Mol. Hum. Reprod. Advance Access originally published online on May 30, 2006
Molecular Human Reproduction 2006 12(8):513-518; doi:10.1093/molehr/gal047
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Involvement of arginase in regulating myometrial contractions during gestation in the rat
1Comprehensive Reproductive Medicine and 2Department of Biosystem Regulation, Institute of Biomaterials and Bioengineering, Graduate School, Tokyo Medical and Dental University, Tokyo, Japan
3 To whom correspondence should be addressed at: Department of Biosystem Regulation, Institute of Biomaterials and Bioengineering, Graduate School, Tokyo Medical and Dental University, 2-3-10 Surugadai, Kanda, Chiyoda-ku, Tokyo 101-0062, Japan. E-mail: azuma.bsr{at}tmd.ac.jp
4 Present address: Department of Obstetrics and Gynecology, Dokkyo Medical University Koshigaya Hospital, 2-1-50 Minamikoshigaya, Koshigaya, Saitama, 343–8555, Japan
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Abstract |
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This study was designed to investigate the role of arginase in regulating myometrial contractions during gestation in the rat. Arginase activity in the myometrium was significantly decreased during the 7th–21st day of gestation, with the lowest value on the 14th day. However, the enzyme activity became significantly higher at term gestation (22nd day) than that in the non-pregnant myometrium. Arginase I protein was undetectable in the non-pregnant myometrium, at 7th and 14th day of gestation and after delivery. A slight positive signal for arginase I was detectable at 21st day of gestation. However, the protein was clearly up-regulated at term gestation (22nd day), although arginase II protein was down-regulated during gestation, with the lowest value on the 14th day. Gestational changes in arginase activity negatively correlated with those in cyclic GMP production, whereas the changes positively correlated with those in endogenous nitric oxide synthase (NOS) inhibitors and endothelin-1 (ET-1) contents. Myometrial arginase activity was inhibited by NG-hydroxy-L-arginine as an intermediate of NO production from L-arginine in a concentration-dependent manner. Both basal and stimulated guanylyl cyclase activities were enhanced at mid- and reduced at term gestation and after delivery, thereby partly increasing cyclic GMP production at mid- and partly decreasing the nucleotide production at term gestation and after delivery. These results suggest that the decreased arginase activity at mid-gestation possibly results from the down-regulation of arginase II protein. Whereas, the enhanced overall arginase activity at term gestation seems to be because of the induced functional arginase I in concert with the attenuated arginase II expression. The enhanced arginase activity at term gestation would be implicated in increasing myometrial contractions mediated by the increased ET-1. The increased peptide production at term gestation is possibly because of the reduced cyclic GMP production resulting from enhanced arginase activity, accumulated endogenous NOS inhibitors and attenuated guanylyl cyclase activity.
Key words: arginase I and II/endothelin-1/gestation/myometrial contraction/nitric oxide
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Introduction |
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Mechanisms of the uterine quiescence during gestation and the initiation of uterine contractions during labour are not fully understood. Because NO has an ability to produce relaxation of the myometrium (Izumi et al., 1993
; Yallampalli et al., 1993; Lees et al., 1994; Buhimschi et al., 1995; Izumi and Garfield, 1995; Kaya et al., 1998), attention has been paid to the role of NO for uterine quiescence. Regarding nitric oxide synthase (NOS) protein expression, it has been reported that inducible NOS in the myometrium may participate in regulating the uterine activity during pregnancy in the human and rat (Ali et al., 1997; Bansal et al., 1997; Riemer et al., 1997). According to Norman et al. (1999), constitutive isoforms of NOS were also up-regulated in human pregnancy and may play a role in the maintenance of myometrial quiescence. Furthermore, it has been reported that NOS activity was decreased on the day of delivery in the rabbit (Sladek et al., 1993). However, whether NO could directly cause relaxation is still controversial. We have previously reported that exogenously applied ET-1 causes myometrial contractions in the non-pregnant state and greatly increases resting tone at term gestation (Sakamoto et al., 1999) and that the decreased NO production because of accumulated endogenous NOS inhibitors possibly results in the increased ET-1 production, thereby increasing myometrial contractions at term gestation (Momohara et al., 2004). Although the NO-cyclic GMP generation system exists in rat myometrium, NO per se does not directly cause myometrial relaxation (Hennan and Diamond, 1998; Word and Cornwell, 1998; Momohara et al., 2004). However, NO possibly plays an important role for regulation of myometrial contractions during gestation through modulating ET-1 production in the myometrium (Momohara et al., 2004).
Arginase metabolizes L-arginine to urea and L-ornithine (Wu and Morris, 1998). Arginase exists in two isoforms, arginase I as the hepatic type localized in the cytosol and arginase II as the extrahepatic type localized in the mitochondrial matrix (Jenkinson et al., 1996). Because NOS shares L-arginine as a common substrate with arginase (Boucher et al., 1999; Mori and Gotoh, 2000), NO production absolutely depends on the availability of L-arginine to NOS. In this regard, the L-arginine catabolism via the arginase pathway can act as an endogenous negative control system to regulate overall NO production. Furthermore, NG-hydroxy-L-arginine (NOHA) as an intermediate of NO production from L-arginine is a potent inhibitor of arginase (Boucher et al., 1994; Daghigh et al., 1994). Therefore, the decreased NOHA production associated with impaired NO production may lead to a vicious circle for the NO generation pathway.
To our knowledge, however, there are no observations whether the changes in arginase activity are involved in modulating myometrial contractions during gestation in relation to NO and ET-1 productions. Thus, these experiments were designed to investigate the role of arginase for modulating myometrial contractions during gestation in the rat.
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Materials and methods |
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Animals and tissues
Female Sprague-Dawley rats, 12–18 weeks of age, were mated in the evening at the proestrus cycle. If sperm was observed in the vaginal smear the next morning, that day was defined as day 0 of gestation. Rats in estrus (non-pregnant) at 7th, 14th, 21st and 22nd day of gestation and after delivery (within 1 day) were sacrificed by exsanguination under anaesthesia with ether and hysterectomized. Immediately after the hysterectomy, both uterine horns were opened longitudinally, and fetuses and placentas were separated. The uteri were immersed in oxygenated and ice-cold modified Krebs solution (NaCl 115.0 mM, KCl 4.7 mM, MgSO4 · 7H2O 1.2 mM, CaCl2 · 2H2O 2.5 mM, KH2PO4 1.2 mM, NaHCO3 25.0 mM and glucose 10 mM, pH 7.4). The tissues were frozen after dissection and stored at –80°C except for measuring cyclic GMP in the fresh specimens. For measuring arginase, soluble guanylyl cyclase, arginase protein expression and endogenous inhibitors and ET-1 contents, endometrium or decidua was removed with the aid of a surgical knife. Histological examination was performed to confirm the successful removal of endometrium or decidua.
The study was conducted in compliance with the Animal Welfare Regulation of Tokyo Medical and Dental University.
Protein preparation for enzyme activities
Frozen tissues were homogenized in a Polytron (Kinematica, Lucerne, Switzerland) at maximum speed for 20 s, each 3 times in a buffer, consisting of 50 mM Tris–HCl, 10 mM CHAPS, 2 mM EDTA, 1 mM phenylmethylsulphonyl fluoride (PMSF), 1 mM dithiothreitol (DTT), 1 µM pepstatin A and 2 µM leupeptin, pH 7.4. Homogenates were centrifuged at 10 000 g for 20 min at 4°C to separate the supernatant, in which protein concentration was determined using BCA protein assay reagent kit (Pierce, Rockford, IL, USA). The supernatant was used for measuring arginase activity. The supernatant was then centrifuged at 50 000 g for 60 min at 4°C, and the resultant supernatant (soluble fraction) was assayed for soluble guanylyl cyclase activity.
Arginase activity
Arginase activity was determined by measuring the conversion of L-[14C-guanido]arginine to [14C]urea according to the method described previously with minor modifications (Sakai et al., 2004). Aliquots of tissue extracts (10 µl) were incubated in a final volume of 100 µl buffer containing 8 mM Tris–HCl, 0.08 µCi/ml of L-[14C-guanido]arginine (specific activity: 51.5 mCi/mmol) and 1 mM MnCl2, pH 9.6, for 2 h at 37°C. Reactions were then terminated by the addition of 450 µl of ice-cold stop solution containing 250 mM sodium acetate and 100 mM urea, pH 4.5. Samples were passed through a column containing 1.5 ml Dowex 50W-X8 resin (Na+ form) to remove unmetabolized L-[14C-guanido]arginine. The columns were then washed with 2.25 ml of distilled water, and [14C]urea was quantified in the flow-through fraction using a liquid scintillation counter (TRI-CARB 2750TR/LL, Packard Instrument, Meriden, CT, USA). The arginase activity was determined as a net activity calculated from the difference of activities in the presence and absence of 10 µM NG-hydroxy-nor-L-arginine (nor-NOHA) as an arginase inhibitor without affecting NOS activity (Custot et al., 1997; Moali et al., 1998). Results were expressed as pmol urea/mg protein/2 h. The effect of NOHA on arginase activity was determined in the presence of various concentrations of NOHA (1–20 µM).
Western blotting
For western blotting, myometrial specimens were homogenized at 4°C in lysis buffer, consisting of 50 mM Tris–HCl, 300 mM NaCl, 1% Triton X 100 and 10% protease inhibitor cocktail, pH 7.5. Homogenates were centrifuged at 10 000 g for 20 min. Then, the supernatant was mixed with an equal volume of sodium dodecyl sulphate (SDS) loading buffer, consisting of 125 mM Tris–HCl, 10% ß-mercaptoethanol, 4% SDS, 30% glycerol and 0.01% bromophenol blue. Mixtures were boiled at 95°C for 5 min. Twenty micrograms each of arginase protein was subjected to electrophoresis on 10% SDS–PAGE and then transferred to a polyvinylidene difluoride membrane (Hybond-P, Amersham Biosciences, Bukinghamshire, UK). After blocking with 5% non-fat dry milk, membranes were incubated overnight at 4°C with the primary antibodies, mouse monoclonal anti-arginase I (1:1500; BD Biosciences, San Jose, CA, USA) and rabbit polyclonal anti-arginase II (1:400; Santa Cruz Biotechnology, Santa Cruz, CA, USA). Membranes were incubated for 1 h at room temperature with a donkey anti-mouse secondary antibody conjugated with horseradish peroxidase for arginase I or a donkey anti-rabbit secondary antibody conjugated with horseradish peroxidase for arginase II and then visualized using enhanced chemiluminescence (ECL plus, Amersham Biosciences). Results of arginase protein expressions were analysed densitometrically using Scion Image and expressed as mean ratio of arginase protein density to glyceraldehyde-3-phosphatedehydrogenase (GAPDH) density. Anti-GAPDH antibody was purchased from Chemicon International (Temecula, CA, USA) (1:200). The ratio in non-pregnant myometrium was defined as 1.00 for arginase II. In the case of arginase I, the ratio at 22nd day of gestation was defined as 1.00.
Soluble guanylyl cyclase activity
Soluble guanylyl cyclase activity was determined by the method described previously with minor modifications (Wheeler et al., 1997; Masuda et al., 2002). Aliquots of soluble fraction (20 µl) pre-incubated with 20 µl of 100 µM sodium nitroprusside (SNP) or distilled water for 10 min at 37°C was added to 60 µl of reaction mixture, consisting of 10 mM Tris–HCl, 1 mM GTP, 4 mM MnCl2, 2 mM DTT, 100 µM 3-isobutyl-1-methylxanthine (IBMX), 16 mM phosphocreatine and 28 units/ml creatine phosphokinase. Mixtures were incubated for 60 min at 37°C. Reactions were stopped by adding 900 µl of ice-cold 5 mM EDTA and then boiling for 5 min at 95°C. Cyclic GMP level was determined by radioimmunoassay kit (Yamasa Shoyu, Tokyo, Japan). SNP-activated activities were calculated from cyclic GMP production in the presence of 100 µM SNP. Results were expressed in pmol/mg protein.
Cyclic GMP production stimulated with L-arginine
Cyclic GMP production in the myometrium was determined according to the method described previously (Masuda et al., 1999). Freshly isolated myometrial specimens were pre-incubated in modified Krebs solution for 60 min at 37°C, transferred into fresh Krebs solution and followed by a further 40-min incubation until the specimens were rapidly transferred into 10% trichloroacetic acid (TCA) with liquid nitrogen to stop the reaction. L-Arginine at a concentration of 300 µM or vehicle was added 20 min after transferring the preparations into the fresh Krebs solution. All experiments were performed in the presence of 10 µM IBMX as a non-selective phosphodiesterase inhibitor. The cyclic GMP level was determined by radioimmunoassay kit (Yamasa Shoyu). The net production of cyclic GMP stimulated with 300 µM L-arginine was expressed as the difference between the production with 300 µM L-arginine alone and that with 300 µM L-arginine plus 100 µM nitroarginine as an inhibitor of NOS.
Endogenous NOS inhibitors in the myometrium
NG-monomethyl-L-arginine (MMA) and asymmetric NG, NG-dimethyl-L-arginine (ADMA) as endogenous NOS inhibitors in the myometrium were determined by means of automated high-performance liquid chromatography (HPLC), according to the method described previously (Azuma et al., 1995). In brief, myometrial specimens were minced with scissors and homogenized in a Polytron at maximum speed for 20 s in 5 mM HEPES buffer. The homogenate was centrifuged at 10 000 g at 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.
Endothelin-1 in the myometrium
Endothelin-1 (ET-1) in the myometrium was determined 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 M acetic acid containing 0.01% Triton X-100 and 1 µM 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 octadecylsilyl silica (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, Osaka, 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 predominantly because of ET-1.
Statistical analysis
Results are given as means ± SEM. Statistical analysis was performed using Student’s t-test for unpaired data, and multiple comparisons were made by one-way analysis of variance (ANOVA). P values <0.05 were considered to be statistically significant.
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Arginase activity
The arginase activity was determined to be 1192.2 ± 65.5 pmol urea/mg protein/2 h (n = 6) in the non-pregnant myometrium, which was significantly (P < 0.05 and P < 0.005) reduced at 7th, 14th and 21st day of gestation, with the lowest value on the 14th day. On the contrary, the enzyme activity was significantly (P < 0.005) enhanced at term gestation (1628.5 ± 77.3 pmol urea/mg protein/2 h at 22nd day). The value after delivery remained higher but not significant compared with the corresponding value in the non-pregnant myometrium. These results are shown in Figure 1.
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The effect of NOHA as an intermediate of NO production from L-arginine on arginase activity was examined in the absence or presence of various concentrations of NOHA (1–20 µM). The enzyme activity was inhibited by NOHA in a concentration-dependent manner. The 50% inhibitory concentration (IC50) of NOHA was determined as 1.50 ± 0.09 µM (n = 5).
Arginase protein expression
Arginase I protein was undetectable in the non-pregnant myometrium, at 7th and 14th day of gestation. A slight positive signal for arginase I was detectable at 21st day of gestation. However, the signal at term gestation (22nd day) was definite and significantly (P < 0.05) stronger than that at 21st day. Interestingly, the positive signal was undetectable after delivery. These results are shown in Figures 2A and 3A. On the contrary, arginase II protein was detectable in all myometrial specimens tested (non-pregnant, 7th, 14th, 21st and 22nd day of gestation and after delivery). Results are shown in Figures 2B and 3B. The arginase II protein expression was significantly (P < 0.005) decreased during gestation and after delivery, with the lowest value on the 14th day of gestation and gradually increasing from the lowest value towards term gestation and after delivery.
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Soluble guanylyl cyclase activity
Both basal guanylyl cyclase activity and stimulated activity with 100 µM SNP as a NO donor were significantly (P < 0.005) increased at 14th day of gestation (from 0.50 ± 0.11 and 1.40 ± 0.10 pmol/mg protein to 2.48 ± 0.33 and 5.85 ± 0.72 pmol/mg protein, respectively, n = 4 each). Values at term (22nd day of gestation) were greatly decreased but significantly (P < 0.05) higher (0.89 ± 0.11 pmol/mg protein for basal level and 2.23 ± 0.16 pmol/mg protein for stimulated level, n = 4 each) than those from non-pregnant myometrium. After delivery, however, these values completely reverted to the normal level in the non-pregnant myometrium (Figure 4).
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Changes in cyclic GMP production and contents of endogenous NOS inhibitors and ET-1 in the myometrium
Net production of cyclic GMP stimulated with 300 µM L-arginine in the non-pregnant myometrium was determined to be 1.01 ± 0.26 pmol/mg protein (n = 4), which was significantly (P < 0.005) increased (3.61 ± 0.25 pmol/mg protein, n = 4) at the middle of gestation (14th day) and greatly decreased at term gestation (22nd day) and after delivery. The changes in cyclic GMP production observed at 14th and 22nd day of gestation and after delivery were associated with significantly (P < 0.005) decreased endogenous NOS inhibitors (MMA plus ADMA) and ET-1 at mid-gestation and with significantly (P < 0.005) accumulated NOS inhibitors and ET-1 at term gestation and after delivery (Figure 5A, B and C).
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Discussion |
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We have reported that the quiescence of myometrium at mid-gestation of the rat is at least partly mediated by the decreased ET-1 production because of the increased cyclic GMP production as a marker of NO production and that the impaired NO production because of the accumulation of endogenous NOS inhibitors and down-regulation of guanylyl cyclase results in the increased ET-1 production within the myometrium, thereby increasing myometrial contractions at term gestation and after delivery (Momohara et al., 2004
). The NO-cyclic GMP generation system exists in rat myometrium, nevertheless NO per se does not cause directly the myometrial relaxation (Hennan and Diamond, 1998; Word and Cornwell, 1998; Momohara et al., 2004). However, NO plays an important role for regulation of myometrial contractions during gestation through modulating ET-1 production in the myometrium (Momohara et al., 2004). Because arginase shares L-arginine as a common substrate with NOS, NO production depends on the availability of L-arginine to NOS. In this regard, the L-arginine catabolism via the arginase pathway can act as an endogenous negative control system to regulate overall NO production. In these experiments, we demonstrated that arginase activity was significantly reduced at mid-gestation but significantly enhanced at term gestation. These changes in arginase activity were negatively correlated to the cyclic GMP production, that is, the ability of myometrium to produce cyclic GMP was increased at mid-gestation but decreased at term gestation, suggesting that the increased cyclic GMP production as a marker of NO production (Figure 5A) at gestation possibly results from reduced arginase activity in addition to the reduced endogenous NOS inhibitors (Figure 5B) and increased guanylyl cyclase activity (Figure 4). Conversely, the decreased production of the nucleotide at term gestation (Figure 5A) possibly results from the enhanced arginase activity in addition to the accumulation of endogenous NOS inhibitors (Figure 5B) and decreased guanylyl cyclase activity (Figure 4). Therefore, it seems likely that arginase plays an important role in regulating NO production in the gestational rat myometrium. However, further work needs to be performed to improve our understanding.
Arginase exists in two isoforms, arginase I as the hepatic type and arginase II as the extrahepatic type. Recent studies in animals demonstrated that expression of arginase isoforms were detectable in various cells and tissues (Mori and Gotoh, 2000). In the non-pregnant myometrium, at 7th and 14th day of gestation and after delivery, arginase I was undetectable despite the abundant expression of arginase II protein. Arginase II expression was significantly decreased during 7th–22nd day of gestation, with the lowest value at 14th day. Thus, the reduced arginase activity at mid-gestation seems to be because of the decreased arginase II expression. Interestingly, although the arginase I protein expression was only slight at 21st day of gestation, the positive signal at term gestation (22nd day) was definite and significantly stronger than that at 21st day. It seems, therefore, likely that the induced functional arginase I in concert with the reduced arginase II expression possibly reflects on the enhanced overall arginase activity at term gestation, because the arginase II protein expression was gradually increased from the lowest value at 14th day of gestation towards term gestation and after delivery.
NOHA inhibited arginase activity in a concentration-dependent manner with IC50 value of 1.5 µM. Similar results have been reported by Boucher et al. (1994) and Daghigh et al. (1994). Because NOHA is an intermediate of NO production from L-arginine, the intermediate may play an inhibitory role for arginase in vivo. The arginase may be under the silent state when NO/NOHA productions are sufficient. The enzyme activity may be enhanced when NO/NOHA productions are impaired by the accumulation of endogenous NOS inhibitors, thereby resulting in a vicious circle for NO generation. At term gestation, MMA and ADMA as endogenous NOS inhibitors were significantly increased in the myometrium. These results fit well with our previous report, and the concentrations of the inhibitors seemed to be sufficient to inhibit NOS (Momohara et al., 2004).
Furthermore, we have reported that exogenously applied ET-1 causes myometrial contractions composed of two types: increases in resting tone and rhythmic contraction under the non-pregnant state. At term gestation, however, ET-1 greatly increased the resting tone with little change in rhythmic contractions (Sakamoto et al., 1999). The apparent concentration of ET-1 at term gestation seems to be sufficient to increase the resting tone. At the middle of gestation, however, the concentration decreased by a threshold or less (Figure 5C) (Momohara et al., 2004). Therefore, gestational changes in the myometrial ET-1 content possibly reflect on those in myometrial contractions. Peri et al. (1992) demonstrated that the concentration of ET-1 receptors progressively decreased during pregnancy, rising again at the time of spontaneous delivery. Taken together, decreased concentrations of ET-1 and ET-1 receptors at mid-gestation may decrease myometrial contractions. At term gestation, on the contrary, increased concentrations of ET-1 and ET-1 receptors may result in the increased myometrial contractions. The gestational changes in ET-1 content were accompanied by changes in cyclic GMP production and content of endogenous NOS inhibitors. The increased cyclic GMP production because of decreased arginase activity and endogenous NOS inhibitors and enhanced guanylyl cyclase activity at mid-gestation may result in the decreased ET-1 production. On the contrary, the decreased nucleotide production because of enhanced arginase activity, accumulated endogenous NOS inhibitors and attenuated guanylyl cyclase activity at term gestation may bring about the increased ET-1 production, because the administration of authentic MMA as an inhibitor of NOS increased myometrial ET-1 content with concomitant decrease in cyclic GMP production in the myometrium (Momohara et al., 2004). Therefore, the changes in cyclic GMP production during gestation of the rat depend on not only the amount of NO production, which is regulated by arginase as well as endogenous NOS inhibitors, but also the guanylyl cyclase activity. Regarding the changes in guanylyl cyclase activity during gestation, the enzyme activity was up-regulated at mid-gestation and down-regulated at term gestation and after delivery (Figure 4). Therefore, we assume that changes in arginase, endogenous NOS inhibitors and guanylyl cyclase are implicated in the changes in cyclic GMP production, which is an important second messenger to modulate ET-1 production (Kelly et al., 2004).
In conclusion, the decreased arginase activity at mid-gestation possibly results from the down-regulation of arginase II protein, whereas the enhanced arginase activity at term gestation seems to be because of the induction of functional arginase I in concert with the attenuated arginase II expression. The enhanced overall arginase activity at term gestation could be implicated in increasing myometrial contractions mediated by the increased ET-1. The increased peptide production is possibly because of the reduced cyclic GMP production resulting from enhanced arginase activity, accumulated endogenous NOS inhibitors and attenuated guanylyl cyclase activity.
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Acknowledgements |
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This study was supported in part by 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 22, 2006; accepted on April 22, 2006.
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