Mol. Hum. Reprod. Advance Access originally published online on May 21, 2004
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Molecular Human Reproduction, Vol. 10, No. 7, pp. 505-512, 2004
Molecular Human Reproduction vol. 10 no. 7 © European Society of Human Reproduction and Embryology 2004; all rights reserved
Roles of endogenous nitric oxide synthase inhibitors and endothelin-1 for regulating myometrial contractions during gestation in the rat
1Comprehensive Reproductive Medicine, Graduate SchoolTokyo Medical & Dental University, Tokyo, Japan 2Department of Biosystem Regulation, Institute of Biomaterials & Bioengineering, Graduate School, Tokyo Medical & Dental University, Tokyo, Japan
3 To whom correspondence should be addressed at: Department of Biosystem Regulation, Institute of Biomaterials & Bioengineering, Graduate School, Tokyo Medical & Dental University, 2-3-10 Surugadai, Kanda, Chiyoda-ku, Tokyo 101-0062, Japan.; Email: azuma.bsr{at}tmd.ac.jp
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Abstract |
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This study investigated the roles of endogenous nitric oxide synthase (NOS) inhibitors and endothelin-1 (ET-1) for regulating myometrial contractions during gestation in the rat. Basal and stimulated cyclic GMP production with L-arginine as a NOS substrate or sodium nitroprusside (SNP) as a NO donor were significantly enhanced at the middle of gestation (14th day), while these were greatly decreased at term (22nd day), suggesting the accelerated NO production and/or up-regulation of guanylate cyclase at the middle of gestation. NOS within the myometrium was mainly Ca2+dependent and partly Ca2 + independent and remained unaffected by aminoguanidine as an inhibitor of inducible NOS in non-pregnant and gestational myometrium. NOS activity per se and endothelial NOS (eNOS) protein expression remained unchanged at the middle and term gestation. Neuronal NOS (nNOS) and inducible NOS (iNOS) proteins were undetectable. SNP at a high concentration of 100 µmol/l failed to modify the spontaneous and ET-1-induced rhythmic contractions in non-pregnant and gestational myometrium. Contents of NG-monomethyl-L-arginine (L-NMMA) plus asymmetric NG,NG-dimethyl-L-arginine (ADMA) as endogenous NOS inhibitors and ET-1 within the myometrium were significantly decreased at 14th and 20th days of gestation, whereas these were significantly increased at term gestation (22nd day) and after delivery. There was a significant and positive correlation between endogenous NOS inhibitor content and ET-1. ET-1 within the myometrium was significantly increased with a concomitant decrease in cyclic GMP production after the intraperitoneal application of authentic L-NMMA for 2 weeks, suggesting that the impaired NO production with endogenous NOS inhibitors would result in increased ET-1 content. These results suggest that endogenous NOS inhibitors such as L-NMMA and ADMA play an important role for regulating NO production in rat myometrium. The impaired NO production due to accumulated endogenous NOS inhibitors possibly results in increased ET-1 content within the myometrium, thereby increasing myometrial contractions at term gestation and after delivery.
Key words: cyclic GMP/endogenous NOS inhibitors/endothelin-1/gestation/NO
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Introduction |
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Mechanisms to maintain pregnancy and to control labour are not yet fully understood. Recently, attention has been paid to the role of nitric oxide (NO) for maintaining pregnancy, since it is reported that NO has an ability to produce relaxation of the myometrium (Izumi et al., 1993
; Lees et al., 1994; Buhimschi et al., 1995; Izumi and Garfield, 1995; Kaya et al., 1998). With regard to the nitric oxide synthase (NOS) protein expression, it is reported that the myometrial inducible NOS (iNOS) may participate in the regulation of uterine activity during pregnancy in human and rat (Ali et al., 1997; Bansal et al., 1997; Riemer et al., 1997). Meanwhile, Norman et al. (1999) demonstrated that constitutive isoforms of NOS were also up-regulated in human pregnancy and may play a role in the maintenance of myometrial quiescence. In addition, Sladek et al. (1993) reported that the NOS activity was decreased on the day of delivery in the rabbit, pulling the trigger for labour.
On the contrary, there are reports that NO does not produce any relaxation of the myometrium (Hennan and Diamond, 1998; Word and Cornwell, 1998), but produces the contraction (Nakanishi et al., 1996). According to Weiner et al. (1994), iNOS mRNA was not found in the myometrium of pregnant and non-pregnant guinea-pig and myometrial calcium-dependent NOS activity declined slowly with advancing gestation, but never significantly differed from the activity in non-pregnant animals. Furthermore, Bartlett et al. (1999) reported the expression of endothelial NOS (eNOS) in myometrial tissues from preterm, term non-labour and active labour at term. However, iNOS and nNOS proteins were not detected at any stage of pregnancy. Messenger RNA for all three NOS isoforms (eNOS, nNOS and iNOS) was detected, although iNOS and (nNOS) mRNA were detectable only with high cycle number, implying a low copy number. Levels of eNOS protein and of eNOS mRNA expression were not correlated with gestational stage, suggesting that endogenously produced NO is not likely to be a modulator of myometrial tone during human pregnancy. Meanwhile, Thomson et al. (1997) reported that each of three isoforms of NOS was localized in the human myometrium, but no differences were found in either the expression or enzyme activity of NOS in the myometrium before and during labour at term. Therefore, the physiological role of NO and changes in NOS expression during gestation are still controversial.
It is well established that NO is not only a vasodilator (Furchgott and Zawadzki, 1980) but also a factor to inhibit endothelin-1 (ET-1) production (Boulanger and Lüscher, 1990). In addition, NO production is regulated by endogenous NOS inhibitors such as NG-monomethyl-L-arginine (L-NMMA) and asymmetric NG,NG-dimethyl-L-arginine (ADMA) in endothelial cells of the rabbit carotid artery (Azuma et al., 1995), bovine ciliary muscle (Azuma et al., 1997), rabbit proximal urethra and corpus cavernosum (Masuda et al., 2001, 2002), and human uterine artery (Beppu et al., 2002). Beppu et al. (2002) demonstrated that ET-1 content within the vessel wall became higher as endogenous NOS inhibitors were accumulated in endothelial cells and as NO production was decreased. In addition, we have demonstrated that exogenously applied ET-1 causes myometrial contractions composed of two types: increases in resting tone and rhythmic contractions under the non-pregnant state. At term gestation, however, ET-1 greatly increased the resting tone with little change in the rhythmic contractions (Sakamoto et al., 1999).
This study was therefore designed to investigate the roles of endogenous NOS inhibitors and ET-1 for regulating myometrial contractions during gestation in the rat.
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Materials and methods |
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Chemicals
The following chemicals were used: L-arginine, aprotinin, calmodulin, dithiothreitol (DTT), flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), 3-isobutyl-1-methylxantine (IBMX), leupeptin, NG-monomethyl-L-arginine (L-NMMA), ß-nicotinamide adenine dinucleotide phosphate reduced form (NADPH), NG-nitro-L-arginine (NOARG), oxytocin, leupeptin, pepstatin A, phenylmethylsulphonyl fluoride (PMSF), sodium nitroprusside (SNP), tetrahydrobiopterin and Triton-X100 (all from Sigma, USA), [14C]L-arginine (specific activity: 313 mCi/mmol) (NEN Life Science Products, Inc., USA), monoclonal endothelial (e)NOS, monoclonal neuronal (n)NOS and polyclonal inducible (i)NOS (all from Becton, Dickinson and Co., USA), radioimmunoassay kit for cyclic GMP (Yamasa Shouyu Co., Japan), trichloroacetic acid (TCA), Tris–HCl and enzyme-linked immunosorbent assay (ELISA) kit for ET-1 (all from Wako Pure Chemical Industries Ltd, Japan), Dowex AG50W-X8; Na+ form (Bio-Rad Laboratories, Inc., USA), Nonidet P-40 and EDTA (all from Nacalai Tesque, Japan), 3-[(3-cholamidpropyl)dimethylammonio]-1-propanesulphonate (CHAPS) (Dojindo Laboratories, Japan), aminoguanidine (Matheson Coleman & Bell Co., USA), octadecylsilyl silica (ODS) (Waters Co., USA) and acetic acid (Kanto Kagaku Co., Japan).
Animals and tissues
Female Sprague–Dawley rats, 10–15 weeks of age, were mated in the evening at the proestrus cycle. If sperm were observed in the vaginal smear the next morning, that day was defined as the day 0 of gestation. Rats in estrus (non pregnant), 7th, 14th, 20th and 22nd days of gestation and after delivery (within 1 day) were killed by exsanguination under anaesthesia with ether and hysterectomized. Immediately after the hysterectomy, the fetuses and placentas were removed from the uteri. The uteri were merged in oxygenated and ice-cold modified Krebs solution (in mmol/l: NaCl 115.0, KCl 4.7, MgSO4·7H2O 1.2, CaCl2·2H2O 2.5, KH2PO4 1.2, NaHCO3 25.0 and glucose 10; pH 7.4). For determinations of endogenous NOS inhibitors, cyclic GMP production, NOS protein expression, NOS activity and ET-1 content within the myometrium, endometrium was removed with surgical knife. Histological examination was performed to confirm the successful removal of the endometrium.
The study was conducted in compliance with the Animal Welfare Regulation of Tokyo Medical and Dental University.
Determination of cyclic GMP production with L-arginine or SNP
Freshly isolated myometrial specimens (longitudinal specimens of 
10 mg wet weight) were preincubated in modified Krebs solution for 60 min at 37°C, transferred into fresh Krebs solution and followed by further 40 min incubation until the specimens were rapidly transferred into 10% TCA with liquid nitrogen in order to stop the reaction. L-Arginine at a concentration of 300 µmol/l or 10 µmol/l SNP was added 20 min after transferring the preparations into the fresh Krebs solution. All experiments were performed in the presence of 10 µmol/l IBMX as a non-selective phosphodiesterase inhibitor. The cyclic GMP level was determined by radioimmunoassay kit for cyclic GMP according to the method described previously (Masuda et al., 1999). The net production of cyclic GMP stimulated with L-arginine was expressed as the difference between the production with 300 µmol/l L-arginine alone and 300 µmol/l L-arginine plus 100 µmol/l NOARG as an inhibitor of NOS (Kobayashi and Hattori, 1990).
Measurement of NOS activity
NOS activity was determined by measuring the conversion of [14C]L-arginine to [14C]L-citrulline according to the method described previously (Masuda et al., 2001, 2002). In brief, myometrial specimens were homogenized at 4°C in a buffer (Tris–HCl 50 mmol/l, CHAPS 10 mmol/l, EDTA 2 mmol/l, dithiothreitol 1 mmol/l, PMSF 1 mmol/l, pepstatin A 1 µmol/l, and leupeptin 2 µmol/l, pH 7.4). Homogenates were centrifuged at 10 000 g for 20 min. Aliquots of supernatant (100 µg of protein) were then incubated in the buffer containing NADPH (1 mmol/l), FAD (4 µmol/l), FMN (4 µmol/l), tetrahydrobiopterin (10 µmol/l), calmodulin (1 mg/l), and [14C]L-arginine (12 µmol/l of L-arginine, 0.1 µCi/ml). Reactions were performed at 37°C for 60 min in a final volume of 120 µl, then terminated by cooling on ice. After addition of 900 µl of water, samples were passed through a column containing 1.5 ml Dowex 50W-X8 resin to remove unmetabolized [14C]L-arginine. The columns were then washed with 1.35 ml of water, and [14C]L-citrulline was quantified in the flow-through fraction using a liquid scintillation counter (TRI-CARB 2750TR/LL; Packard Instrument Co., USA). The NOS activity was expressed as nmol L-citrulline/mg protein.
Western blotting
For western blot analysis, myometrial specimens were homogenized at 4°C in an extraction buffer (Tris–HCl 50 mmol/l, CHAPS 10 mmol/l, EDTA 2 mmol/l, DTT 1 mmol/l, PMSF 1 mmol/l, pepstatin A 1 µmol/l, and leupeptin 2 µmol/l, pH 7.4). Homogenates were centrifuged at 10 000 g for 20 min. One volume of supernatant was mixed with 1 volume of sodium dodecyl sulphate (SDS) loading buffer (125 mmol/l Tris–HCl pH 6.8, 40% glycerol, 20% SDS, 10% ß-mercaptoethanol and 0.05% Bromophenol Blue), which had been diluted x5 with the extraction buffer described above and boiled at 95°C for 5 min. Fifty microgram of samples were separated on a 9.0% SDS–polyacrylamide gel electrophoresis and transferred to Hybond-P PVDF membranes (Amersham Biosciences, UK). Membranes were blocked with 5% non-fat dry milk and then incubated with the primary antibodies (mouse monoclonal eNOS, rabbit polyclonal nNOS, rabbit polyclonal iNOS; Becton, Dickinson & Co., USA). Bound antibodies were visualized using a donkey anti-mouse secondary antibody conjugated with horseradish peroxidase (for eNOS,) or donkey anti-rabbit secondary antibody conjugated with horseradish peroxidase (for iNOS, nNOS) and detected by enhanced chemiluminescence (Amersham Biosciences). Results of eNOS protein expression were analysed densitometrically using a scanner (Epson ES-2200; Seiko-Epson Co., Japan) and expressed as mean ratio of eNOS protein density to GAPDH density. The ratio in non-pregnant myometrium was defined as 1.00. Human endothelial cell lysate, mouse pituitary lysate and mouse macrophage lysate stimulated with interferon-
and LPS (Becton, Dickinson & Co.) were used as positive control for eNOS, nNOS and iNOS respectively.
Effects of SNP on myometrial contractions
The mechanical responses were determined according to the method described previously (Sakamoto et al., 1999). In brief, a longitudinal strip (2 mm in width and 5 mm in length) of the uterus was mounted vertically in an organ chamber containing 5 ml of modified Krebs solution, continuously bubbled with 95% O2 and 5% CO2 at 37°C. One end of each strip was secured to the bottom of the organ chamber, and the other was attached to a force-displacement transducer (TB-611T; Nihon Kohden Kogyo Co., Japan). Isometric changes in tension were recorded on a pen-writing oscillograph (R-64; Rikadenki Kogyo Co., Japan). All strips were allowed to equilibrate for 
60 min in the bathing solution and during this period the bathing solution was replaced every 20 min with fresh solution. For observation of the effects of SNP on ET-1-induced myometrial contraction, 100 µmol/l SNP was added 10 min after the application of 10 nmol/l ET-1. For observation of SNP effect on the spontaneous contractions, 100 µmol/l SNP was added after the contractions became constant.
Determination of L-NMMA, ADMA and SDMA in the myometrium
L-NMMA, ADMA and symmetric NG,NG-dimethyl-L-arginine (SDMA) within the myometrium were determined by means of automated high-performance liquid chromatography (HPLC) according to the method described previously (Azuma et al., 1995, 1997; Hamasaki et al., 1997). In brief, myometrial specimens were minced with scissors and homogenized in a Polytron at maximum speed for 20 s in 5 mmol/l HEPES buffer. The homogenate was centrifuged at 10 000 g, 4°C for 20 min. TCA in a final concentration of 5% was added to the supernatant to precipitate proteins. This solution was centrifuged at 3000 g, 4°C for 15 min. The supernatant (100 µl) was used as a sample for HPLC.
Determination of ET-1 content within the myometrium
ET-1 within the myometrium was extracted according to the method described previously (Beppu et al., 2002). In brief, myometrial specimens were minced with scissors and homogenized in a Polytron at maximum speed for 20 s to a 25% homogenate in extracting buffer (1 mol/l acetic acid containing 0.01% Triton X-100 and 10 µg/ml pepstatin A). The homogenate was centrifuged at 25 000 g for 30 min at 4°C after boiling in water for 10 min to inactivate neutral endopeptidase. Supernatant was collected and ODS suspension was added to adsorb ET-1. This solution was centrifuged at 3000 g, 4°C for 3 min. The solvent (4% acetic acid and 86% ethanol) was added to the precipitate and centrifuged at 3000 g, 4°C for 3 min. Supernatant was evaporated and the residue was dissolved in 100% ethanol and evaporated again. The residue was finally dissolved in the buffer provided in the assay kit.
ET-1 concentration was determined by ELISA kit for ET-1 (Wako Pure Chemical Industries, Japan). Cross-reactivity has been estimated to be 100% against ET-1, 160% against ET-2 and <0.4% against ET-3 and big ET-1 (Instruction from Wako Pure Chemical Industries. Because the cross-reactivity was high (160%) against ET-2, we determined whether the supernatant contains ET-2 according to the method described previously (Ishizaka et al., 1999). The HPLC analysis revealed that ET-2 was undetectable in the supernatant. Therefore, we assumed that the positive signal detectable by the present ELISA method was due predominantly to ET-1.
Administration of authentic L-NMMA by osmotic pump
For observation of the effect of L-NMMA administration on ET-1 contents and cyclic GMP production in the myometrium, authentic L-NMMA was administrated in a dose of 2 mg/100 g body weight/day for 2 weeks with the aid of osmotic miniature pump (Alza Co., USA), which was implanted into the peritoneal cavity of non-pregnant Sprague–Dawley female rats. For the control, saline was administrated instead of L-NMMA.
Statistical analysis
All data are given as mean±SEM. Multiple comparisons between two groups were made by one-way analysis of variance (ANOVA). P<0.05 was considered statistically significant.
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Results |
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Cyclic GMP production
The ability of myometrial specimens to produce cyclic GMP was compared among three groups: non-pregnant, 14th and 22nd day of gestation rats. Results are shown in Figure 1a, b
. In the non-pregnant myometrium, the basal cyclic GMP level was determined to be 0.36±0.09 pmol/mg protein (n=6). The cyclic GMP production was increased by 0.99±0.31 pmol/mg protein (n=6) in the presence of 300 µmol/l L-arginine, which was significantly attenuated by 100 µmol/l NOARG as a NOS inhibitor (0.26±0.07 pmol/mg protein, n=6). Net production, which was expressed as the difference between production with L-arginine alone and L-arginine plus NOARG, was estimated to be 0.73±0.24 pmol/mg protein (n=6). The basal and net production of the nucleotide was significantly (P<0.005) increased at 14th day of gestation, while these were greatly decreased at the term gestation. Values at term were, however, significantly higher than those from non-pregnant myometrium (P<0.05 and P<0.005) respectively (Figure 1a).
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Both basal and stimulated production with 10 µmol/l SNP as a NO donor was significantly (P<0.005) increased at 14th day of gestation, whereas the production was greatly decreased at 22nd day of gestation. Values at the term were again significantly (P<0.005) higher than those from non-pregnant myometrium (Figure 1b
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NOS activity and NOS protein expression
Changes in myometrial NOS activity and NOS protein expression during gestation were determined. The NOS activity was detectable and significantly attenuated in the presence of 20 mmol/l EDTA or 100 µmol/l NOARG, while it remained unaffected by 30 µmol/l aminoguanidine as an inhibitor of inducible NOS (Wildhirt et al., 1999), indicating that the constitutive NOS was dominant in the myometrium. Calcium-dependent and -independent NOS activities remained unchanged in non-pregnant preparations and 14th and 22nd days of gestation (Figure 2).
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eNOS, but not nNOS or iNOS, protein was detectable in the non-pregnant and gestational myometrium, and in the myometrium after delivery. The eNOS protein expression also remained unchanged in all myometrial specimens tested (non-pregnant, 7th, 14th and 22nd days of gestation and post delivery). Results of the densitometric analyses of eNOS and western blotting of iNOS and nNOS are shown in Figure 3A, B
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Mechanical responses of the myometrium to SNP
SNP was used as a NO donor to test whether the agent produces relaxation response in the myometrial specimens in vitro. SNP even in a high concentration of 100 µmol/l failed to modify the spontaneous rhythmic contractions and the contraction caused by 10 nmol/l of ET-1 in the non-pregnant and gestational (7th, 14th, 20th and 22nd days of gestation) myometrial specimens. Representative tracings are shown in Figure 4
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Changes in L-NMMA, ADMA and SDMA contents within the myometrium
Three different methylarginines were detectable in the myometrial specimens isolated from non-pregnant rats. The content was determined to be 1.2±0.1 nmol/g wet weight for L-NMMA (n=4), 5.7±0.3 nmol/g wet weight for ADMA (n=4) and 0.5±0 nmol/g wet weight for SDMA (n=4). Based on the tissue water content of 82.2±0.2% (n=5) in the non-pregnant myometrium, the apparent concentrations were calculated to be 1.5±0.1 µmol/l for L-NMMA, 6.9±0.3 µmol/l for ADMA and 0.6±0 µmol/l for SDMA.
As shown in Figure 5A, contents of L-NMMA plus ADMA as endogenous NOS inhibitors were significantly (P<0.005) decreased at 14th and 20th days of gestation, whereas these were significantly (P<0.005) increased at 22nd day of gestation and after delivery. The apparent concentrations of L-NMMA and ADMA at 22nd day of gestation were calculated to be 4.4±0.3 and 10.6±0.5 µmol/l respectively on the basis of tissue water content of 84.8±0.4% (n=5). The SDMA content (0.5±0 nmol/g wet weight in the non-pregnant myometrium, n=4) remained unchanged until 20th day of gestation and significantly increased at term (1.1±0.2 nmol/g wet weight, n = 4) and after delivery (1.4±0.2 nmol/g wet weight, n=4).
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ET-1 content within the myometrium and relationship between endogenous NOS inhibitors
ET-1 was detectable at a level of 991±57 pg/g wet weight in the non-pregnant myometrium (n=4). The content remained unchanged at 7th day of gestation, whereas it was significantly (P<0.005) decreased at 14th and 20th days of gestation. On the contrary, the ET-1 content was significantly (P<0.005) increased at term gestation and after the delivery (Figure 5B
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As shown in Figure 6, changes in ET-1 content within the myometrium positively correlated with those of endogenous NOS inhibitors (L-NMMA plus ADMA) within the myometrium, indicating that the ET-1 content became higher as endogenous NOS inhibitors were accumulated.
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In order to examine the relationship between accumulation of endogenous NOS inhibitors in myometrium and changes in cyclic GMP production and ET-1 content, we investigated whether or not the exogenously applied authentic L-NMMA changes the ET-1 content and cyclic GMP production in the non-pregnant myometrium of the rat. The ET-1 content was significantly (P<0.005) increased after administration of authentic L-NMMA at a dose of 2 mg/100 g body weight/day for 2 weeks with the aid of an osmotic pump (Figure 7
), which had been implanted into the peritoneal cavity. Basal and stimulated cyclic GMP production with 300 µmol/l l-arginine were significantly (P<0.005) reduced in the myometrium previously treated with authentic L-NMMA [0.22±0.06 versus 0.44±0.07 pmol/mg protein for basal production (n=4) and 0.50±0.12 versus 1.14±0.30 pmol/mg protein for the stimulated production (n=4)]. The L-NMMA level in myometrial specimens was determined to be 1.2±0.2 nmol/g wet weight for the saline control (n=4) and 6.0±0.7 nmol/g wet weight after the authentic L-NMMA (n=4). Contents of ADMA and SDMA remained unchanged (data not shown).
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Discussion |
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We demonstrated in the present study that the NO–cyclic GMP generation system exists in rat myometrium, since the stimulated cyclic GMP production with L-arginine was significantly attenuated by NOARG as a NOS inhibitor, SNP as a NO donor increased cyclic GMP production, and NOS activity and eNOS protein were detectable in the myometrium. Furthermore, the basal cyclic GMP production as well as the production stimulated by L-arginine or SNP were greatly enhanced at 14th day of gestation and significantly decreased at term. In spite of the detectable NO–cyclic GMP generation system in the myometrium, SNP at a concentration of 100 µmol/l failed to modify the spontaneous rhythmic contractions and contractions caused by ET-1 in the non-pregnant and gestational myometrium in vitro.
The effect of SNP on the myometrial contraction of pregnant animals has been evaluated by several investigators (Yallampalli et al., 1993; Izumi and Garfield, 1995; Hennan and Diamond, 1998; Word and Cornwell, 1998). Although Hennan and Diamond (1998) and Word and Cornwell (1998) reported no suppression of contractile activity in response to SNP, Izumi and Garfield (1995) reported that SNP eliminated spontaneous contractility but did not affect KCl-induced contractions. In another paper, Yallampalli et al. (1993) reported that SNP was ineffective at low concentrations (>1 mmol/l) and required a relatively high concentration (5 mmol/l) to produce relaxation in the pregnant rat myometrium, and that it had a long lag period (20 min) to onset. They attributed the long lag period to onset of relaxation and the need for high concentrations by poor biotransformation of SNP to NO (Kowaluk et al., 1992) in the rat uterus. In each case, however, extremely high concentrations of SNP were required to produce an effect. Word and Cornwell (1998) demonstrated that SNP-induced ‘relaxation’ was irreversible and that spontaneous contractile activity did not resume after removal of the agent from the bathing solution, and concluded that responses to high concentrations of SNP (>1 mmol/l) are not likely to be mediated by cyclic GMP. On the other hand, it is generally accepted that nitrovasodilator-induced relaxation of vascular smooth muscle involves elevation of cyclic GMP and activation of specific cyclic GMP-dependent protein kinase (PKG). However, several types of smooth muscle, including rat myometrium, are not relaxed by SNP in spite of the significant elevation in cyclic GMP and PKG activation (Hennan and Diamond, 2001). The failure of the uterus to relax in the face of cyclic GMP elevation and PKG activation was suggested to be due to a lack of phosphorylation of the PKG substrate identified in the rat aorta (Hennan and Diamond, 2001). In addition, Word and Cornwell (1998) clearly demonstrated that myometrial tissues from pregnant rats were not sensitive to relaxation by SNP and that the insensitivity to SNP was accompanied by progesterone-mediated decreases in the level of PKG expression.
If the finding that rat myometrium is not relaxed by SNP in spite of the cyclic GMP elevation is accepted, what is the physiological role of the NO–cyclic GMP system in the gestational myometrium?
Sakamoto et al. (1999) have reported that exogenously applied ET-1 (0.3–30 nmol/l) causes myometrial contractions composed of two types: increases in resting tone and rhythmic contractions under the non-pregnant state. At term gestation, however, ET-1 greatly increased the resting tone with little change in the rhythmic contractions. In the present experiments, we demonstrated that ET-1 content in the myometrium was decreased at the middle of gestation, but significantly increased at term gestation (22nd day) and after delivery. The apparent myometrial concentration of ET-1 at term gestation (22nd day) was calculated to be 1 nmol/l on the basis of the tissue water content (84.8±0.4%). The apparent concentration of ET-1 seems to be sufficient to increase the resting tone. At the middle of gestation, however, the concentration decreased by a threshold of 0.3 nmol/l. Therefore, gestational changes in myometrial ET-1 content possibly reflect the gestational changes in the myometrial contractions. Peri et al. (1992) demonstrated that the concentration of ET-1 receptors progressively decreased during the pregnancy, rising again at the time of spontaneous delivery, and suggested a paracrine role for endometrial ET-1 during delivery, because giant cells immunoreactive for ET-1 were detectable in close proximity to the myometrial cells, which was accompanied by increased ET-1 receptors in the myometrium of parturient rabbits. Taken together, decreased concentrations of ET-1 and ET-1 receptors at the middle of gestation may decrease myometrial contractions. At term gestation, on the other hand, increased concentrations of ET-1 and ET-1 receptors may result in the increased myometrial contractions. This assumption seems to be supported by the findings that the contractile sensitivity and maximum contraction of pregnant rat myometrium for ET-1 increased during the progress of gestation (Izumi et al., 1995).
Changes in myometrial ET-1 content positively correlated with those in endogenous NOS inhibitor (L-NMMA plus ADMA) content within the myometrium, indicating that ET-1 content became higher as endogenous NOS inhibitors were accumulated, and that conversely ET-1 content became lower as endogenous NOS inhibitors were decreased. The increased cyclic GMP generation with L-arginine at 14th day of gestation was associated with the decreased endogenous NOS inhibitors, decreased ET-1 level and the increased cyclic GMP generation with SNP as an activator of soluble guanylate cyclase. According to Boulanger and Lüscher (1990), NO inhibits ET-1 release from porcine aorta. Furthermore, we demonstrated in the present experiments that the administration of authentic L-NMMA increased the myometrial ET-1 content with concomitant decrease in cyclic GMP production in the myometrium. If these findings are considered together, the enhanced cyclic GMP production at middle of gestation (14th day) seems to be brought about by the increased NO production due to the decreased endogenous NOS inhibitors and/or up-regulation of soluble guanylate cyclase (Ignarro, 1990; Chinkers and Garbers, 1991), thereby decreasing ET-1 content in the myometrium. At term gestation, on the other hand, the high ET-1 content would result from the impaired cyclic GMP generation, which is probably due to the significantly accumulated endogenous NOS inhibitors in the myometrium and/or down-regulation of soluble guanylate cyclase. We should consider the possibility that the down-regulation of guanylate cyclase may also be involved in the decreased cyclic GMP generation at term gestation, since the cyclic GMP generation in response to SNP was greatly decreased at term compared to mid gestation (Figure 1a, b). However, further work needs to be performed to improve our understanding.
Although there are numerous reports describing the involvement of NOS and NO in gestation and active labour at parturition, results are controversial at present. Bansal et al. (1997) demonstrated that expression of iNOS was highest in myometrium of preterm non-labour patients. At term, however, iNOS expression fell by 75%, and was barely detectable in preterm in labour or term in-labour specimens. Bansal et al. (1997), Ali et al. (1997) and Riemer et al. (1997) concluded that the myometrial iNOS expression probably participated in the regulation of uterine activity during pregnancy in humans and rats. Meanwhile, Norman et al. (1999) reported that eNOS and nNOS protein concentrations were higher in the preterm pregnant myometrium than non-pregnant myometrium. Endothelial NOS, but not nNOS, protein concentration was lower in myometrial samples obtained at term compared with those obtained at preterm. They concluded that constitutive isoforms of NOS were also up-regulated in human pregnancy and may play a role in the maintenance of myometrial quiescence. In addition, Purcell et al. (1997) showed the changes in NO production by the uterine placental tissues throughout gestation, which may be important in regulating NO delivery to the myometrium.
On the contrary, Weiner et al. (1994) reported that iNOS mRNA was not found in the myometrium of pregnant or non-pregnant guinea-pig and that myometrial calcium-dependent NOS activity declined slowly with advancing gestation, but never significantly differed from the activity in non-pregnant animals. Furthermore, Bartlett et al. (1999) reported the expression of eNOS in myometrial tissues from preterm, term non-labour and active labour at term. However, iNOS and nNOS proteins were not detected at any stage of pregnancy. Messenger RNA for all three NOS isoforms (eNOS, nNOS and iNOS) was detected, although iNOS and nNOS mRNA were detectable only with high cycle number, implying a low copy number. Levels of eNOS protein and eNOS mRNA expression were not correlated with gestational stage, suggesting that endogenously produced NO is not likely to be a modulator of myometrial tone during human pregnancy. According to Thomson et al. (1997), each of three isoforms of NOS were localized in the human myometrium, but no differences were found in either the expression or enzyme activity of NOS in the myometrium before and during labour at term. In the current experiment, we demonstrated that NOS activity per se and eNOS protein expression remained unchanged during the gestation and after delivery. In addition, NOS activity was greatly attenuated in calcium-free medium containing EDTA and by NOARG as a non-selective NOS inhibitor, but unaffected by aminoguanidine as an inhibitor of inducible NOS (Wildhirt et al., 1999). Meanwhile, nNOS and iNOS proteins were undetectable in non-pregnant and gestational myometrium, and in the myometrium after delivery. Therefore, we concluded that changes in NO production during gestation were not due to the changes in NOS activity per se and NOS protein expression. However, further studies should be performed to confirm whether NOS activity and NOS protein expression change, and which isoforms of NOS play a physiological role during the gestation.
Recently, it has been reported that the accumulation of endogenous NOS inhibitors such as L-NMMA and ADMA in plasma (Böger et al., 1997) and tissues (Azuma et al., 1995, 1997; Masuda et al., 1999, 2001, 2002) might explain in part the mechanism of impaired NO production. The apparent concentrations of L-NMMA and ADMA which had been calculated on the basis of tissue water content were 4.4±0.3 and 10.6±0.5 µmol/l at term gestation (22nd day) respectively. The inhibition of NOS was determined to be 35.4% by 4.4 µmol/l of authentic L-NMMA and 52.8% by 10.6 µmol/l of authentic ADMA (Y.Momohara, unpublished observation). Thus, the detected concentrations of L-NMMA and ADMA in the myometrium seem to be high enough to inhibit NOS activity.
Mechanisms of accumulating L-NMMA and ADMA are not clarified accurately in the present experiments. Dimethylarginine dimethylaminohydrolase (DDAH), an enzyme that hydrolyses L-NMMA and ADMA to L-citrulline and methylamines (Ogawa et al., 1989), is widely distributed in tissues (Kimoto et al., 1995) probably including the myometrium. DDAH activity may be enhanced at 14th and 20th days of gestation, leading to the decreased L-NMMA and ADMA levels. This possibility seems to be partly supported by the findings that the level of SDMA, which is not a substrate for DDAH (Ogawa et al., 1989), remained unchanged at 14th and 20th day of gestation. On the other hand, contents of L-NMMA, ADMA and SDMA were significantly increased at term gestation (22nd day) and after delivery. These three methylarginines enter cells through the cationic amino acid transporter known collectively as system y+ (Bogle et al., 1995). Therefore, these results lead us to assume that the transmembrane transport of three methylarginines would be enhanced at term gestation and after delivery. However, further experiments should be performed to understand the detailed mechanism.
In conclusion, endogenous NOS inhibitors such as L-NMMA and ADMA play an important role for regulating NO production in the rat myometrium. The impaired NO production due to accumulated endogenous NOS inhibitors possibly results in the increased ET-1 content within the myometrium, thereby increasing the myometrial contractions at the term gestation and after delivery.
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This study was supported in part by Grants-in-Aid for Scientific Research (08457436 to T.A. and 14572152 to H.A.) from the Ministry of Education, Culture, Sports, Science and Technology, Japan, the Smoking Research Foundation, Japan (to H.A.) and the New Drug Research (NDR) Foundation, Japan (to H.A.).
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Submitted on March 6, 2004; resubmitted on April 11, 2004; accepted on April 27, 2004.
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