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REPRODUCTION-DEVELOPMENT |
Osaka University Graduate School of Medicine, Department of Obstetrics and Gynecology, Suita, Osaka 565-0871, Japan
Address all correspondence and requests for reprints to: Masahiro Tahara, Osaka University Graduate School of Medicine, Department of Obstetrics and Gynecology, 2-2 Yamadaoka, Suita, Osaka, 565-0871, Japan. E-mail: . taharam{at}gyne.med.osaka-u.ac.jp
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Introduction |
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In recent years, there have been remarkable advances in our knowledge of the biochemical mechanisms underlying smooth muscle contractility. It is widely accepted that a rise in the concentration of intracellular free Ca2+ ([Ca2+]i) is the major trigger for smooth muscle contraction (5, 6), and that the degree of myosin light chain (MLC) phosphorylation by the Ca2+/calmodulin-dependent enzyme MLC kinase is the essential factor that determines the extent of smooth muscle contraction (7). MLC phosphorylation results in smooth muscle contraction, whereas MLC dephosphorylation by myosin phosphatase results in muscle relaxation (8). However, studies using Ca2+ indicators to measure [Ca2+]i revealed that the Ca2+ concentration does not always parallel the degree of MLC phosphorylation and contraction (5). The extent of MLC phosphorylation or force of contraction induced by agonist stimulation is higher than that of increase in [Ca2+]i, and this phenomenon is known as "Ca2+ sensitization" (5, 7).
Although the mechanism underlying Ca2+ sensitization has not been fully elucidated, several lines of evidence indicated that the small GTPase Rho, a member of the Rho subfamily of the Ras superfamily of monomeric GTPases, is responsible for the Ca2+ sensitization induced by agonist stimulation (9, 10). The small GTPase Rho is involved in various cellular functions, including stress-fiber and focal-adhesion formation, cell morphology, cell motility, membrane ruffling, and smooth muscle contraction (11). The activated Rho increased the MLC phosphorylation at a constant Ca2+ concentration, which suggested that Rho was involved in the regulatory mechanism of the Ca2+ sensitivity (12). While several proteins have been identified as effectors of Rho, including protein kinase N, peroxidase-coupled antigoat antibody (Rho-kinase), rhophilin, rhotekin, citron, p140 mDia, and citron kinase (13), recent analyses revealed that the signaling pathway that involves Rho-kinase plays a crucial role in the Ca2+ sensitization of smooth muscle contraction by inhibiting the activity of myosin phosphatase (14, 15). Smooth muscle myosin phosphatase consists of a 38-kDa catalytic subunit, the 130-kDa myosin-binding subunit (MBS), and a 21-kDa subunit (16, 17). MBS was originally identified as a substrate of Rho-kinase (18), and myosin phosphatase binds to the phosphorylated MLC via MBS and dephosphorylates MLC. These studies showed that RhoA regulates MLC phosphorylation through its target protein, Rho-kinase, which phosphorylates the MBS of myosin phosphatase and thereby inhibits the activity of this phosphatase (14, 19).
Although the heteromeric G proteins of the myometrium have been reported to change during pregnancy (20, 21), the expression and functional status of the monomeric GTPase in myometrium during pregnancy have not been characterized. Recently the detection of RhoA and Rho-kinase in the pregnant myometrium has been reported (22, 23), which suggests that the RhoA/Rho-kinase system plays a role in uterine smooth muscle contraction. However, the expressions and functions of these proteins in myometrium have not been fully examined at the pharmacological or molecular level.
In the present study, we examined the potential role of the RhoA/Rho-kinase pathway in oxytocin-induced uterine contraction by using a specific Rho-kinase inhibitor, Y-27632. Y-27632 has been developed and reported to inhibit the kinase activity of Rho-kinase specifically in a manner competitive with ATP (24). This inhibitory probe for Rho-kinase is very useful and enables the evaluation of the physiological roles of Rho-kinase in intact smooth muscle. We demonstrated here whether Y-27632 inhibits oxytocin-induced uterine contraction. Furthermore, the involvement of MLC and MBS phosphorylation in the effect of oxytocin and Y-27632 was investigated. We also determined the expression of RhoA and Rho-kinase in the rat uterus during the course of pregnancy compared with the nonpregnant uterus. These results suggest that the RhoA/Rho-kinase pathway may have an important role in the enhancement of contractility.
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Materials and Methods |
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Isometric recordings of myometrial contraction
Experiments were performed on uteri from 8-wk-old Wistar female rats (nonpregnant, 200 g; pregnant, primipara, d 21 of pregnancy). All protocols were in accordance with the National Institutes of Health Guidelines and approved by the Animal Care and Use Committee of Osaka University School of Medicine. Animals were anesthetized with ether and killed by cervical dislocation. Uterine horns were obtained from pregnant (d 21) or nonpregnant rats and were cut into longitudinal strips approximately 1 cm long and 1 mm wide. These strips were mounted for isometric recording under 1 g tension in normal Tyrode’s solution at 37 C, gassed with 95% O2/5% CO2 and equilibrated under these conditions for 1 h. The mechanical responses were measured as integrated tension by means of a force displacement transducer. Uterine strips were exposed to Tyrode’s solution containing oxytocin (0.2 nM) to induce phasic contractions. After 20 min of equilibration, Y-27632 was added in a cumulative manner to the bath at 20-min intervals. Control experiments were run in parallel in which the same amount of the vehicle (distilled water) for Y-27632 was added to the strips at the same intervals as the Y-27632 application. The relaxant effect was assessed by expressing the integrated tension during the 20-min period after the addition of each drug concentration as a percentage of the tension in the 20-min period before drug addition.
Measurement of intracellular Ca2+
Uterine myocytes were prepared by the method of Masumoto and colleagues (27, 28). Longitudinal muscles of pregnant (d 21) rats were cut into several pieces (1 x 1 mm), and incubated in HBSS containing 0.1% collagenase at 37 C for 25 min. Dispersed cells were suspended in DMEM with 10% FBS and plated on poly-L-lysine-coated 35-mm glass-bottomed dishes (MatTek Co., Ashland, MA). Cells were loaded with the membrane-permeant Ca2+ indicator dye fura-2 AM (1 µM), then rinsed and maintained in fura-2-free solution before beginning data acquisition. Cells on coverslips were excited with a mercury lamp through 340- and 380-nm band-pass filters, and emitted fluorescence at 510 nm was detected with a digital imaging fluorescence microscope (Hamamatsu Photonics, Hamamatsu, Japan). After subtracting the background signal, the ratio of 340/380 emissions at 510 nm was used as an indicator of [Ca2+]i. The effects of oxytocin with and without Y-27632 on the [Ca2+]i were determined in ten cells.
Immunoblot analysis for detection of MLC and MBS phosphorylation
The levels of phosphorylation of MLC and MBS were assessed in frozen strips to avoid the influence of cyclical phosphorylation-dephosphorylation reactions accompanying myometrial contractions as follows; uterine horns were obtained from pregnant (d 21) Wistar rats and were cut into longitudinal strips approximately 1 cm long and 1 mm wide. The strips were mounted for isometric recording as described above. The contraction induced by oxytocin (0.2 nM) was then examined, and the specimens were removed from the hook at the time of maximal contraction and immediately frozen by immersion in 90% acetone-10% trichloroacetic acid (TCA) containing 10 mM dithiothreitol (DTT) cooled with dry ice, and then homogenized in 10% TCA containing 10 mM DTT. The homogenate was centrifuged at 15,000 x g at 4 C for 5 min and the supernatant was carefully removed. The pellet was washed with ice-cold acetone containing 10 mM DTT three times, and after the third wash, the remaining acetone was allowed to completely evaporate for 10 min, while the sample was kept on ice. The dried samples were cut into small pieces and mixed with SDS sample buffer, sonicated for 10 sec on ice three times, heated at 100 C for 5 min, and centrifuged at 15,000 x g for 5 min. An aliquot was retained for protein assay using the method of Lowry.
To evaluate the effects of the Rho-kinase inhibitor Y-27632 on phosphorylation of MLC or MBS, the uterine strips were preincubated with Y-27632 (10-5 M) for 10 min and then stimulated without removal of Y-27632. The specimens were removed from the hook and immediately frozen at the same time as the corresponding specimens without Y-27632 were frozen, and treated as described above.
For the detection of the degree of MLC phosphorylation, the extracted samples (20 µg protein in each sample) were separated by SDS-PAGE and blotted onto polyvinylidenedifluoride membranes, and then the membranes were immunologically probed separately with antiphosphorylated MLC antibody (25) and with anti-MLC20 antibody. The signals were detected with ECL reagents, and quantitative densitometric analysis of immunoblots was performed using Fluor Chem IS-8000 (Alpha Innotech Corp., San Leandro, CA). The degree of MLC phosphorylation were calculated by dividing the densitometric values of the phosphorylated MLC signal by those of MLC20 signal. For the detection of the degree of MBS phosphorylation, the membranes were immunologically probed separately with anti-pS854 antibody and anti-MBS antibody. One of the major sites for phosphorylation of MBS by Rho-kinase both in vitro and in vivo has been identified as Ser-854, and the antibody we used specifically recognizes MBS phosphorylated at Ser-854 (26). The degree of MBS phosphorylation was calculated by dividing the densitometric values of pS854 signal by those of MBS signal.
Identification of RhoA/Rho-kinase proteins by immunoblot analysis
For detection of RhoA and Rho-kinase proteins in rat myometrium, samples were prepared as follows: myometrial tissues were obtained from nonpregnant and pregnant (d 12, 18, 21) rats, and were cut into longitudinal strips approximately 1 cm long and 1 mm wide. These tissues were immediately frozen and extracted by TCA/acetone as described above. Samples (20 µg protein in each sample) were separated by SDS-PAGE (7.5 or 12%). Proteins were transferred to polyvinylidenedifluoride membranes and probed with anti-RhoA (26C4) and anti-Rock-2 (C-20) antibodies. Rock-2 (C-20, Santa Cruz Biotechnology, Inc.) is raised against epitope mapping at the carboxy terminus of rat Rock-2 (also designated Rho-kinase). Immunoblots were developed using ECL reagents. The same membranes were re-probed with anti-GAPDH antibody (29) and used as a control to ascertain that equivalent amounts of proteins had been transferred. Quantitative densitometry of immunoblots was performed using Fluor Chem IS-8000 (Alpha Innotech Corp.). For the quantitative analysis, the densitometric values were normalized to GAPDH, and then the ratios of RhoA or Rho-kinase/GADPH were expressed as the fold increases from those of nonpregnant samples. The results from five studies were analyzed.
Identification of RhoA/Rho-kinase mRNA by RT-PCR and Northern blot analysis
Myometrial tissues were obtained from nonpregnant and pregnant (d 12, 18, 21) rats, and were immediately frozen. RNA was extracted from the tissues using TRIzol reagent, and any contaminating genomic DNA was digested by RNase-free DNase. Total RNA (1 µg) was used as the template for the reverse transcriptase (RT) reaction by using SuperScript II following the manufacturer’s instructions. An aliquot (5 µl) of RT product was used for PCR amplification in a total volume of 50 µl. The primers used in RT-PCR for RhoA, Rho-kinase and GADPH were as follows: RhoA sense 5'-ACC AGT TCC CAG AGG TTT ATGT-3', antisense 5'-TTT GGT CTT TGC TGA ACACT-3'; Rho-kinase sense 5'- GCA CAT GTA TGA AAA TGG ATG AAAC-3', antisense 5'- CAT AAT TTT GCT GTA GGT TCC TAC AAGT-3'; GADPH sense 5'-ACC ACA GTC CAT GCC ATC AC-3', antisense 5'- TCC ACC ACC CTG TTG CTG TA-3'. The thermal cycle profile used in this study was (1) denaturing for 30 sec at 94 C (2), annealing primers for 90 sec at 55 C, and (3) extending the primers for 30 sec at 72 C. The sequence of the primer for RT-PCR analysis of RhoA and Rho-kinase used in this study was reported previously by Nishimura et al. (30). PCR amplification for RhoA and Rho-kinase was performed for 30 cycles and that for GAPDH (as an internal control) was performed for 25 cycles. These amplifications were performed in the range of the linear relationship between the cycle number and the intensity of RT-PCR product (data not shown). A portion (10 µl) of the PCR mixture was electrophoresed in a 1.5% agarose gel in TAE buffer (40 mM Tris-acetate, pH 8.5, 2 mM EDTA). The gel was stained with ethidium bromide and then photographed.
For Northern blot analysis, poly(A)+ RNAs were isolated using the PolyA tract mRNA Isolation System (Promega Corp., Madison, WI), and 10 µg of poly(A)+ RNA were electrophoresed on a 1% agarose/formaldehyde gel. After transfer to a Hybond-XL nylon membrane (Amersham Pharmacia Biotech, Arlington Heights, IL) and UV cross-linkage, poly(A)+ RNAs were hybridized at 65 C in UltaHyb solution (Ambion, Inc. Austin, TX). cDNA probes for RhoA and Rho-kinase were kindly provided by Dr. K. Kaibuchi (Nagoya University). A BamHI fragment (900-bp) from pEXV vector containing the full-length RhoA cDNA and a BamHI- and BglII-digested fragment (1.3 kb) from pEXV vector containing full-length Rho-kinase cDNA were labeled with [32P]deoxy-CTP by random-primed synthesis. GAPDH cDNA probe was used as a control to ascertain that equivalent amounts of mRNA had been transferred. Blots were washed under standard high-stringency conditions (final wash: 42 C, 0.1% SSC, 0.1% SDS) and exposed to Fuji Photo Film Co., Ltd. Medical x-ray Film with an intensifying screen at -70 C. The radioactivity of each band was quantified using a BAS2000 imaging system (Fuji Photo Film Co., Ltd. Film, Tokyo, Japan). For the quantitative analysis, the radioactivities of RhoA and Rho-kinase bands were normalized to those for GAPDH, and then the ratios of RhoA or Rho-kinase/GADPH were expressed as the fold increases from those of nonpregnant samples. The results from three studies were analyzed.
Statistical analysis
The results are expressed as means ± SEM. Statistical analysis was performed with unpaired t test in the organ chamber experiment, and with one-way ANOVA and subsequent Scheffé’s post hoc test in the other experiments. P < 0.05 was considered to indicate a statistical difference.
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Results |
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shows a recording of oxytocin-induced myometrial contractility, and the effect of Y-27632. Oxytocin (0.2 nM) increased contractile activity compared with spontaneous activity, and Y-27632 reduced force in all preparations studied. The inhibitory effect of Y-27632 was observed within 5 min, and both of amplitude and frequency of contraction were decreased dose dependently. With 10-4 M Y-27632, contractile activity was essentially abolished. Under control condition in which the same amount of the vehicle (distilled water) for Y-27632 was added to the strips at the same intervals as the Y-27632 application, consistent amplitude and frequency were recorded for several hours (data not shown). After application of the maximum dose of Y-27632 (10-4 M), the contractility was reversed immediately by washing the strips with medium containing oxytocin (Fig. 1A). Figure 1B shows the dose-response relationship of Y-27632 and oxytocin-induced uterine contraction: Y-27632 inhibited phasic contraction induced by oxytocin in a concentration-dependent manner. These results indicate that Rho-kinase was involved in oxytocin-induced uterine contraction. We also examined the effect of Y-27632 in the nonpregnant uterus. The sensitivity to Y-27632 in nonpregnant uterus was significantly decreased by 3 x 10-5 M Y-27632 compared with pregnant uterus, and the concentration-response curve was shifted rightwards (Fig. 1B). The ID50 values for relaxation with Y-27632 in pregnant and nonpregnant uterus were 5.2 ± 0.8 and 723.9 ± 2.8 µM, respectively.
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, the addition of 0.2 µM oxytocin to myometrial (d 21) cells caused a rapid and transient increase, followed by a sustained phase, in the ratio of 340/380 nm fura-2 fluorescences, indicating an increase of [Ca2+]i (first peak). The initial component is thought to be caused by Ca2+ release from internal stores, whereas the sustained phase is thought to be due to Ca2+ influx across the cell membrane (31). Pretreatment of cells with Y-27632 (10-4 M) for 10 min did not affect the oxytocin-induced increase of [Ca2+]i (second peak) in Fig. 2. This was the case for pretreatment with Y-27632 for more than 10 min (data not shown). These results suggest that Y-27632 inhibited oxytocin-induced uterine contraction without a reduction of [Ca2+]i. As a negative control, identical cells were superfused in Ca2+-free, 0.5 mM EGTA- containing solution before activation by oxytocin. As shown in the third peak, the initial Ca2+ transient was still present with about 80% magnitude, whereas the sustained Ca2+ rise was absent. This is coincidence with the previous report by Arnaudeau et al. (32).
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, the phosphorylation of MLC was increased by oxytocin (0.2 nM), without any change in the level of immunoreactive MLC. Y-27632 (10-5 M) reduced the extent of MLC phosphorylation induced by oxytocin. Quantitative analysis showed that oxytocin induced significant increase of MLC phosphorylation, which was reduced significantly by Y-27632 (Fig. 3B). As Y-27632 did not inhibit oxytocin-induced Ca2+ mobilization in myometrial cells (Fig. 2
), it is likely that the reduction of the phosphorylation of MLC by Y-27632 occurred without changing the [Ca2+]i level. Next, to examine how Y-27632 affected MLC phosphorylation, we evaluated the effects of oxytocin and Y-27632 on MBS phosphorylation of myosin phosphatase. One of the major sites of phosphorylation of MBS by Rho-kinase has been identified as Ser854, and we used an antibody that specifically recognizes MBS phosphorylated at Ser854 (26). Immunoblotting with this antibody revealed that the phosphorylated form of MBS increased with oxytocin (0.2 nM) treatment compared with the untreated control (Fig. 4A). Y-27632 (10-5 M) reduced the extent of MBS phosphorylation induced by oxytocin without any change in the level of immunoreactive MBS. Quantitative analysis indicated that oxytocin caused a significant increase of MBS phosphorylation and that this enhancement of MBS phosphorylation was significantly inhibited by Y-27632 (Fig. 4B). It seemed that there was a highly positive correlation between the extent of MBS phosphorylation and that of oxytocin-induced contraction.
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, RhoA and Rho-kinase were clearly expressed in the myometrium. Quantitative analysis showed that the amount of Rho-kinase protein expression showed a significant increase at d 18 and 21 (Fig. 5B), whereas RhoA protein expression during pregnancy remained constant at the nonpregnant level. This result is consistent with the previous report by Moore et al. (23).
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shows the expression of RhoA, Rho-kinase, and GAPDH mRNAs in the rat myometrium as determined by RT-PCR using the total RNA prepared from the uteri of nonpregnant and pregnant (d 12, 18, 21) rats. Bands with the expected sizes for the RhoA-specific product were clearly detected at similar levels in nonpregnant and pregnant uterine tissues. The Rho-kinase-specific product could hardly be detected in the nonpregnant myometrium but was increased and could clearly be detected at d 18 and 21. To confirm these changes of mRNA expression determined by RT-PCR analysis, we further analyzed the level of expression of RhoA and Rho-kinase mRNAs by Northern blot analysis. As shown in Fig. 6B, RhoA (2.3 and 1.8 kb) and Rho-kinase (7.6, 6.6, and 5.6 kb) transcripts were detected, and the sizes of these transcripts were the same as previously reported elsewhere (35, 36, 37). Quantitative analysis showed that the level of expression of mRNA hybridizing with the Rho-kinase-specific probe in nonpregnant uterus was quite low, but it increased significantly during the course of pregnancy, especially at d 18 (4.6 ± 0.7-fold) and d 21 (7.1 ± 0.6-fold) (Fig. 6C). However, the level of expression of mRNA hybridizing with the RhoA probe did not change during pregnancy (Fig. 6, B and C). These results of Northern blot analysis showed that the expression of Rho-kinase mRNA in the myometrium is minimal in the nonpregnant state, and up-regulated during pregnancy, which is in agreement with the immunoblot (Fig. 5) and RT-PCR (Fig. 6A) data. These data indicate that the expression of Rho-kinase is up-regulated during pregnancy at the transcriptional level.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The novel findings of the present study are that (1) oxytocin-induced contraction was associated with enhanced MBS phosphorylation, which presumably resulted in the inhibition of myosin phosphatase and thereby induced uterine smooth muscle contraction (2), Rho-kinase mediated this MBS phosphorylation, and (3) the expression of Rho-kinase was up-regulated during pregnancy.
We used Y-27632, a pyrimidine derivative that is a specific inhibitor of the Rho-kinase (44), to determine the role of the RhoA/Rho-kinase cascade in oxytocin-induced myometrial contraction. Y-27632 inhibited oxytocin-induced pregnant rat uterine contraction in a concentration-dependent manner. In accordance with the inhibitory effect on oxytocin-induced myometrial contraction, immunoblot analysis showed that the oxytocin-induced increase in MLC and MBS phosphorylation was significantly inhibited by Y-27632. The concentration of Y-27632 used in this study was similar to that showing antihypertensive effects in hypertensive rat models (44). These results indicate that the activation of Rho-kinase and the phosphorylation of MBS and MLC occur concomitantly during oxytocin-induced uterine contraction, and suggest that the RhoA/Rho-kinase pathway may have an important role in enhancing contractility by increasing the phosphorylation of MLC as a result of the inhibition of myosin phosphatase by its phosphorylation.
The mechanisms controlling the transition to the increased uterine contractility at the onset of labor are still largely undefined. Many compounds such as PGs, oxytocin, and ß-adrenoceptor agonists have been implicated in the control of uterine contractility. These agonist-receptor interactions require the participation of a member of the heteromeric G protein family. Previous reports have suggested that a change in the expression of heteromeric G proteins might be involved in the control of uterine activity in humans and guinea pigs (20, 21). However, it is so far unknown whether low molecular weight G proteins may also be involved in the mechanism that increases the uterine contractility.
In the present study, we demonstrated that the expression of Rho-kinase mRNA was significantly up-regulated in the myometrium during pregnancy. The relaxant effects of Rho-kinase inhibitor Y-27632 were increased significantly in the d 21-pregnant rat uterus compared with the nonpregnant uterus (Fig. 1B). Niiro et al. (22) reported that the expression of RhoA and Rho-kinase mRNAs in the pregnant rat myometrium increased in comparison with that in the nonpregnant myometrium. It was also reported that stimulation of RhoA signaling in human pregnant myometrial tissue with lysophosphatic acid increased the level of MLC phosphorylation, which was inhibited by preincubation of the tissue with C3 transferase, a specific inhibitor of Rho (23). These results, together with our present data, suggest that RhoA/Rho-kinase pathway has an important role in oxytocin-induced uterine contraction. Moreover, the up-regulation of Rho-kinase in late pregnancy raises the possibility that not only the heteromeric G proteins (20, 21) but also RhoA/Rho-kinase pathway, one of low molecular weight G proteins, may be involved in the mechanism underlying the increased contractility of the pregnant myometrium.
No rescue experiment was done on Y-27632-treated uterine strips in the present study. Also, no direct methods are available to assay Rho-kinase activity in living myometrial cells or tissues. Thus, it remains possible that inhibition of other serine-threonine protein kinases in addition to Rho-kinase may also have contributed to the effects of Y-27632 observed in the present study. It is also possible that upstream pathways other than RhoA activate Rho-kinase in myometrial cells, because RhoA is not the only possible activator of Rho-kinase (45). These issues should be addressed in future studies.
If it is assumed that oxytocin induces uterine contraction through Ca2+-dependent and Ca2+-independent pathway, our results suggests that the latter pathway is, at least in part, induced through RhoA/Rho-kinase cascade. However, to conclude that RhoA/Rho-kinase cascade is responsible for this Ca2+-independent effect of oxytocin, our findings should be strengthened by additional analysis with permeabilized tissues in which [Ca2+]i can be manipulated freely. Future studies will be directed toward clarifying this specificity.
Spontaneous preterm labor remains a major obstetric problem because of the high incidence of associated perinatal mortality and morbidity (46). The drugs available for the management of preterm labor are only poorly effective and have potentially serious side effects for the mother or fetus. ß-adrenoceptor agonists, the drugs most widely used for the suppression of preterm uterine contraction, do not significantly modify the ultimate perinatal outcome, because of insufficient tissue selectivity and the resultant metabolic and cardiovascular side effects (47, 48, 49). Therefore, it is necessary to search for new drugs that are more effective and selective. For that purpose, new developments in our understanding of the cellular mechanisms involved in uterine contractions of preterm labor are essential and will lead to more selective therapy. The present study may provide insight into possible treatment strategies involving RhoA/Rho-kinase pathway. Hitherto, little has been known about the significance of the RhoA/Rho-kinase system in vivo because specific Rho inhibitors, such as botulinum C3 exoenzyme and Rho GDP dissociation inhibitor, are difficult to use, or not permitted for in vivo use. Y-27632 is very promising in that sense because its clinical application for the treatment of asthma, glaucoma and cancer metastasis is now under investigation (50, 51, 52). After application of the maximum dose of Y-27632 (10-4 M), we observed that contractility was reversed immediately by washing the strips with medium containing oxytocin. This reversibility seems promising for in vivo applications. This agent may be useful as a uterine relaxant for the prevention of premature delivery, which is a major cause of perinatal mortality and morbidity, although much research is needed to further define the cellular, biochemical, and molecular bases for these physiological processes involved in the regulation of uterine smooth muscle contraction and relaxation. Future studies should evaluate whether Y-27632 is effective in in vivo models of labor or preterm labor.
In summary, we demonstrated that Rho-kinase expression is up-regulated in the myometrium during pregnancy and mediates uterine contractility by inhibiting myosin phosphatase through MBS phosphorylation. The detailed molecular mechanism of the up-regulation of Rho-kinase must be further examined in future studies.
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Acknowledgments |
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Footnotes |
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Abbreviations: [Ca2+]i, Intracellular free Ca2+; DTT, dithiothreitol; ECL, enhanced chemiluminescence; MBS, myosin-binding subunit; MLC, myosin light chain; RhoA, peroxidase-coupled antimouse IgG antibody; Rho-kinase, peroxidase-coupled antigoat IgG antibody; TCA, trichloroacetic acid.
Received August 30, 2001.
Accepted for publication November 16, 2001.
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References |
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S-induced enhancement of phosphorylation of 20 kDa myosin light chain in vascular smooth muscle cells: inhibition of phosphatase activity. FEBS Lett 367:246–250[CrossRef][Medline]
s in pregnancy. Endocrinology 132:2484–2490s in human myometrium in term and preterm labor: a mechanism for parturition. J Clin Endocrinol Metab 79:1835–1839[Abstract]
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 C Cario-Toumaniantz, G Reillaudoux, V Sauzeau, F Heutte, N Vaillant, M Finet, P Chardin, G Loirand, and P Pacaud Modulation of RhoA--Rho kinase-mediated Ca2+ sensitization of rabbit myometrium during pregnancy -- role of Rnd3 J. Physiol., October 15, 2003; 552(2): 403 - 413. [Abstract] [Full Text] [PDF]
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 A. P. SOMLYO and A. V. SOMLYO Ca2+ Sensitivity of Smooth Muscle and Nonmuscle Myosin II: Modulated by G Proteins, Kinases, and Myosin Phosphatase Physiol Rev, October 1, 2003; 83(4): 1325 - 1358. [Abstract] [Full Text] [PDF]
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 S. Wrayzx, K. Jones, S. Kupittayanant, Y. Li, A. Matthew, E. Monir-Bishty, K. Noble, S. J. Pierce, S. Quenby, and A. V. Shmygol Calcium Signaling and Uterine Contractility Reproductive Sciences, July 1, 2003; 10(5): 252 - 264. [Abstract] [PDF]
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10-4 M). Maximal inhibition was achieved at 10-4 M. The contractility was reversed immediately by washing out Y-27632 with medium containing oxytocin. B, Concentration- effect relationship of Y-27632 on pregnant (
) and nonpregnant (
) myometrial contraction (n = 8). y-Axis, % contractility is the residual contractility as a percent of that before addition of Y-27632; x-axis, molar concentration of Y-27632. Data were expressed as the means ± SEM. *, P < 0.05 compared with pregnant (
