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The Role of Mitogen-Activated Protein Kinase in Oxytocin-Induced Contraction of Uterine Smooth Muscle in Pregnant Rat
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
229, 938–944 (1996)
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The Role of Mitogen-Activated Protein Kinase in Oxytocin-Induced Contraction of Uterine Smooth Muscle in Pregnant Rat Ataru Nohara, Masahide Ohmichi,1 Koji Koike, Nobuyuki Masumoto, Mamoru Kobayashi,* Masuo Akahane,* Hiromasa Ikegami, Kenji Hirota,† Akira Miyake, and Yuji Murata Department of Obstetrics and Gynecology, Osaka University Medical School, 2-2, Yamadaoka, Suita, Osaka 564, Japan; *Kissei Pharmaceutical Co. Ltd., 4365-1, Hotaka, Minami-azumi, Nagano 399-83, Japan; and †Department of Obstetrics and Gynecology, Nissei Hospital, 6-3-8, Itachibori, Nishi-ku, Osaka 550, Japan Received November 13, 1996 Oxytocin causes the rapid tyrosine phosphorylation of mitogen-activated protein (MAP) kinase in both human and rat puerperal uterine myometrial cultured cells. The potential role of the MAP kinase pathway in oxytocin action was investigated with the specific MAP kinase kinase (MEK) inhibitor, PD98059. Oxytocin stimulation of the tyrosine phosphorylation of MAP kinase in both human and rat cultured puerperal uterine cells was abolished by pretreatment of the cells with MEK inhibitor in a dose-dependent manner. Although MEK inhibitor had no effect on oxytocin-induced intracellular Ca2/ mobilization in either pregnant human or pregnant rat uterine cells, it partly inhibited oxytocin-induced pregnant rat uterine contraction in a dose-dependent manner. These results suggest that MAP kinase pathway may have some important roles in oxytocin-induced uterine contraction. q 1996 Academic Press
Diverse extracellular stimuli, including growth factors, hormones, osmolar shock, stress and elevated temperature, result in stimulation of the activity of a number of serine/threonine kinases, including the mitogen-activated protein (MAP) kinase (1-8). The activation of this kinase is catalysed by MAP kinase kinase or MEK, which phosphorylates MAP kinase on threonine and tyrosine residues (9,10). MEK is itself phosphorylated and activated by an upstream kinase or kinases that appear to require activation of the ras protooncogene (11,12). One of these MEK kinases may be the product of the raf protooncogene (13-15), although other MEK kinases have been identified (16-19). While we clarified recently that oxytocin stimulates MAP kinase activity in cultured human puerperal uterine myometrial cells (20), the precise role of the MAP kinase pathway in the biological action of oxytocin remains unclear. A recently identified specific inhibitor of MEK is thought to an invaluable tool that will aid in elucidating the role of the MAP kinase cascade in a variety of biological settings (21-23). In this report, we describe the use of this selective inhibitor of specific components of MAP kinase cascade to determine the physiological role of this pathway in oxytocin-induced uterine contraction. MATERIALS AND METHODS
Materials Anti-MAP kinase and anti-phosphotyrosine antisera were obtained from Upstate Biotechnology (Lake Placid, NY). RPMI1640 medium was obtained from GIBCO (Grand Island, NY). Collagenase CLS3 was obtained from Worthington
1 Address all correspondence and requests for reprints to Dr. Masahide Ohmichi, Osaka university Medical School, 2-2, Yamadaoka, Suita, Osaka 564, Japan. Fax: 81-6-879-3359. E-mail: [email protected]. Abbreviations used: MAPK, mitogen-activated protein kinase; MEK, mitogen-activated kinase or extracellularactivated kinase kinase; GST, glutathione S-transferase; SDS, sodium dodecylsulfate; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; MBP, myelin basic protein.
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Biochemical Corporation (Freehold, NJ). MEK inhibitor, PD98059, was a gift from the Department of Physiology, University of Michigan School of Medicine (Ann Arbor, MI). ECL Western blotting detection reagents were obtained from Amersham (Buckinghamshire, England).
Methods Preparation of puerperal uterine myometrial cells. Rats at 21 days of pregnancy were stunned and bled in the morning. The uterus was removed and the fetuses were gently expelled. In other experiments, human uterine myometrial tissues were obtained from women who had undergone selective cesarean delivery after informed consent was granted for their use. Cells were prepared by the modified method of Palmberg and Thygerg (24). The tissues were cut into 1-2 mm3 fragments and digested with 0.1% trypsin for 1h at 377 C in calcium-magnesium (Ca-Mg) free Hanks’ solution. The tissues were then digested with 0.1% collagenase and 0.1% deoxyribonuclease for 30 min at 377 C in Ca-Mg free Hanks’ solution. Cell aggregates were isolated by gentle pipetting. Nondispersed fragments were separated by filtration through gauze cloth. The cells were maintained at 377 C in an atmosphere of 95% air and 5% CO2 in RPMI1640 medium containing 10% fetal bovine serum supplemented with penicillin (200 U/ml) and streptomycin (200 mg/ml). They were used for experiments after 5 days. Assay of protein tyrosine phosphorylation of MAP kinase by immunoblots. For analysis of tyrosine phosphorylation of MAP kinase, cells were grown in 100-mm dishes. After hormone treatment, the cells were washed once with icecold PBS, and then 100 ml 1%SDS was added. Lysates were heated for 5 min at 1007 C and diluted to 1:10 with HNTG buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl2 , 1 mM EDTA, 10 mM sodium pyrophosphate, 100 mM sodium orthovanadate, 100 mM NaF, 10 mg/ml aprotinin, 10 mg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride) (25), followed by incubation with anti-MAP kinase antiserum. Immune complexes were precipitated with protein A-Sepharose, and isolated proteins were analyzed by electrophoresis on 8% SDS-PAGE. Proteins were transferred to a nitrocellulose membrane and immunoblotted with antiphosphotyrosine antiserum, as described previously (26). Measurement of contraction of pregnant rat uterine tissue. Rats at 21 days of pregnancy were stunned and bled in the morning. The uterus was removed and the fetuses were gently expelled. A uterine muscle strip (15 mm long, 5 mm wide) was longitudinally dissected and suspended vertically in a 10-ml chamber containing modified LockeRinger solution (the composition of which was as follows: NaCl 154 mM; NaHCO3 4.8 mM; KCl 5.4 mM; CaCl2 0.36 mM; MgCl2 0.19 mM; KH2PO4 0.15 mM and glucose 3.1 mM) gassed with 95% O2/5% CO2 and maintained at 267C to suppress the spontaneous contractions. The contractions was measured isometrically using a mechanoelectric transducer (NEC San-ei, 45196A, Tokyo, Japan) which in turn passed it to a potentiometric pen-recorder (NEC San-ei, 8K-23, Tokyo, Japan). The initial tension was set at about 1.0 g. In the absence of spontaneous contractions, 10-mU/ml of oxytocin was added to the chamber and the effect of MEK inhibitor on uterine contractions was evaluated. Uterine activity was calculated as the sum of the amplitudes of each contraction during 30 minutes, and the percent changes before and after the drug application were compared. Measurement of intracellular Ca2/ concentration ([Ca2/]i). [Ca2/]i was monitored with a digital imaging fluorescence microscope using a Ca2/-sensitive fluorescent dye, fura-2AM, as described previously (27, 28). Briefly, uterine cells were incubated at 377 C for 60 min in medium 199 containing 5 mmol/L fura-2AM. The cells were then rinsed with Hanks 1 Balanced Salt Solution and placed on the microscope stage. [Ca2/]i was measured at 100-ms intervals with an M-500 digital imaging microscopic system (Scholar Tech Corp., Osaka, Japan). [Ca2/]i was determined as the ratio of the intensities of fluorescent emission at 510 nm with excitation at 340 and 380 nm. The excitation beam was targeted on the cells, and emission images were recorded on video film and analyzed with a computer. The effects of oxytocin with or without MEK inhibitor, PD98059, on the [Ca2/]i in 10 cells were determined.
RESULTS
Oxytocin Stimulation of Tyrosine Phosphorylation of MAP Kinase Cultured pregnant rat uterine myometrial cells obtained at day 21 (before the onset of labor) were treated with the indicated concentrations of oxytocin or with 10 nM epidermal growth factor (EGF) for 5 min (Fig.1A, lines 1-5). The cell lysates were immunoprecipitated with antiMAP kinase antiserum, followed by SDS-PAGE and immunoblotting with antiphosphotyrosine antiserum. Oxytocin caused a dose-dependent increase in tyrosine phosphorylation of MAP kinase. For evaluation of the effect of MEK inhibitor PD98059, cells were preincubated with 100 mM MEK inhibitor for 10 min prior to addition of the indicated concentrations of oxytocin or EGF for 5 min (Fig.1A, lines 6-10). Following the treatment, lysates were assayed for tyrosine phosphorylation of MAP kinase. Pretreatment of cells with 100 mM MEK inhibitor alone was without effect, but this compound completely inhibited both oxytocin- and EGF939
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FIG. 1. Effect of MEK inhibitor on oxytocin-induced tyrosine phosphorylation of MAP kinase. Rat (A,B) or human (C) puerperal uterine myometrial cells were grown in 100-mm dishes. (A) Cells were treated for 10 minutes with (lines 6-10) or without (lines 1-5) 100 mM of MEK inhibitor, followed by incubation with the indicated concentrations of oxytocin (lines 2-4, 7-9) or 10 nM of EGF (lines 5 and 10). (B) Cells were treated for 10 min with the indicated concentrations of MEK inhibitor (lines 2-4), followed by incubation with 1 mM of oxytocin for 5 min (lines 1-4). (C) Cells were treated for 10 min with 100 mM of MEK inhibitor, followed by incubation with 1 mM of oxytocin for 5 min. Cells were lysed in 100 ml 1% SDS, and the lysates were heated for 5 minutes in 1007C water, followed by immunoprecipitation with anti-MAP kinase antiserum. The immunoprecipitated MAP kinase was then subjected to SDS-PAGE, followed by immunoblotting with antiphosphotyrosine antiserum, as described in Material and Methods.
induced tyrosine phosphorylation of MAP kinase. The dose dependence of this inhibitory response to MEK inhibitor was also evaluated (Fig.1B). MEK inhibitor inhibited the oxytocininduced tyrosine phosphorylation of MAP kinase dose-dependently. Moreover, the effect of MEK inhibitor on oxytocin-induced tyrosine phosphorylation of MAP kinase in cultured human puerperal myometrial cells was examined (Fig.1C). MEK inhibitor also completely inhibited the oxytocin-induced tyrosine phosphorylation of MAP kinase. Effect of MEK Inhibitor on Oxytocin-Induced Contraction of Pregnant Rat Uterine Smooth Muscle For evaluation of the specific role of the MAP kinase pathway in oxytocin-induced pregnant rat uterine contraction, uterine smooth muscle strips were stretched to optimal length, and active force was measured after treatment with oxytocin followed by incubation with the indicated dose of MEK inhibitor. Whereas 1% DMSO (which is the final DMSO concentration of each solution of MEK inhibitor) alone had no effect on oxytocin-induced uterine contraction, 100 mM MEK inhibitor immediately inhibited the oxytocin-induced uterine contraction (Fig.2A). Figure 2B shows the dose-response relationship for MEK inhibitor and oxytocin-induced uterine contraction: 100 mM MEK inhibitor partly inhibited oxytocin-induced uterine contraction. It was reported that oxytocin elevates the intracellular Ca2/ concentration in puerperal uterine myometrial cells (27). To examine why MEK inhibitor does not inhibit oxytocin-induced uterine contraction completely, the effect of MEK inhibitor on oxytocin-induced Ca2/ mobilization was evaluated in rat (Fig.3A) and human (Fig.3B) puerperal uterine myometrial cells by a digital imaging fluorescence microscope. The intracellular Ca2/ concentration was elevated by oxytocin showing a peak after 30 s, and then returned to nearly the basal level after 200 s in both rat and human puerperal uterine myometrial cells. However, pretreatment of 100 mM MEK inhibitor had no effect on the oxytocin-induced change of intracellular Ca2/ concentration. 940
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FIG. 2. Effect of MEK inhibitor on oxytocin-induced contraction of isolated pregnant rat uterus. (A) The isolated uterine tissues were suspended at 267 C in modified Locke-Ringer solution under aeration with 95% O2 and 5% CO2 and weighted with 1 gram. The activity of the uterus was measured with a pressure transducer (45196A, NEC Sanei, Japan) and a rectigram (8K, NEC San-ei, Japan). The tissues were added of 1% DMSO or 100 mM MEK inhibitor before treatment with oxytocin for 30 minutes. (B) The dose-dependency of MEK inhibitor on oxytocin-induced uterine activity of isolated pregnant rat uterus was determined with Magnus Method (nÅ5).
DISCUSSION
Primary regulation of contraction in smooth muscle involves phosphorylation of the myosin light chain by Ca2//calmodulin-dependent myosin light chain kinase (29,30). However, oxyto941
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FIG. 3. Effect of MEK inhibitor on oxytocin-induced [Ca2/]i mobilization. The [Ca2/]i change induced by 10 nM of oxytocin in rat (A) or human (B) puerperal uterine myometrial cells was measured with a digital imaging fluorescence microscope before (1st change) and after (2nd change) the treatment with 100 mM MEK inhibitor for 30 min.
cin-induced Ca2/-free contraction of the uterus is not accompanied by phosphorylation of myosin light chains (31). Therefore, it has been speculated that there is involvement of Ca2/independent protein kinase in the Ca2/-free contraction (32). Indeed, it was reported that phosphorylation of cytoskeletal elements is consistent with sustained contraction in bovine tracheal smooth muscle (33,34). Therefore, it was hypothetized that phosphorylation of cytoskeletal or related materials is the cause of Ca2/-free contraction. MAP kinase is Ca2/independent protein kinase, and the substrate of MAP kinase is microtubule-associated protein2 which is one of the cytoskeletal proteins (35). Recently, we clarified that oxytocin stimulates MAP kinase activity in cultured human puerperal uterine myometrial cells (20). In this study, a specific inhibitor of MEK PD98059 partly inhibited oxytocin-induced uterine contraction. PD98059 had no effect on Ca2/ mobilization induced by oxytocin. These data lead us to speculate that the mechanism of oxytocin-induced uterine contraction involves both the MAP kinase pathway and Ca2/ mobilization. Ritodrine hydrochloride (ritodrine) is known to relax uterine contraction. The mechanism of which is thought to increase intracellular cAMP through activation of adrenergic b2-receptors (36, 37). The agents which increase intracellular cAMP also inhibit uterine contraction (38). Moreover, it has been reported that the relaxation of smooth muscle caused by b-stimulants is correlated with intracellular Ca2/ changes through cyclic AMP systems (39). Scheid et al. (40) suggested that adrenergic b-stimulants induced cyclic AMP production, triggering the activation of the Na// Ca2/ pump and enhanced Ca2/ intake into intracellular storage. However, the minimum concentration of these drugs required to produce muscular relaxation was lower than that required to increase in the amount of cyclic AMP. It appears that b-adrenoceptor stimulants do not act only through the cyclic AMP system. Therefore, the reason that elevation of intracellular cAMP inhibits uterine contraction is still unknown. Recently, it was reported that cAMP, possibly acting through protein kinase A, blocks transmission of signals from Ras to Raf-1 and thereby prevents activation of MAP kinase (41-43). Indeed, we reported that 1 mM ritodrine completely attenuated oxytocininduced MAP kinase activity (20). These data provide additional support for the concept that the MAP kinase pathway is essential in smooth muscle contraction. 942
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In conclusion, the experiments presented in the present paper suggest that MAP kinase pathway has some role in oxytocin-induced pregnant rat uterine muscle contraction. ACKNOWLEDGMENT We thank Dr. Alan R. Saltiel for the gift of the MEK inhibitor PD98059.
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41. Wu-J Pdent Jelinek, T., Wolfman, A., Weber, M. J., and Sturgill, T. W. (1993) Science 262, 1065–1069. 42. Sevetson, B. R., Kong, X., and Lawrence, J. C., Jr. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 10305–10309. 43. Hordijk, P. L., Verioan, I., Jalink, K., Corven, E. J., and Moolenarr, W. H. (1994) J. Biol. Chem. 269, 3532– 3538.
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The Role of Mitogen-Activated Protein Kinase in Oxytocin-Induced Contraction of Uterine Smooth Muscle in Pregnant Rat Ataru Nohara, Masahide Ohmichi,1 Koji Koike, Nobuyuki Masumoto, Mamoru Kobayashi,* Masuo Akahane,* Hiromasa Ikegami, Kenji Hirota,† Akira Miyake, and Yuji Murata Department of Obstetrics and Gynecology, Osaka University Medical School, 2-2, Yamadaoka, Suita, Osaka 564, Japan; *Kissei Pharmaceutical Co. Ltd., 4365-1, Hotaka, Minami-azumi, Nagano 399-83, Japan; and †Department of Obstetrics and Gynecology, Nissei Hospital, 6-3-8, Itachibori, Nishi-ku, Osaka 550, Japan Received November 13, 1996 Oxytocin causes the rapid tyrosine phosphorylation of mitogen-activated protein (MAP) kinase in both human and rat puerperal uterine myometrial cultured cells. The potential role of the MAP kinase pathway in oxytocin action was investigated with the specific MAP kinase kinase (MEK) inhibitor, PD98059. Oxytocin stimulation of the tyrosine phosphorylation of MAP kinase in both human and rat cultured puerperal uterine cells was abolished by pretreatment of the cells with MEK inhibitor in a dose-dependent manner. Although MEK inhibitor had no effect on oxytocin-induced intracellular Ca2/ mobilization in either pregnant human or pregnant rat uterine cells, it partly inhibited oxytocin-induced pregnant rat uterine contraction in a dose-dependent manner. These results suggest that MAP kinase pathway may have some important roles in oxytocin-induced uterine contraction. q 1996 Academic Press
Diverse extracellular stimuli, including growth factors, hormones, osmolar shock, stress and elevated temperature, result in stimulation of the activity of a number of serine/threonine kinases, including the mitogen-activated protein (MAP) kinase (1-8). The activation of this kinase is catalysed by MAP kinase kinase or MEK, which phosphorylates MAP kinase on threonine and tyrosine residues (9,10). MEK is itself phosphorylated and activated by an upstream kinase or kinases that appear to require activation of the ras protooncogene (11,12). One of these MEK kinases may be the product of the raf protooncogene (13-15), although other MEK kinases have been identified (16-19). While we clarified recently that oxytocin stimulates MAP kinase activity in cultured human puerperal uterine myometrial cells (20), the precise role of the MAP kinase pathway in the biological action of oxytocin remains unclear. A recently identified specific inhibitor of MEK is thought to an invaluable tool that will aid in elucidating the role of the MAP kinase cascade in a variety of biological settings (21-23). In this report, we describe the use of this selective inhibitor of specific components of MAP kinase cascade to determine the physiological role of this pathway in oxytocin-induced uterine contraction. MATERIALS AND METHODS
Materials Anti-MAP kinase and anti-phosphotyrosine antisera were obtained from Upstate Biotechnology (Lake Placid, NY). RPMI1640 medium was obtained from GIBCO (Grand Island, NY). Collagenase CLS3 was obtained from Worthington
1 Address all correspondence and requests for reprints to Dr. Masahide Ohmichi, Osaka university Medical School, 2-2, Yamadaoka, Suita, Osaka 564, Japan. Fax: 81-6-879-3359. E-mail: [email protected]. Abbreviations used: MAPK, mitogen-activated protein kinase; MEK, mitogen-activated kinase or extracellularactivated kinase kinase; GST, glutathione S-transferase; SDS, sodium dodecylsulfate; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; MBP, myelin basic protein.
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Biochemical Corporation (Freehold, NJ). MEK inhibitor, PD98059, was a gift from the Department of Physiology, University of Michigan School of Medicine (Ann Arbor, MI). ECL Western blotting detection reagents were obtained from Amersham (Buckinghamshire, England).
Methods Preparation of puerperal uterine myometrial cells. Rats at 21 days of pregnancy were stunned and bled in the morning. The uterus was removed and the fetuses were gently expelled. In other experiments, human uterine myometrial tissues were obtained from women who had undergone selective cesarean delivery after informed consent was granted for their use. Cells were prepared by the modified method of Palmberg and Thygerg (24). The tissues were cut into 1-2 mm3 fragments and digested with 0.1% trypsin for 1h at 377 C in calcium-magnesium (Ca-Mg) free Hanks’ solution. The tissues were then digested with 0.1% collagenase and 0.1% deoxyribonuclease for 30 min at 377 C in Ca-Mg free Hanks’ solution. Cell aggregates were isolated by gentle pipetting. Nondispersed fragments were separated by filtration through gauze cloth. The cells were maintained at 377 C in an atmosphere of 95% air and 5% CO2 in RPMI1640 medium containing 10% fetal bovine serum supplemented with penicillin (200 U/ml) and streptomycin (200 mg/ml). They were used for experiments after 5 days. Assay of protein tyrosine phosphorylation of MAP kinase by immunoblots. For analysis of tyrosine phosphorylation of MAP kinase, cells were grown in 100-mm dishes. After hormone treatment, the cells were washed once with icecold PBS, and then 100 ml 1%SDS was added. Lysates were heated for 5 min at 1007 C and diluted to 1:10 with HNTG buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl2 , 1 mM EDTA, 10 mM sodium pyrophosphate, 100 mM sodium orthovanadate, 100 mM NaF, 10 mg/ml aprotinin, 10 mg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride) (25), followed by incubation with anti-MAP kinase antiserum. Immune complexes were precipitated with protein A-Sepharose, and isolated proteins were analyzed by electrophoresis on 8% SDS-PAGE. Proteins were transferred to a nitrocellulose membrane and immunoblotted with antiphosphotyrosine antiserum, as described previously (26). Measurement of contraction of pregnant rat uterine tissue. Rats at 21 days of pregnancy were stunned and bled in the morning. The uterus was removed and the fetuses were gently expelled. A uterine muscle strip (15 mm long, 5 mm wide) was longitudinally dissected and suspended vertically in a 10-ml chamber containing modified LockeRinger solution (the composition of which was as follows: NaCl 154 mM; NaHCO3 4.8 mM; KCl 5.4 mM; CaCl2 0.36 mM; MgCl2 0.19 mM; KH2PO4 0.15 mM and glucose 3.1 mM) gassed with 95% O2/5% CO2 and maintained at 267C to suppress the spontaneous contractions. The contractions was measured isometrically using a mechanoelectric transducer (NEC San-ei, 45196A, Tokyo, Japan) which in turn passed it to a potentiometric pen-recorder (NEC San-ei, 8K-23, Tokyo, Japan). The initial tension was set at about 1.0 g. In the absence of spontaneous contractions, 10-mU/ml of oxytocin was added to the chamber and the effect of MEK inhibitor on uterine contractions was evaluated. Uterine activity was calculated as the sum of the amplitudes of each contraction during 30 minutes, and the percent changes before and after the drug application were compared. Measurement of intracellular Ca2/ concentration ([Ca2/]i). [Ca2/]i was monitored with a digital imaging fluorescence microscope using a Ca2/-sensitive fluorescent dye, fura-2AM, as described previously (27, 28). Briefly, uterine cells were incubated at 377 C for 60 min in medium 199 containing 5 mmol/L fura-2AM. The cells were then rinsed with Hanks 1 Balanced Salt Solution and placed on the microscope stage. [Ca2/]i was measured at 100-ms intervals with an M-500 digital imaging microscopic system (Scholar Tech Corp., Osaka, Japan). [Ca2/]i was determined as the ratio of the intensities of fluorescent emission at 510 nm with excitation at 340 and 380 nm. The excitation beam was targeted on the cells, and emission images were recorded on video film and analyzed with a computer. The effects of oxytocin with or without MEK inhibitor, PD98059, on the [Ca2/]i in 10 cells were determined.
RESULTS
Oxytocin Stimulation of Tyrosine Phosphorylation of MAP Kinase Cultured pregnant rat uterine myometrial cells obtained at day 21 (before the onset of labor) were treated with the indicated concentrations of oxytocin or with 10 nM epidermal growth factor (EGF) for 5 min (Fig.1A, lines 1-5). The cell lysates were immunoprecipitated with antiMAP kinase antiserum, followed by SDS-PAGE and immunoblotting with antiphosphotyrosine antiserum. Oxytocin caused a dose-dependent increase in tyrosine phosphorylation of MAP kinase. For evaluation of the effect of MEK inhibitor PD98059, cells were preincubated with 100 mM MEK inhibitor for 10 min prior to addition of the indicated concentrations of oxytocin or EGF for 5 min (Fig.1A, lines 6-10). Following the treatment, lysates were assayed for tyrosine phosphorylation of MAP kinase. Pretreatment of cells with 100 mM MEK inhibitor alone was without effect, but this compound completely inhibited both oxytocin- and EGF939
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FIG. 1. Effect of MEK inhibitor on oxytocin-induced tyrosine phosphorylation of MAP kinase. Rat (A,B) or human (C) puerperal uterine myometrial cells were grown in 100-mm dishes. (A) Cells were treated for 10 minutes with (lines 6-10) or without (lines 1-5) 100 mM of MEK inhibitor, followed by incubation with the indicated concentrations of oxytocin (lines 2-4, 7-9) or 10 nM of EGF (lines 5 and 10). (B) Cells were treated for 10 min with the indicated concentrations of MEK inhibitor (lines 2-4), followed by incubation with 1 mM of oxytocin for 5 min (lines 1-4). (C) Cells were treated for 10 min with 100 mM of MEK inhibitor, followed by incubation with 1 mM of oxytocin for 5 min. Cells were lysed in 100 ml 1% SDS, and the lysates were heated for 5 minutes in 1007C water, followed by immunoprecipitation with anti-MAP kinase antiserum. The immunoprecipitated MAP kinase was then subjected to SDS-PAGE, followed by immunoblotting with antiphosphotyrosine antiserum, as described in Material and Methods.
induced tyrosine phosphorylation of MAP kinase. The dose dependence of this inhibitory response to MEK inhibitor was also evaluated (Fig.1B). MEK inhibitor inhibited the oxytocininduced tyrosine phosphorylation of MAP kinase dose-dependently. Moreover, the effect of MEK inhibitor on oxytocin-induced tyrosine phosphorylation of MAP kinase in cultured human puerperal myometrial cells was examined (Fig.1C). MEK inhibitor also completely inhibited the oxytocin-induced tyrosine phosphorylation of MAP kinase. Effect of MEK Inhibitor on Oxytocin-Induced Contraction of Pregnant Rat Uterine Smooth Muscle For evaluation of the specific role of the MAP kinase pathway in oxytocin-induced pregnant rat uterine contraction, uterine smooth muscle strips were stretched to optimal length, and active force was measured after treatment with oxytocin followed by incubation with the indicated dose of MEK inhibitor. Whereas 1% DMSO (which is the final DMSO concentration of each solution of MEK inhibitor) alone had no effect on oxytocin-induced uterine contraction, 100 mM MEK inhibitor immediately inhibited the oxytocin-induced uterine contraction (Fig.2A). Figure 2B shows the dose-response relationship for MEK inhibitor and oxytocin-induced uterine contraction: 100 mM MEK inhibitor partly inhibited oxytocin-induced uterine contraction. It was reported that oxytocin elevates the intracellular Ca2/ concentration in puerperal uterine myometrial cells (27). To examine why MEK inhibitor does not inhibit oxytocin-induced uterine contraction completely, the effect of MEK inhibitor on oxytocin-induced Ca2/ mobilization was evaluated in rat (Fig.3A) and human (Fig.3B) puerperal uterine myometrial cells by a digital imaging fluorescence microscope. The intracellular Ca2/ concentration was elevated by oxytocin showing a peak after 30 s, and then returned to nearly the basal level after 200 s in both rat and human puerperal uterine myometrial cells. However, pretreatment of 100 mM MEK inhibitor had no effect on the oxytocin-induced change of intracellular Ca2/ concentration. 940
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FIG. 2. Effect of MEK inhibitor on oxytocin-induced contraction of isolated pregnant rat uterus. (A) The isolated uterine tissues were suspended at 267 C in modified Locke-Ringer solution under aeration with 95% O2 and 5% CO2 and weighted with 1 gram. The activity of the uterus was measured with a pressure transducer (45196A, NEC Sanei, Japan) and a rectigram (8K, NEC San-ei, Japan). The tissues were added of 1% DMSO or 100 mM MEK inhibitor before treatment with oxytocin for 30 minutes. (B) The dose-dependency of MEK inhibitor on oxytocin-induced uterine activity of isolated pregnant rat uterus was determined with Magnus Method (nÅ5).
DISCUSSION
Primary regulation of contraction in smooth muscle involves phosphorylation of the myosin light chain by Ca2//calmodulin-dependent myosin light chain kinase (29,30). However, oxyto941
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FIG. 3. Effect of MEK inhibitor on oxytocin-induced [Ca2/]i mobilization. The [Ca2/]i change induced by 10 nM of oxytocin in rat (A) or human (B) puerperal uterine myometrial cells was measured with a digital imaging fluorescence microscope before (1st change) and after (2nd change) the treatment with 100 mM MEK inhibitor for 30 min.
cin-induced Ca2/-free contraction of the uterus is not accompanied by phosphorylation of myosin light chains (31). Therefore, it has been speculated that there is involvement of Ca2/independent protein kinase in the Ca2/-free contraction (32). Indeed, it was reported that phosphorylation of cytoskeletal elements is consistent with sustained contraction in bovine tracheal smooth muscle (33,34). Therefore, it was hypothetized that phosphorylation of cytoskeletal or related materials is the cause of Ca2/-free contraction. MAP kinase is Ca2/independent protein kinase, and the substrate of MAP kinase is microtubule-associated protein2 which is one of the cytoskeletal proteins (35). Recently, we clarified that oxytocin stimulates MAP kinase activity in cultured human puerperal uterine myometrial cells (20). In this study, a specific inhibitor of MEK PD98059 partly inhibited oxytocin-induced uterine contraction. PD98059 had no effect on Ca2/ mobilization induced by oxytocin. These data lead us to speculate that the mechanism of oxytocin-induced uterine contraction involves both the MAP kinase pathway and Ca2/ mobilization. Ritodrine hydrochloride (ritodrine) is known to relax uterine contraction. The mechanism of which is thought to increase intracellular cAMP through activation of adrenergic b2-receptors (36, 37). The agents which increase intracellular cAMP also inhibit uterine contraction (38). Moreover, it has been reported that the relaxation of smooth muscle caused by b-stimulants is correlated with intracellular Ca2/ changes through cyclic AMP systems (39). Scheid et al. (40) suggested that adrenergic b-stimulants induced cyclic AMP production, triggering the activation of the Na// Ca2/ pump and enhanced Ca2/ intake into intracellular storage. However, the minimum concentration of these drugs required to produce muscular relaxation was lower than that required to increase in the amount of cyclic AMP. It appears that b-adrenoceptor stimulants do not act only through the cyclic AMP system. Therefore, the reason that elevation of intracellular cAMP inhibits uterine contraction is still unknown. Recently, it was reported that cAMP, possibly acting through protein kinase A, blocks transmission of signals from Ras to Raf-1 and thereby prevents activation of MAP kinase (41-43). Indeed, we reported that 1 mM ritodrine completely attenuated oxytocininduced MAP kinase activity (20). These data provide additional support for the concept that the MAP kinase pathway is essential in smooth muscle contraction. 942
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In conclusion, the experiments presented in the present paper suggest that MAP kinase pathway has some role in oxytocin-induced pregnant rat uterine muscle contraction. ACKNOWLEDGMENT We thank Dr. Alan R. Saltiel for the gift of the MEK inhibitor PD98059.
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