extracellular Ca2+ or the addition of nifedipine inhibited (35%) InsP production due to bombesin and ET-1. The inhibitory effects of genistein and tyrphostins were abolished in Ca2+-depleted medium, were not additive with that of nifedipine, and (as for nifedipine) were counteracted by the Ca2+ channel agonist Bay K 8644. The data are consistent with a PTK-mediated process in the activation of the voltage-gated Ca2+ influx that is involved in the production of InsP by stimulated G protein-coupled receptors.
Signaling pathways associated with the hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP2) play a key role in the regulation of cell function (Berridge, 1993). We have previously shown that in myometrium, PIP2breakdown mediated by various contractile agonists is associated with the stimulation of phospholipase C (PLC) via the activation of specific G protein-coupled receptors (Marc et al., 1988; Leiber et al., 1990;Amiot et al., 1993; Dokhac et al., 1994). PLC stimulation is insensitive to pertussis toxin, suggesting that a member of the Gq family is involved (Lajat et al., 1996). There is evidence that in the myometrium, at least two distinct mechanisms underlie the activation of PIP2-PLC in response to carbachol and oxytocin (Dokhac et al., 1992). One mechanism concerns the well recognized agonist-induced activation of receptor-Gq protein-PLCβ3 cascade (Lajat et al., 1996), which is insensitive to elevation of intracellular Ca2+ and contributes predominantly to the increased production of inositol phosphates. A second Ca2+-dependent pathway is responsible for the additional, although modest (35%), receptor- and G protein-mediated stimulation of a PLC activity through an increased influx of Ca2+ after the activation of voltage-operated Ca2+ channels (Dokhac et al., 1992, 1996).
Protein tyrosine kinases (PTKs) play a critical role in regulating various cellular processes, including PIP2hydrolysis, through tyrosine phosphorylation and the activation of PLC-γ in a number of cell systems and tissues (van der Geer and Hunter, 1994; Malarkey et al., 1995; Post and Heller Brown, 1996). We recently demonstrated (Palmier et al., 1996) that pervanadate, a protein tyrosine phosphatase (PTP) inhibitor, elicits uterine contraction via a PTK-dependent process associated with the generation of InsP3, a major determinant of myometrial contractility. The increased degradation of PIP2was demonstrated to be due to the pervanadate-mediated PLC-γ1 phosphorylation on tyrosine residues. PLC-γ phosphorylation is stimulated by epidermal growth factor and platelet-derived growth factor whose receptors display intrinsic PTK activity (van der Geer and Hunter, 1994). However, recent work has shown that the increase in PIP2 hydrolysis induced by activation of G protein-coupled receptors is partly regulated via a PTK pathway. This pathway appears, in some cases (Leeb-Lundberg and Song, 1991; Gusovsky et al., 1993; Marrero et al., 1994; Piper et al., 1994), to be concomitant with increased tyrosine phosphorylation of PLC-γ (Gusovsky et al., 1993; Marrero et al., 1994). Data from the literature also provide evidence for the existence of a PTK-linked signal transduction pathway in the regulation of smooth muscle contraction induced by agonists acting through G protein-linked receptors (Saifedine et al., 1992; Di Salvo et al., 1994; Hollenberg, 1994; Gould et al., 1995).
The aim of this study was to evaluate the potential role of a tyrosine phosphorylation pathway in the regulation of both phosphoinositide metabolism and contractility of the rat myometrium induced by activation of G protein-coupled receptors. The results demonstrate that the production of inositol phosphates and the attendant tension elicited by bombesin, ET-1, and carbachol were partially attenuated by the PTK inhibitors genistein and active tyrphostins. The PTK-dependent production of inositol phosphates could not be ascribed to an enhanced tyrosine phosphorylation of PLC-γ1, which appeared to be quite modest. Instead, our data suggest that PTK activities stimulate voltage-operated Ca2+ channels involved in the Ca2+-associated production of inositol phosphates, triggered by activated G protein-coupled receptors.
Experimental Procedures
Materials.
myo-[2-3H]Inositol (10–20 Ci/mmol) was purchased from Amersham International (Amersham, Bucks, U.K.). Carbamoylcholine chloride (carbachol), oxytocin, bombesin, β-estradiol 3-benzoate, tyrphostins, nifedipine, and atropine were from Sigma Chemical Co. (St. Louis, MO). Genistein and (±)-Bay K 8644 were from ICN Biomedicals France (Orsay, France). [d-Phe6]Bombesin-(6–13) methyl ester was a generous gift from Dr. David H. Coy (Peptide Research Laboratories, Tulane University School of Medicine, New Orleans, LA). Endothelin-1 (ET-1) and BQ-123 were from Neosystem (Strasbourg, France). Fura-2/AM was from Molecular Probes (Interchim, Montluçon, France). Monoclonal mouse anti-phosphotyrosine (clone 4G10), monoclonal mouse anti-PLC-γ1, and polyclonal rabbit anti-PLC-γ1 antibodies were from Upstate Biotechnology Incorporated (Lake Placid, NY). Horseradish peroxidase-conjugated goat antibody to mouse IgG was from Bio-Rad S.A. (Ivry Sur Seine, France). NitroBind nitrocellulose, 0.45-μm pore size, was from Micron Separations Inc. (Westborough, MA). Western blot enhanced chemiluminescence reagent, the Renaissance product line, was obtained from Dupont NEN (Les Ulis, France). Other chemicals were of the highest grade commercially available.
Animals and Tissue Processing.
Immature female rats (Wistar, 4 weeks old) were treated with 30 μg of estradiol for 2 days and used on the next day. Animals were sacrificed by decapitation, their uteri were removed immediately, and the myometrium was prepared free of endometrium as previously described (Marc et al., 1988; Amiot et al., 1993).
Measurement of [3H]Inositol Phosphates.
Myometrial strips (about 25 mg) were allowed to equilibrate at 37°C for 25 min in 5 ml of Krebs-Ringer-bicarbonate buffer (pH 7.4) containing (117 mM NaCl, 4.7 mM KCl, 1.1 mM MgSO4, 1.2 mM KH2PO4, 24.7 mM NaHCO3, 0.8 mM CaCl2, and 1 mM glucose; gas phase O2/CO2, 19:1) under constant agitation. Tissues were then incubated with 5 μCi ofmyo-[2-3H]inositol (0.4 μM) in 0.8 ml of fresh buffer for 4 h, by which time the incorporation of3H into inositol lipids has reached a plateau (Amiot et al., 1993). Myometrial strips were washed 3 times with nonradioactive Krebs’ buffer and transferred into 1 ml of fresh buffer and incubated for 20 min before the addition of 10 mM LiCl. After 10 min, the agents to be tested were added at the indicated concentration, and incubation was further continued for the time indicated for the specific experiment. Reactions were stopped by immersing the tissue strips in 1.5 ml of cold 7% (w/v) trichloroacetic acid, followed by homogenization and centrifugation at 3000g for 15 min at 4°C. The trichloroacetic acid-soluble supernatants were extracted with diethyl ether, neutralized with Tris base, and applied to a column of the anion exchange resin (AG 1-X8; formate form; 200–400 mesh) for the separation of the individual inositol phosphates as described previously (Marc et al., 1988; Amiot et al., 1993). Alternatively, total inositol phosphates [i.e., inositol trisphosphate (InsP3) plus inositol bisphosphate (InsP2) plus inositol monophosphate (InsP1)] were eluted together in a single step with 12 ml of 1 M ammonium formate plus 0.1 M formic acid. The3H content of the fractions was determined by scintillation counting in Quicksafe A (Zinsser analytic). Results were expressed as cpm/100 mg of tissue or, alternatively, as a percentage of stimulation over the basal values obtained before the addition of the stimulatory agonist.
Methods for Recording Uterine Contractile Responses.
The contractile activity of isolated myometrial strips was measured with an isometric transducing device. The segments were loaded at a basal tension of 0.2 to 0.3g and were bathed at 37°C in 10 ml of Krebs’ buffer (95% O2/5% CO2) of the same salt composition as used for the above incubations. Contractile activity was integrated during a 2-min exposure to the indicated agent (Marc et al., 1988; Amiot et al., 1993;Palmier et al., 1996).
Fura-2/AM Loading and Ca2+ Imaging in Isolated Uterine Myocytes.
The enzymatic dispersion procedure for isolating single myometrial cells from estrogen-treated rats was performed as described previously (Amédée et al., 1986; Dokhac et al., 1996). Uterine myocytes (8 × 105 cells/ml) suspended in minimal essential medium with Earle’s balanced salts containing 10% (w/v) FCS were plated on collagen-coated glass coverslips and were incubated at 37°C in a humidified atmosphere of 5% CO2/95% air for 20 to 24 h. Fura-2/AM loading and Ca2+ imaging of cells were carried out essentially as detailed elsewhere (Sauvadet et al., 1996). Briefly, cells attached to collagen were loaded for 20 min at 25°C with 2 μM Fura-2/AM in balanced salt solution (130 mM NaCl, 5.0 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 10 mM glucose and 50 mM HEPES, pH 7.4) containing 1 mg/ml BSA. Cells were then rinsed twice with the balanced salt solution and allowed to incubate in the same buffer for 15 min at 25°C to facilitate hydrolysis of intracellular Fura-2/AM. For Ca2+imaging, light from a 100-W xenon lamp was filtered alternately through 360- and 380-nm filters to determine the fluorescence ratio (F360/F380). Fura-2 fluorescence (Nikon UV-fluor ×40 objective) was filtered at 510 nm and recorded by an intensified CCD Photonic Science camera (Sauvadet et al., 1996). Each fluorescence image was the average of two images to improve the signal-to-noise ratio, and one average image was recorded every 3 s. Data are reported as the fluorescence ratio (F360/F380) after subtraction of the respective backgrounds. Tracings of fluorescence ratio are representative of at least six cells and were performed on two different cell isolations.
Immunoblotting and Immunoprecipitation.
Myometrial strips (about 50 mg wet weight) were allowed to equilibrate for 20 min at 37°C in 5 ml of Krebs-Ringer-bicarbonate buffer, pH 7.4 (gas phase O2/CO2, 19:1) under constant agitation. Tissue strips were then transferred into 1 ml of fresh buffer and further allowed to equilibrate for 10 min. The agents to be tested were added at the indicated concentration, and incubation was continued for the time indicated for the specific experiment. Reactions were stopped by immersion of the myometrial strips in liquid nitrogen. Frozen tissues were extracted in 600 μl of cold solubilization buffer (1% Triton X-100, 10% glycerol, 150 mM NaCl, 100 mM NaF, 10 mM Na4P2O7, 200 mM Na3VO4, 10 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, and 10 μg/ml leupeptin in 50 mM HEPES, pH 7.4), with an Ultra Turrax homogenizer as described previously (Palmier et al., 1996), with minor modifications. After 30 min at 4°C, the lysates were clarified by centrifugation (10,000g, 20 min at 4°C), and the protein content of the supernatant was determined (Lowry et al., 1951). In some experiments, detergent-extracted proteins (50 μg) were treated with Laemmli’s sample buffer (Laemmli, 1970) and resolved by SDS-polyacrylamide gel electrophoresis (7.5% w/v acrylamide) The separated proteins were transferred to a nitrocellulose membrane for immunoblotting.
For immunoprecipitation experiments, detergent-extracted proteins (500 μg) were incubated with 5 μl of anti-PLC-γ1 rabbit polyclonal antibody overnight at 4°C and then with protein A-Sepharose (20 mg) for 2 h at 4°C. Immune complexes were collected by centrifugation at 10,000g for 60 s and washed five times with cold solubilization buffer and then once in cold PBS. Immunoprecipitated proteins were dissolved in 20 μl of 5% SDS and 25 mM dithiothreitol, heated for 10 min at 95°C, treated with Laemmli’s sample buffer, and subjected to 7.5% SDS-polyacrylamide gel electrophoresis. The separated proteins were then transferred to nitrocellulose for immunoblotting.
The nitrocellulose membranes were blocked for 1.5 h at 37°C with 3% BSA in TTBS (Tris-buffered saline: 20 mM Tris·HCl, pH 7.5, 500 mM NaCl, containing 0.1% Tween 20). The sheets were then washed with TTBS. The blocked nitrocellulose sheets were blotted with the monoclonal anti-phosphotyrosine antibody (1:7500, 0.13 μg protein/ml) overnight at 4°C, followed by three washes with TTBS. The immunoreactive bands were visualized by enhanced chemiluminescence system after sequential incubation with horseradish peroxidase-conjugated goat anti-mouse IgG for 60 min at room temperature. In certain experiments, the blots were reprobed after stripping in 62.5 mM Tris·HCl, pH 6.7, 2% SDS, and 100 mM β-mercaptoethanol for 60 min at 57°C. Nitrocellulose sheets were rinsed in TBS and then reblocked with 3% BSA in TTBS and reprobed with monoclonal anti-PLC-γ1 antibody (0.5 μg/ml). Quantification of the developed blots was performed using a densitometer (Molecular Dynamics).
Data Analysis.
The results are expressed as mean ± S.E.M. and were analyzed statistically using Student’s ttest. P ≤ .05 was considered to be significant.
Results
Effect of Protein Tyrosine Kinase Inhibitors Genistein and Tyrphostins on Accumulation of Inositol Phosphates Triggered by Bombesin.
Results in Table 1 show that the production of [3H]inositol phosphates, triggered by bombesin (Amiot et al., 1993), was attenuated (35–40%) in the presence of genistein, a PTK inhibitor (Akiyama et al., 1987). In experiments not reported (n = 3), it was found that genistein did not affect the potency of bombesin (EC50 values for bombesin were 8.8 ± 0.9 and 9 ± 1 nM in control and genistein-treated tissues, respectively). Data in Fig. 1A show the inhibitory curve to increasing concentrations of genistein against bombesin-mediated inositol phosphate generation. Inhibition by genistein was dose dependent (IC50 = 5 μM; maximal 40% inhibition at 50 μM). Pretreatment of the myometrium with 50 μM genistein resulted in an attenuation in the accumulation for each of the three inositol phosphates (values for InsP3, InsP2, and InsP1 were 10,000 ± 950, 98,000 ± 9000, and 130,000 ± 12,000 cpm/100 mg tissue without genistein and 6160 ± 620, 59,000 ± 5000, and 78,000 ± 6000 cpm/100 mg tissue in the presence of genistein, respectively), indicating that the inhibitory effect of genistein was operating at the level of PLC degrading PIP2. Data in Fig. 1B illustrate the effects of tyrphostins (Tyr), a second class of PTK inhibitors (Levitzki, 1992). Inclusion of the active Tyr25 resulted in a dose-dependent inhibition of the production of inositol phosphates mediated by bombesin (IC50 = 52 ± 6 μM), with a maximal (38%) inhibition at 100 μM, similar to that elicited by genistein. Inhibition was similarly observed with Tyr47, another active tyrphostin, but not with the inactiveTyr63.
Effects of Genistein on Oxytocin-, Carbachol-, ET-1-, and Fluoroaluminate-Mediated Inositol Phosphate Accumulation.
Genistein inhibited not only the production of inositol phosphates triggered by bombesin (Table 1) but also that caused by oxytocin, carbachol, and ET-1, which activate the PLC pathway in the myometrium via their respective G protein-coupled receptors (Marc et al., 1988;Leiber et al., 1990; Amiot et al., 1993; Dokhac et al., 1994). The attenuation by 50 μM genistein of the generation of inositol phosphates for oxytocin, carbachol, and ET-1 averaged 32%, 37%, and 39%, respectively. Genistein (50 μM) was similarly able to inhibit by 44% the inositol phosphate response elicited by AlF4 −, a direct activator of G proteins.
Effects of Genistein on Myometrial Contractions Triggered by Bombesin and Carbachol.
Treatment of myometrial strips with 20 μM genistein caused a rightward shift in the bombesin dose-response curve (Fig. 2), with an EC50 value of 2 ± 0.3 and 7 ± 0.6 nM in the absence and presence of genistein, respectively. Maximal tension was achieved by increasing bombesin concentration, consistent with previous observations (Marc et al., 1988; Leiber et al., 1990; Amiot et al., 1993) that a suboptimal generation of InsP3is sufficient to cause maximal contraction. Data in Fig.3A show the inhibitory curve to increasing concentration of genistein against carbachol-elicited contraction. The muscarinic agonist was used at 6 μM, a concentration that triggers almost maximal contractile activity but no more than 30% of the inositol phosphate response (Marc et al., 1988; Leiber et al., 1990). Genistein antagonized carbachol-elicited contraction at concentrations between 3 and 100 μM (IC50 = 20 μM and 80% maximal inhibition at 80 μM). We further tested whether by attenuating PTP activities (Palmier et al., 1996), pervanadate potentiated the action of the contractile agonist, which appeared to be regulated via a PTK-dependent process. This proved to be the case. A concentration of pervanadate (0.8 μM), which by itself caused no appreciable contractile response (10 ± 1%), markedly potentiated contraction triggered by a suboptimal concentration (1 μM) of carbachol. The muscarinic contractile effect averaged 15 ± 2% and 50 ± 4% in the absence and presence of pervanadate, respectively (Fig. 3B). As expected (Palmier et al., 1996), prior treatment with genistein decreased the magnitude of contraction elicited by each agent added alone and similarly abrogated the potentiated tension effect detected by the combined addition of pervanadate and carbachol. Both the stimulation and the potentiation of tension caused by pervanadate (Fig. 3B) were not abolished by genistein if the latter was added after pervanadate. This is consistent with a rapid tyrosine phosphorylation process that could not be reversed when PTP activity was eliminated by pervanadate (Pumiglia et al., 1992;Palmier et al., 1996).
Effects of Bombesin on Tyrosine Phosphorylation of Proteins in Rat Myometrium.
Myometrial strips were stimulated with bombesin, and the pattern of protein tyrosine phosphorylation in detergent extracts was analyzed by Western blotting with anti-phosphotyrosine antibodies. Results from a typical kinetic experiment (Fig.4A) show that bombesin caused a rapid increase in the tyrosine phosphorylation of proteins, particularly noticeable in the 70- to 80-kDa and the 120- to 130-kDa ranges. Phosphorylation peaked as early as 30 s, with a mean 20-fold (n = 3) increase in tyrosine phosphorylation. Phosphorylation gradually returned to almost basal levels within 1 min of bombesin exposure. Pervanadate at 3 μM caused a barely detectable increase in tyrosine phosphorylation (Fig. 4B). However, when bombesin was added with pervanadate, tyrosine phosphorylation peaked at 30 s (not shown) but persisted for at least 10 min (mean 30-fold increase in tyrosine phosphorylation, n = 3). Maximal phosphorylation levels after a 30-s stimulation with bombesin alone averaged 66% those obtained with bombesin and pervanadate. Data in Fig. 4B further show that pretreatment of myometrial strips with [d-Phe6]bombesin-(6–13) methyl ester, a specific antagonist of GRP-preferring bombesin receptors (Amiot et al., 1993), abolished protein tyrosine phosphorylation triggered by bombesin.
Effects of Fluoroaluminates and Pertussis Toxin on Protein Tyrosine Phosphorylation.
As shown in Fig. 5, the increase in tyrosine phosphorylation triggered by bombesin was totally insensitive to pertussis toxin, similar to the inability of the toxin to affect bombesin-mediated PLC activation (Amiot et al., 1993). The contribution of a G protein to the protein tyrosine phosphorylation process is illustrated in Fig. 5. AlF4 − was able to induce enhanced protein tyrosine phosphorylation, which was potentiated by 3 μM pervanadate. The profile of tyrosine phosphorylated proteins was strikingly similar to that observed with agonists acting through seven transmembrane receptors.
Effects of Bombesin and ET-1 on Tyrosine Phosphorylation of PLC-γ1 in Rat Myometrium.
To investigate whether PLC-γ1 was among the proteins that undergo tyrosine phosphorylation, myometrial strips were incubated in the presence of 100 nM bombesin added alone or in combination with 3 μM pervanadate. Equal amounts of protein from detergent-extracted myometrium were immunoprecipitated with anti-PLC-γ1, and Western blots of the precipitated proteins were probed with both the anti-phosphotyrosine and anti-PLC-γ1 antibodies. Immunoblot analysis with anti-PLC-γ1 antibodies (Fig.6, B and D) showed that similar amounts of PLC-γ1 were present in the control and in the differentially stimulated preparations. Bombesin alone induced a transient increase in tyrosine phosphorylation of PLC-γ1 that peaked (less than 4-fold) at 30 s and then declined and reached basal levels after 1 min (Fig.6A). In additional experiments, it was found that the increase in tyrosine-phosphorylated PLC-γ1 was similar in bombesin-treated myometrial strips, either unloaded or loaded under the conditions used for recording tension. A 3.6- and 2-fold stimulation of tyrosine-phosphorylated PLC-γ1 was obtained for loaded strips versus 3.2- and 1.2-fold stimulation for unloaded strips exposed to bombesin for 30 s and 1 min, respectively. Notably, the phosphotyrosine content of PLC-γ1 was augmented 5-fold when myometrial strips were stimulated for 30 s with bombesin in the presence of 3 μM concentration of the PTP inhibitor pervanadate and was maintained for at least 10 min (Fig. 6C). The phosphorylation of PLC-γ1 was strongly reduced by the two protein tyrosine kinase inhibitors genistein at 50 μM and Tyr47 at 100 μM (not shown). Similar results were obtained for myometrial strips stimulated by ET-1 (100 nM), in which a 6- to 7-fold increase in tyrosine phosphorylation of PLC-γ1 was observed.
Data in Fig. 7 attempt to further correlate the level of tyrosine-phosphorylated PLC-γ1 with the extent of inositol phosphate production mediated by pervanadate, bombesin, and ET-1. There seems to be a close correlation between the gradual increase in tyrosine-phosphorylated PLC-γ1 with increasing pervanadate concentration (3, 10, and 25 μM) and the corresponding ability of the protein tyrosine phosphatase inhibitor to gradually increase the production of inositol phosphates. For both bombesin- and ET-1-stimulated myometrium, the production of inositol phosphates which was sensitive to genistein was associated with a rather modest increase in the amount of tyrosine-phosphorylated PLC-γ1. Thus, for a PTK-dependent increase in inositol phosphate production higher to that achieved with 25 μM pervanadate, a small (5- to 6-fold) augmentation of tyrosine-phosphorylated PLC-γ1 was obtained with both bombesin and ET-1 versus a 56-fold increase in the level of tyrosine-phosphorylated PLC-γ1 associated with the PTP inhibitor. The data suggested that factors other than PLC-γ1 are probably involved in the genistein-sensitive production of inositol phosphates triggered by G protein-coupled receptors.
Dihydropyridine-Sensitive Ca2+ Channels as Targets for Inhibitory Effect of Genistein and Tyr47.
Data in Fig.8 show that omission of Ca2+ from the incubation medium resulted in a decrease (35%) in the amount of inositol phosphates generated by bombesin. The addition of nifedipine 1 min before bombesin caused a similar reduction in the agonist-mediated inositol phosphate response. The degree of inhibition caused by Ca2+withdrawal or the addition of nifedipine was similar (40%) to that elicited by the tyrosine kinase inhibitors genistein and Tyr47 at their maximum effective concentrations. Both genistein and Tyr47 could no longer attenuate the inositol phosphate response when incubations were carried out in a Ca2+-poor medium. Similarly, the simultaneous addition of both nifedipine and genistein or of nifedipine and Tyr47 gave inhibition no greater than that obtained with either agent alone. In addition, the inhibitory effects of genistein and Tyr47 on the increase in the generation of inositol phosphates due to bombesin were prevented by Bay K 8644, similar to the antagonistic effect exerted by the Ca2+ channel agonist on the nifedipine-induced inositol phosphate response. These data imply that the ability of the PTK inhibitors to attenuate the generation of inositol phosphates resulted from an inhibition of bombesin-mediated Ca2+ influx via nifedipine-sensitive Ca2+ channels. Similar findings were obtained with ET-1 (data not shown).
The importance of a PTK-regulated, voltage-gated Ca2+-entry process in the contractile effect of bombesin is illustrated in Table 2. Inhibition caused by 20 μM genistein (Fig. 2 and Table 2) was comparable to that of 10 nM nifedipine. Contractions triggered by 0.5 and 2.5 nM bombesin were attenuated in the presence of nifedipine by 76 ± 8% and 57 ± 6%, respectively, and in the presence of genistein by 90 ± 9% and 64 ± 6%, respectively. Of interest, inhibitions by both nifedipine and genistein were abrogated by the Ca2+ channel agonist Bay K 8644.
As illustrated in Fig. 9A, the application of bombesin (100 nM) caused a transient intracellular Ca2+ concentration ([Ca2+]i) peak followed by a lower but sustained increase in [Ca2+]i (“plateau phase”). When genistein (20 μM) was applied 5 min before bombesin, the peptide evoked a smaller and briefer [Ca2+]i response (Fig.9B). The pattern of genistein inhibition was very similar to [Ca2+]i attenuations that were previously observed with other contractile agonists in Ca2+-free medium (Arnaudeau et al., 1994; Dokhac et al., 1996). The data are consistent with the inhibition of Ca2+ influx by the PTK inhibitor.
Discussion
In the present study, we demonstrated in estradiol-treated rat myometrium that the stimulation of the PIP2-PLC pathway by activated G protein-coupled receptors, as assessed by the increased production of InsP (InsP3, InsP2, and InsP1) and the attendant increase in muscle tension (Marc et al., 1988; Leiber et al., 1990; Amiot et al., 1993; Dokhac et al., 1994), were in part regulated by a tyrosine phosphorylation-dependent process. The production of inositol phosphates stimulated by bombesin was decreased in a dose-dependent manner by two sets of PTK inhibitors, genistein and active tyrphostins, with a maximal inhibition of 35% to 40%. Genistein similarly reduced inositol phosphate generation triggered by oxytocin, carbachol, and ET-1, acting via their respective receptors (Marc et al., 1988; Leiber et al., 1990; Dokhac et al., 1994), as well as by AlF4 −, a direct G protein activator (Marc et al., 1988). This is consistent with a PTK regulatory process operating downstream from receptor activation. A potential PTK-linked signal transduction pathway for the regulation of smooth muscle contraction has been suggested (Hollenberg, 1994) and was demonstrated to operate for uterine contraction triggered by pervanadate, a potent PTP inhibitor (Palmier et al., 1996). The contractile action of G protein-coupled receptors such as angiotensin, histamine, and α-adrenergic receptors in various smooth muscle systems is also inhibited by PTK inhibitors (Di Salvo, 1994;Hollenberg, 1994; Gould et al., 1995). Our data are in line with these observations because they illustrate the opposing effects (i.e., inhibitory and stimulatory) for genistein and pervanadate, respectively, on agonist-mediated myometrial contraction.
An increase in cellular PTK activities triggered by bombesin was demonstrated by the ability of the peptide to cause a transient increase in the tyrosine phosphorylation of several proteins in the 70- to 80-kDa and 120- to 130-kDa range. The potentiated increase in tyrosine phosphorylation observed if pervanadate was also present is consistent with a potential role for PTPs (Fischer et al., 1991) in controlling phosphotyrosine levels in the myometrium. Pharmacological evidence was further provided that both PLC activation and enhanced protein tyrosine phosphorylation were triggered by the same subclass of bombesin receptors. Our finding that AlF4 − gave a similar pattern of enhanced tyrosine protein phosphorylation revealed the involvement of heterotrimeric G proteins. The tyrosine phosphorylation of proteins triggered by bombesin was insensitive to pertussis toxin. The toxin also has no effect on PLC activation, indicating that the putative G protein that is involved in both receptor-mediated effects is, at least in part, represented by Gq/G11 (Lajat et al., 1996).
It is well known that PLC-γ isoforms are activated by phosphorylation on specific tyrosine residues (Carpenter et al., 1993; van der Geer and Hunter, 1994). PLC-γ has been identified as a potential substrate for several receptor tyrosine kinases, as well as for nonreceptor tyrosine kinases (Liao et al., 1993; van der Geer and Hunter, 1994; Marrero et al., 1995). It has also been shown that the increase in PIP2 hydrolysis triggered by G protein-coupled receptors such as the M5 muscarinic (Gusovsky et al., 1993) or the angiotensin AT1 receptor (Marrero et al., 1994,1995) is in part regulated via a PTK pathway that appears to be concomitant with phosphorylation of tyrosines in PLC-γ1. We recently identified myometrium PLC-γ1 as one of the proteins that is tyrosine phosphorylated on stimulation with pervanadate in association with both increased generation of inositol phosphates and enhancement of muscle tension (Palmier et al., 1996). In this study, we provide further evidence for a close correlation between the tyrosine phosphorylation status of PLC-γ1 and the level of inositol phosphate production triggered by different concentrations of pervanadate, supporting the idea that the two events may be causally related. Stimulation by bombesin and ET-1, particularly if combined with a low concentration of pervanadate, led to an increase in the phosphotyrosine content of PLC-γ1. However, compared with phosphorylation obtained with high doses of pervanadate, a very low level of tyrosine-phosphorylated PLC-γ1 was associated with activation of G protein-coupled receptors and accounted at best for no more than 5% to 10% of the PTK-dependent production of inositol phosphates. Our data strongly suggest that in the rat myometrium, factors other than phosphorylated PLC-γ1 are involved in the PTK-dependent production of inositol phosphates mediated by G protein-coupled receptors.
Previous results from our laboratory (Dokhac et al., 1992, 1996) showed that at least two distinct mechanisms underlie the activation of PLC in myometrium by carbachol and oxytocin. One mechanism concerns the well recognized agonist-induced activation of the receptor-Gq protein-PLCβ3 cascade (Lajat et al., 1996), which appears to be insensitive to increases in intracellular Ca2+. A second, Ca2+-dependent pathway involves the stimulation of PLC activity via an increase in Ca2+ influx, resulting from the activation of voltage-gated Ca2+ channels. Both the Ca2+-dependent and -independent processes are involved in the rapid breakdown of PIP2 with the concomitant production of active Ins(1,4,5)P3(Dokhac et al., 1992). This study extends these observations to bombesin and ET-1 and provides evidence for PTK interference in the Ca2+ entry-dependent process involved in PLC activation. The increased generation of inositol phosphates and the uterine contractions triggered by activated bombesin receptors were attenuated to the same extent by either genistein or nifedipine, and both inhibitory effects were abolished by the Ca2+ channel agonist Bay K 8644 (Triggle and Rampe, 1989). Our findings that genistein decreased both the peak and sustained [Ca2+]itriggered by bombesin are consistent with a number of observations reporting the inhibitory effects of genistein on agonist-mediated Ca2+ mobilization (Di Salvo et al., 1994; Gould et al., 1995; Liu and Sturek, 1996). The possibility of a direct blocking action of genistein on Ca2+ channels cannot be excluded (Kusaka and Sperelakis, 1995), but the similarity, noted here, between the effects of genistein and another, structurally unrelated PTK inhibitor (Levitzki, 1992), Tyr47 (and Tyr25), provides evidence for a tyrosine phoshorylation-dependent effect. Collectively, the data support the contention that voltage-sensitive Ca2+ channel activity is the predominant target for the PTK-dependent regulatory process that contributes to agonist-mediated inositol phosphate production and contraction and that there is no major role for PTK regulation at a step distal to the channel. Our finding that phosphorylated PLC-γ1 palys a minor role in the agonist-mediated production of inositol phosphates is consistent with a recent report by Di Salvo and Nelson (1998) demonstrating in vascular smooth muscle cells that the tyrosine phosphorylation of PLC-γ1 is not required for PTK-dependent increases in intracellular calcium concentration triggered by the stimulation of diverse G protein-linked receptors.
PTK activity has been suggested to control Ca2+entry induced by G protein-coupled receptors in numerous cells (Gusovsky et al., 1993; Liu and Sturek, 1996). Evidence has also been provided that PTKs exert a stimulatory modulation of L-type Ca2+ channels in pituitary GH3 cells (Cataldi et al., 1996) and in various smooth muscle cell preparations (Wijetunge and Hughes, 1995; Hatakeyama et al., 1996), including pregnant rat myometrial cells (Kusaka and Sperelakis, 1995). It is unclear whether the tyrosine residues of the channel itself become phosphorylated or whether some intermediate messenger regulates the activity of the channel.
In summary, the data presented here are consistent with two cascades of events for GRP-preferring bombesin receptors: 1) bombesin receptor stimulation→activation of Gq/G11→stimulation of PLC-β3→stimulation of inositol phosphate production and 2) bombesin receptor stimulation→activation of Gq/G11→stimulation of PTK or PTKs→opening of voltage-gated Ca2+ channels with an increase in the influx of Ca2+→stimulation of PIP2-PLC activity. Three isoforms of PIP2-PLC (PLC-β3, PLC-γ1, and PLCδ) have been identified in rat myometrium (Ku et al., 1995; Lajat et al., 1996;Palmier et al., 1996). The PLC isoform that is regulated via an increase in Ca2+ influx remains to be identified. Cascades 1 and 2 account for 65% and 35% of bombesin-mediated inositol phosphate production, respectively. Both cascades appear to operate for two other G protein-coupled receptors: endothelin and muscarinic receptors. Although it is well accepted that tyrosine phosphorylation events are induced by G protein activation (Lev et al., 1995; Malarkey et al., 1995; Post and Heller Brown, 1996), worth mentioning are two recent studies (Liu et al., 1996; Umemori et al., 1997) that have shown that the tyrosine phosphorylation of Gqα/G11α subunits by protein tyrosine kinases may increase their ability to convey agonist-mediated activation of PLC. The possibility for a tyrosine phosphorylation step operating at the level of Gqα/G11α and its potential contribution to the PTK-dependent regulation have not been addressed in this report and would be worth considering. It will also be interesting to identify the PTK or PTKs that trigger the observed protein tyrosine phosphorylation and activation processes in the myometrium and to determine their mechanism of activation by G protein-coupled receptors. These and other concerns are the subject of our current studies.
Acknowledgments
We are grateful to Dr. Françoise Pecker for extremely helpful advice and assistance with the Ca2+measurement studies and to Dr. Philippe Jourdon for critical discussions. We also thank Gisèle Thomas for scanning analysis and help with the figures.
Footnotes
-
Send reprint requests to: Dr. Denis Leiber, Laboratoire de Signalisation et Régulations Cellulaires, CNRS, EP 1088, Bâtiment 430, Université Paris-Sud, 91405 Orsay Cedex, France.
-
↵1 This work was supported by grants from the Centre National de la Recherche Scientifique (ERS 0570 and EP1088) and by a contribution from the Association de la Recherche contre le Cancer (1355).
- Abbreviations:
- ET-1
- endothelin-1
- InsP3
- inositol trisphosphate
- InsP2
- inositol bisphosphate
- InsP1
- inositol monophosphate
- PIP2
- phosphatidylinositol-4,5-bisphosphate
- PTK
- protein tyrosine kinase
- PTP
- protein tyrosine phosphatase
- PLC
- phospholipase C
- Tyr
- tyrphostin
- Fura-2/AM
- acetoxy methyl ester (AM) form of fura-2
- Received May 14, 1998.
- Accepted January 13, 1999.
- The American Society for Pharmacology and Experimental Therapeutics