Introduction

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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, PIP₂ breakdown 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 G_q 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-G_q protein-PLC3 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 PIP₂ hydrolysis, through tyrosine phosphorylation and the activation of PLC- 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 PIP₂ was demonstrated to be due to the pervanadate-mediated PLC-1 phosphorylation on tyrosine residues. PLC- 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- (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-1, 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

Top Abstract Introduction Experimental Procedures Results Discussion References

Materials. myo-[2-³H]Inositol (10-20 Ci/mmol) was purchased from Amersham International (Amersham, Bucks, U.K.). Carbamoylcholine chloride (carbachol), oxytocin, bombesin, -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-1, and polyclonal rabbit anti-PLC-1 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 of myo-[2-³H]inositol (0.4 µM) in 0.8 ml of fresh buffer for 4 h, by which time the incorporation of ³H 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. The ³H 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.

Data Analysis. The results are expressed as mean ± S.E.M. and were analyzed statistically using Student's t test. P  .05 was considered to be significant.

	Results

Top Abstract Introduction Experimental Procedures Results Discussion References

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.

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Dose-dependent inhibitory effects of genistein and tyrphostins on bombesin-mediated inositol phosphate accumulation. [3H]Inositol-labeled myometrial strips were incubated for 10 min in the absence or presence (A) of genistein and (B) of tyrphostins Tyr25 (), Tyr47 (), and Tyr63 () at the indicated concentrations and then were stimulated for 10 min with 50 nM bombesin. Total inositol phosphates were eluted from AG1-X8 columns in a single step. Results are expressed as a percentage of the response to bombesin alone (278,673 ± 23,409 cpm/100 mg of tissue = 100%). Values are mean ± S.E.M. for four independent experiments, each performed in duplicate. *P < .05,**P < .01, NS not significantly different from bombesin alone.

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 InsP₃ is 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).

View larger version (25K): [in this window] [in a new window]   Fig. 2.   Inhibitory effects of genistein on myometrial contraction triggered by bombesin. Cumulative dose-response curves for bombesin-mediated contractions were obtained before () and after () exposure to 20 µM genistein for 5 min. Contractions were recorded during a 2-min exposure of loaded myometrial segments to the indicated concentrations of agonist. The degree of contraction is expressed as a percentage of the response to a maximally effective concentration of bombesin alone (100%). Values are mean ± S.E.M. for three separate experiments.

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Opposing effects of genistein and pervanadate on carbachol-mediated myometrial contraction. A, contractions were induced by an almost maximal concentration (6 µM) of carbachol, and increasing concentrations of genistein were added at 2-min intervals. , Carbachol-mediated contraction in the presence of 0.11% dimethyl sulfoxide, which is the concentration of the vehicle used at the maximal dose of genistein. The degree of contraction is expressed as a percentage of the response due to carbachol alone (100%). B, contractile activity displayed by 1 µM carbachol (CB), 0.8 µM pervanadate (PV), or 1 µM carbachol plus 0.8 µM pervanadate without (open bars) or with (dotted bars) a 5-min prior treatment with 20 µM genistein. Where indicated (hatched bars), myometrial strips were exposed for 2 min to pervanadate (PV) before incubation with 20 µM genistein for 5 min, followed by the addition of carbachol or buffer. The degree of the contractile response is expressed as a percentage of the response to a maximally effective concentration (25 µM) of carbachol (=100%). Values are mean ± S.E.M. for three independent experiments.

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.

View larger version (31K): [in this window] [in a new window]   Fig. 4.   Effect of bombesin on protein tyrosine phosphorylation in rat myometrium. A, myometrial strips were treated with 100 nM bombesin (Bn) for the times indicated (lanes a-h). B, tissues were incubated for 20 min in the absence or presence of 3 µM pervanadate (PV), without or with the addition of 100 nM bombesin (Bn), for the times indicated (lanes a-e). When used, the specific antagonist [D-phe6]bombesin-(6-13) methyl ester (1 µM) was added 10 min before the agonist. Phosphorylated proteins in detergent extracts were detected by immunoblotting with anti-phosphotyrosine antibodies. The positions of molecular mass markers (×103) are shown. Bottom, densitometric quantification of tyrosine-phosphorylated proteins in the 120- to 130-kDa range (hatched bars) and in the 70- to 80-kDa range (open bars), determined with a densitometer (Molecular Dynamics). Absorbance is expressed in arbitrary units relative to control (= 1). Data represent one of four similar experiments.

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.

View larger version (33K): [in this window] [in a new window]   Fig. 5.   Effects of AlF4 and pertussis toxin on protein tyrosine phosphorylation in rat myometrium. Left, myometrial strips were incubated in the absence or presence of 3 µM pervanadate (PV) for 10 min and then were treated for an additional 10 min without or with 20 mM NaF plus 10 µM AlCl3. Right, tissues were incubated for 6 h in the absence or presence of 400 ng/ml pertussis toxin (PTX). Tissues were then washed with fresh buffer. Rechallenge incubations were conducted with 3 µM PV plus 100 nM bombesin (Bn) as described in the legend to Fig. 4. In both cases, phosphorylated proteins in detergent extracts were detected by immunoblotting with anti-phosphotyrosine antibodies. The antibodies were used alone or in combination with 0.5 mM phosphotyrosine (pY). Positions of molecular mass markers (×103) are shown Data represent one of three similar experiments.

Effects of Bombesin and ET-1 on Tyrosine Phosphorylation of PLC-1 in Rat Myometrium. To investigate whether PLC-1 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-1, and Western blots of the precipitated proteins were probed with both the anti-phosphotyrosine and anti-PLC-1 antibodies. Immunoblot analysis with anti-PLC-1 antibodies (Fig. 6, B and D) showed that similar amounts of PLC-1 were present in the control and in the differentially stimulated preparations. Bombesin alone induced a transient increase in tyrosine phosphorylation of PLC-1 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-1 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-1 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-1 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-1 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-1 was observed.

View larger version (22K): [in this window] [in a new window]   Fig. 6.   Effects of bombesin on tyrosine phosphorylation of PLC-1 in rat myometrium. Left, myometrial strips were treated with 100 nM bombesin (Bn) for the times indicated. Right, tissues were incubated for 20 min in the presence of 3 µM pervanadate (PV), without or with the addition of 100 nM bombesin (Bn), for the times indicated. Equal amounts of detergent-extracted proteins (500 µg) were immunoprecipitated with the anti-PLC-1 polyclonal antibody. A and C, immunoblotting was performed using anti-phosphotyrosine antibodies. B and D, blot was stripped and reprobed with an anti-PLC-1 monoclonal antibody. Positions of molecular mass markers (×103) are shown. Densitometric scanning of phosphorylated PLC-1 was performed using a densitometer from Molecular Dynamics. Numbers in parentheses represent absorbance, expressed in arbitrary units relative to the control (= 1). Data illustrate one of three similar experiments.

View larger version (21K): [in this window] [in a new window]   Fig. 7.   Correlation between the level of tyrosine-phosphorylated PLC-1 and the extent of PTK-dependent inositol phosphate production in the myometrium. For the determination of total inositol phosphates, 3H-prelabeled myometrial strips were treated for 30 s without or with pervanadate at the concentrations indicated, in the absence or presence of 100 nM bombesin (Bn) or 200 nM ET-1, each added alone or in combination with 3 µM pervanadate. Genistein (50 µM), when used, was added 10 min before the agonist. Genistein-sensitive inositol phosphate production is the increase from basal values in [3H]inositol phosphates in the absence of genistein minus inositol phosphate generation in the presence of genistein. Data are expressed as cpm/100 mg of tissue. **P < .01 compared with basal levels of inositol phosphates. For the tyrosine phosphorylation of PLC-1, unlabeled myometrial strips were treated as above. Detergent-extracted proteins were immunoprecipitated with anti-PLC-1 polyclonal antibodies. Precipitated proteins were analyzed by immunoblotting with anti-phosphotyrosine antibodies. Densitometric quantification of tyrosine phosphorylated PLC-1 was performed using a densitometer from Molecular Dynamics. Absorbance is expressed in arbitrary units relative to the control (= 1). Values are mean ± S.E.M. for three independent experiments. **P < .01 and *P < .05 versus basal levels of tyrosine-phosphorylated PLC-1.

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).

View larger version (44K): [in this window] [in a new window]   Fig. 8.   Effects of extracellular calcium, nifedipine, and Bay K 8 644 on the inhibition of inositol phosphate production triggered by genistein and Tyr47. [3H]Inositol-prelabeled myometrial strips were transferred to fresh (0.8 mM Ca2+) Krebs-bicarbonate buffer or to Ca2+-depleted medium. Tissues were allowed to equilibrate for 5 min and then were incubated for 10 min in the absence or presence of genistein (50 µM) and Tyr47 (100 µM). Incubations were continued for 10 min with 10 nM bombesin. When used, nifedipine (250 nM) was added 4 min before bombesin, whereas Bay K 8644 (5 µM) was added 1 min before nifedipine, genistein, and Tyr47. Total [3H]inositol phosphates were determined as described in Materials and Methods. Values are mean ± S.E.M. for three independent experiments, each done in duplicate. **P < .01 compared with bombesin. a, not significantly different from bombesin plus nifedipine. b, not significantly different from bombesin. c, not significantly different from bombesin in Ca2+-free medium.

View larger version (10K): [in this window] [in a new window]   Fig. 9.   Effect of genistein on the rise of intracellular Ca2+ level induced by bombesin in single cells of rat myometrium. Cells were loaded with Fura-2/AM as described in Materials and Methods. A, cells were perfused with balanced salt solution containing 100 nM bombesin (Bn). B, cells were treated with 20 µM genistein for 5 min and then perfused with balanced salt solution containing 20 µM genistein and 100 nM bombesin (Bn). Tracings are representative of at least 6 cells from two different cell isolations.

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

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 -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 G_q/G₁₁ (Lajat et al., 1996).

It is well known that PLC- isoforms are activated by phosphorylation on specific tyrosine residues (Carpenter et al., 1993; van der Geer and Hunter, 1994). PLC- 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-1. We recently identified myometrium PLC-1 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-1 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-1. However, compared with phosphorylation obtained with high doses of pervanadate, a very low level of tyrosine-phosphorylated PLC-1 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-1 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-G_q protein-PLC3 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⁺]_i triggered 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-1 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-1 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 stimulationactivation of G_q/G₁₁stimulation of PLC-3stimulation of inositol phosphate production and 2) bombesin receptor stimulationactivation of G_q/G₁₁stimulation of PTK or PTKsopening of voltage-gated Ca²⁺ channels with an increase in the influx of Ca²⁺stimulation of PIP₂-PLC activity. Three isoforms of PIP₂-PLC (PLC-3, PLC-1, and PLC) 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 G_q/G₁₁ 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 G_q/G₁₁ 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.