Molecular Human Reproduction, Vol. 6, No. 8, 735-742,
August 2000
© 2000 European Society of Human Reproduction and Embryology
Uterine physiology |
Human myometrial cells in culture express specific binding sites for urinary trypsin inhibitor
Department of Obstetrics and Gynecology, Hamamatsu University School of Medicine, Handacho 3600, Hamamatsu, Shizuoka, 431-3192, Japan
Abstract
Urinary trypsin inhibitor (UTI), which is present in amniotic fluid, prevents uterine contractility during pregnancy possibly via specific binding protein mechanisms. To test for the presence of UTI binding sites on the cell surface, we prepared cultured myometrial cells obtained at biopsy from 12 pregnant women and performed binding, competition, and cross-linking experiments using a specific radiolabelled UTI as a ligand. We report for the first time two classes of binding sites of differing affinities. Scatchard analysis at 4°C, using radioiodinated UTI, revealed that UTI binds to 35 000 high affinity binding sites/cell (Kd = 9.1x10–9 mol/l) and 450 000 lower affinity binding sites/cell (Kd = 3.5x10–7 mol/l) in cultured myometrial cells. It appears to be the low affinity site that is internalized, and this has been identified as a protein of ~45 kDa by cross-linking and immunoaffinity labelling studies. Monoclonal antibodies against the NH2-terminal fragment of UTI abrogated specific binding of this protein to the cells. Treatment of the cells with hyaluronidase resulted in >80% inhibition of the [125I]-labelled UTI binding to the cells. These data show that the UTI binding site, which is hyaluronidase sensitive, is expressed on the surface of human uterine myometrial cells to accumulate the UTI molecule during pregnancy.
myometrium/parturition/urinary trypsin inhibitor/UTI/UTI-binding protein
Introduction
Urinary trypsin inhibitor (UTI) is a Kunitz-type protease inhibitor that is found abundantly in human amniotic fluid and urine and in lesser amounts in plasma (El Maradny et al., 1996
; Kobayashi et al., 1999). UTI efficiently inhibits trypsin, chymotrypsin, plasmin and granulocyte elastase (Bost et al., 1998). UTI, as well as being a protease inhibitor, plays an important inhibitory role in normal and pathological processes where signal transduction is involved, such as calcium influx (Kanayama et al., 1995a) and cytokine production (Maehara et al., 1995; Kaga et al., 1996a,b; Kakinuma et al., 1997; Futamura et al., 1999) in a variety of types of cells. Even though the liver is the major source of UTI (Bourguignon et al., 1983; Salier et al., 1987; Diarra-Mehrpour et al., 1989), several other human organs including kidney produce this molecule (Schreitmuller et al., 1987; Shikimi et al., 1992; Bratt et al., 1993; Chan and Salier, 1993; Itoh et al., 1996; Mizushima et al., 1998). UTI is released into the medium during the short-term culture of cells dispersed from amnion; its presence in amnion has been detected also by immunohistochemical techniques (El Maradny et al., 1994, 1996). Thus, the fetal kidney and amniotic epithelial cells are considered to represent the source of amniotic fluid UTI.
It has been reported that UTI showed a strong inhibitory effect on myometrial contractility stimulated by uterotonins (e.g. lipopolysaccharide, oxytocin and prostaglandins) both in an in-vitro experiment and an in-vivo animal model (Maehara et al., 1995; Kanayama et al., 1995a,b, 1996; El Maradny et al., 1996; Kaga et al., 1996a,b; Kakinuma et al., 1997; Katsuki et al., 1997; Futamura et al., 1999). It has been demonstrated that the addition of UTI to human myometrial strips suppressed myometrial contractility in vitro (El Maradny et al., 1996), suggesting the existence of a local ligand-receptor (or binding protein) interaction in uterine myometrium during pregnancy. We recently reported that the concentration of immunoreactive UTI is significantly decreased in the amniotic fluid during the third trimester of human pregnancy (Kobayashi et al., 1999). These results support the hypothesis that parturition may occur through the down-regulation of UTI which decreases uterine contractility and maintains the uterus in a state of quiescence during pregnancy.
The present study aimed to prepare cultured myometrial cells and perform binding experiments using radiolabelled UTI as a ligand. We have established the presence of specific binding sites for UTI in the human uterine myometrial cells at term.
Materials and methods
Subjects
Pregnant uterine myometrial tissue was obtained from 12 women undergoing elective Caesarean section (37–41 weeks gestation; not in labour) at term at Hamamatsu University Hospital and its related Hospitals (Shizuoka, Japan). The myometrial biopsy site was standardized to the upper margin of the lower segment of the uterus in the midline. Specimens obtained from women in labour were not included in this study. The indication for elective Caesarean section was a previous Caesarean section or breech presentation in otherwise uncomplicated pregnancies. All tissues were obtained from informed volunteer patients routinely seen by the physicians in the Department of Obstetrics and Gynecology, as approved by the Institutional Review Board. All women gave their informed consent to participate in this study which was approved by Hamamatsu University Hospital Ethical Committee.
Immediately upon collection, tissues were placed in sterile Ca2+ and Mg2+-free Hanks' balanced salt solution (Sigma Chemical Co, St Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS; Life Technology, Grand Island, NY, USA) for subsequent cell dispersion.
Myometrial cell culture
Tissue (~0.5 g) was minced and placed in HEPES-dissociated buffer (20 ml) together with 0.4% collagenase type II (Sigma) and 3000 IU deoxyribonuclease type IV (Sigma). After 1 h of stirring at 37°C, 0.4% trypsin (Sigma) was added and stirred continuously for an additional 10 min. Dissociated cells were collected as previously described (Vallet-Strouve et al., 1984). Myometrial smooth muscle cells were isolated and maintained in primary and first-passage monolayer cultures in Ham's F12/Dulbecco's minimal essential medium supplemented with 25 mmol/l HEPES buffer, 2.5 mmol/l glucosamine, 3 mmol/l glutamine, 0.03 mmol/l sodium pyruvate (Gibco, Rockville, MD, USA), 2.5 mmol/l sodium lactate, 5 mmol/l glucose (Yoneyama Chemical Co., Tokyo, Japan), 100 IU/ml penicillin, 100 µg/ml streptomycin, 0.25 mg/ml amphotericin B (Gibco) and 10% FBS. Cells were plated in 35 mm 6-well multidishes (Costar, Cambridge, MA, USA) in 2.5 ml culture medium/well (5.0–6.0x105 cells/well). The cultures were maintained at 37°C in a water-saturated atmosphere containing 5% CO2.
The purity of the cell cultures was immunohistochemically assessed with a variety of several protein markers. Antibodies to cytokeratin (for epithelial cells, ICN Pharmaceuticals Inc, Irvine, CA, USA); vimentin (for stromal cells, Biomeda, Foster, CA, USA); and desmin (for myometrial cells, Dako, Copenhagen, Denmark) were used to assess the cells using the avidin–biotin immunostaining method (Guang et al., 1999). In the present study, the majority (>80%) of cultured cells derived from the human uterine tissue were of the smooth muscle phenotype.
Myometrial cell extracts
For the preparation of myometrial cell extracts, the harvested cells (2x107 cells) were resuspended in 200 µL of PBS in the presence of inhibitor cocktail [1 µg/ml approtinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin; (Boehringer Mannheim [Roche Diagnostics], Mannheim, Germany); 1 mmol/l benzamidine, and 1 mmol/l phenylmethylsulphonyl fluoride (PMSF; Sigma )], sonicated on ice at 50 W for 30 s and clarified by centrifugation at 12 000 g for 15 min at 4°C. Protein concentration was determined with the Pierce protein assay kit (Pierce Chemical Company, Rockford, IL, USA).
We reported previously (Kobayashi et al., 1998a,b) that UTI is able to bind to specific binding sites which also bind hyaluronic acid on the surface of tumour cells. Therefore, Streptomyces hyaluronidase solution (50 IU/ml, 2 ml) was added to the cells (2x107 cells). After 10 min of incubation at room temperature, the hyaluronidase solution was carefully aspirated after cell centrifugation, and the cells were then washed and extracted as described above. Then sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and Western blotting using anti-UTI polyclonal antibodies (pAb UTI) were carried out.
Production of monoclonal antibodies raised against UTI
Monoclonal antibodies (mAbs) against UTI were produced as previously described (Kobayashi et al., 1999). mAbs from four clones were designated 2A6, 5C12, 4D1, and 8H11. 8H11 showed the strongest reactivity for UTI and was used for cell binding and competition assays.
Preparations of polyclonal antibodies raised against UTI
A highly purified preparation of human UTI was supplied by Mochida Pharmaceutical Co, Tokyo, Japan. Affinity-purified pAb UTI was prepared as previously described (Kobayashi et al., 1999). pAb UTI was reactive with the 240 kDa inter-
-inhibitor (II), the 120 kDa pre--inhibitor (PI), the 40 kDa UTI and the 8 kDa carboxyl-terminal domain of UTI (HI-8) in enzyme-linked immunosorbent (ELISA) or Western blotting assays.
Radiolabelling of UTI
Mixtures of 30–50 µg UTI in 50 µl 1 mol/l NH4HCO3, 200 µl PBS and 10 µl Na125I (1 mCi, 0.45 nmol iodine) were added to 1.5 mmol/l polyethylene tubes containing 100 µg dry Iodogen. The specific activity obtained with the Na [125I]-labelled material ranged from 0.8x106 to 3.1x106 cpm/µg.
Binding and dissociation experiments on myometrial cells
Binding of [125I]-labelled UTI to the cells was performed in the cell suspension or on cell monolayers grown to confluence. Binding was measured essentially as described previously (Blay and Brown, 1985). Binding and competition assays were performed at 4°C to avoid internalization of UTI. Samples of the cells were incubated with 200 ng/ml (= 5 nmol/l) [125I]-labelled UTI in the absence or presence of increasing concentrations of unlabelled ligand or other purified proteins (1-antitrypsin; AAT; plasminogen activator inhibitor type-1, and 2-macroglobulin; Cosmo Bio Co Ltd, Tokyo, Japan) for 3 h at 4°C. After the incubation mixtures were removed and the cells were washed rapidly five times, bound [125I]-UTI was counted in a Beckman 
4000 counter. Non-specific binding was determined in the presence of a 100-fold molar excess of unlabelled UTI. Specific binding was obtained after subtraction of the non-specific binding from the total binding. Each value was the average of triplicate determinations. Individual subcultures for the binding assays were seeded independently from stock flasks and were grown in separate batches for separate binding assays.
Binding and dissociation studies were performed under continuous shaking. Multiple independently grown confluent myometrial cell monolayers (n = 5) were incubated for 3 h at 4°C with 5 nmol/l of [125I]-UTI in binding buffer either with or without an excess of unlabelled UTI. At indicated incubation time points at 37°C, the integrity of radioactive material in supernatant was evaluated by precipitation with trichloroacetic acid (TCA, 10% w/v) in the presence of 1% bovine serum albumin (BSA). Radioactivity remaining bound to the rinsed monolayer was first released with 0.2 mol/l sodium acetate buffer containing 50 IU/ml Streptomyces hyaluronidase (10 min at room temperature): this procedure releases membrane-bound [125I]-UTI) and residual radioactivity (internalized [125I]-UTI) was solubilized with 1 mol/l NaOH (60 min at room temperature). Each value is expressed as the percentage total radioactivity bound to cell monolayers immediately before beginning of dissociation time course. Data points represent mean values determined for five independently grown myometrial cells. SD was <12% of each point. Analysis of the data was performed using the computer program LIGAND (Munson and Rodbard, 1980).
Molecular weight determination of UTI binding proteins on myometrial cells
This was accompanied by affinity chromatography. Coupling of UTI or BSA to cyanogen bromide-activated Sepharose 4B was performed following the manufacturer's protocol (Pharmacia). Cells were radiolabelled with 1 mCi of Na125I according to the manufacturer's protocol (Pierce), and lysates prepared as previously described (Becherer and Lambris, 1998; Kobayashi et al., 1998b). The lysates from 2x107 [125I]-labelled myometrial cells were first precleared with 100 µl of BSA–Sepharose 4B and then incubated with 100 µl of UTI–Sepharose 4B for 1 h at 23°C with rotation, followed by three washes. The beads resuspended in Laemmli's buffer without 2-mercaptoethanol were analysed by SDS–PAGE (12% acrylamide).
Immunochemical reactivity of UTI and its derivatives
The immunoreactivity of purified UTI, the COOH-terminal fragment of UTI (HI-8), and UTI reduced with 2-mercaptoethanol as well as amniotic fluid, urine and plasma was assessed by Western blotting (Kobayashi et al., 1999) and ELISA. The ELISA was essentially performed as previously described (Esparza et al., 1991) in a 96-well microtitre plate wells that had been precoated with 5 µg/ml proteins.
Cross-linking of [125I]-labelled UTI to its binding proteins
Monolayer cells were incubated for 3 h at 4°C with 200 ng [125I]-labelled UTI in the absence or presence of 20 µg unlabelled UTI. At the end of the incubation, after washing, succinimidyl 3-(2-pyridyldithio)propionate (Cosmo Bio) freshly dissolved in dimethyl sulphoxide was diluted in PBS at 23°C to a final concentration of 1 mmol/l. One ml of this solution was added to each dish and the dishes were incubated for 20 min at 23°C. Then SDS–PAGE analysis and autoradiography of the UTI–UTI-binding protein complexes was carried out (Presta et al., 1989).
Statistical analysis
All statistical analysis was performed using StatView for Macintosh. Dunnett's t-test was used to analyse statistically significant differences between groups by the multiple-comparison procedure. For comparison of two groups, the Mann–Whitney U-test or Dunnett's t-test were used. P < 0.05 was considered to be statistically significant. Numerical data from each experiment were expressed as mean ± SD.
Results
Characterization of purified UTI and related proteins and specificity of the antibodies
Using Western blot analysis, pAb UTI reacted with purified UTI, HI-8, amniotic fluid-derived UTI, urine-derived UTI, and with UTI-related proteins in plasma (II, PI, and UTI; see Salier, 1990) (Figure 1A). Amniotic fluid UTI corresponded in electrophoretic mobility to the 40 kDa purified UTI, urine UTI, and plasma UTI. mAb 8H11 did not react with HI-8 (Figure 1B), suggesting that the epitope resided in the NH2-terminal domain of UTI (since it was missing in HI-8). 8H11 also showed no reactivity with II and PI. Although II comprises two heavy chains and one UTI, it is likely that 8H11 failed to react with UTI within the II molecule, possibly by steric hindrance.
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In a parallel experiment, enzyme immunoassay analysis of the specificity of the pAb UTI and mAb 8H11 was performed (Figure 2
). pAb UTI recognized a determinant present on a wide variety of UTI preparations (UTI, HI-8, and UTI reduced with 2-mercaptoethanol). Contrasting these results, mAb 8H11 did not recognize a determinant present on the COOH-terminal fragment of UTI after the epitope was destroyed by reduction of the disulphide bonds. Thus, we conclude that the 8H11 determinant is sequested in the NH2-terminal structure of UTI.
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= pAb UTI and 
= mAb 8H11). Each column represents the mean of three observations ± SD (bar). Data were analysed by Mann–Whitney U-test. *Significant difference between polyclonal and monoclonal antibodies (P < 0.05).UTI is present in human myometrial cells
To evaluate the presence of UTI in myometrial cell cultures, extracts of the cells were analysed by Western blotting using pAb UTI (Figure 3
). The pAb UTI identified a major 40 kDa polydispersed band. Several minor immunoreactive proteins (22, 15 and 10 kDa) were also observed in the cell extracts. To confirm that the UTI present in myometrial cell cultures was due to UTI-binding proteins which bind to hyaluronic acid, the cells were treated with hyaluronidase. The pAb UTI also identified double bands of 10 kDa in the hyaluronidase-treated cells. The 40 kDa minor band (UTI) was observed in these cell extracts. It is likely that the 10 kDa double bands are UTI fragments endocytosed by the cells and that the 22 and 15 kDa bands were degradation products of UTI. These results suggest that UTI was mainly bound to the surface of myometrial cells via hyaluronic acid.
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Quantification of UTI binding to myometrial cells
UTI binding sites of the cells were defined in ligand binding and competition experiments. Labelled and unlabelled UTI bound with the same affinity to myometrial cells. At 4°C, the binding of labelled UTI to myometrial cells increased steadily, reaching a maximum after 3 h of incubation (data not shown). The UTI binding to the cells as a function of increasing concentrations of labelled UTI appeared saturable, approaching a maximum at 150~445 nmol/l [352 ± 71 nmol/l (mean ± SD), n = 8]. These results suggest the presence of apparent binding sites for UTI on the myometrial cells. Figure 4
shows that labelled UTI bound to myometrial cells in a specific manner. Of the total binding, >80% was specific, since it was blocked in the presence of a 20-fold excess of unlabelled UTI. The concentration of unlabelled UTI that reduced labelled UTI (200 ng) binding by 50% (IC50) was ~400 ng. This value was obtained from three independently conducted binding competition curves. Other protease inhibitors, e.g. 1-anti- trypsin (AAT), plasminogen activator inhibitor type-1 and 2-macroglobulin (data not shown), did not compete.
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We extended these observations to determine the number of UTI binding sites and the affinity constant. Scatchard plot analysis performed on five preparations of myometrial cells demonstrated the presence of 35 000 ± 8200 (mean ± SD) high affinity binding sites/cell (Kd = 9.1x10–9 mol/l) and of 450 000 ± 70 000 lower affinity binding sites/cell (Kd = 3.5x10–7 mol/l) (Figure 5
). Even though some variability was observed among the different cell preparations, no relationship was observed in the number and/or affinity constant of low and high affinity sites with respect to time in culture.
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8H11 inhibits labelled UTI binding to myometrial cells
Incubation of myometrial cells with labelled UTI in the presence of mAb 8H11 (>25 µg/ml) resulted in inhibition of >90% of the labelled UTI binding to the cells (Figure 6A
). Non-immune anti-mouse immunoglobulin G (IgG), however, displayed no inhibitory activity on the binding reaction. The labelled UTI binding to the cells was negated by the treatment of the cells with hyaluronidase (Figure 6B), suggesting that the UTI-binding sites were directly or indirectly bound to hyaluronic acid (see Figure 3
).
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Binding, dissociation and internalization of labelled UTI
In view of the above findings, all subsequent binding assays were performed at 4°C with an equilibration time of 3 h. Only a small proportion of the radiolabel was internalized by the cells (<10%). In contrast, at 37°C, binding was ~80% complete at 30 min, and maximal after 1 h of incubation (data not shown). The binding of [125I]-labelled UTI to the cells was reversible. Dissociation and internalization was readily monitored if labelled UTI was allowed to dissociate spontaneously from, and internalize into, the cells at 37°C (Figure 7
). During dissociation at 37°C for 10 min, 25% of the radiolabelled materials appearing in the incubation medium could be precipitated with TCA and, therefore, probably represented non-degraded labelled UTI. A large proportion of the radiolabel was internalized by the cells (~50%). Upon dissociation and internalization at 37°C, intact, non-degraded labelled UTI released into the incubation medium matched exactly the reduction in cell surface-bound protein (hyaluronidase elutable). Only a small proportion (<20%) of the labelled UTI was resistant to precipitation by TCA. Thus we were able to conclude that at 37°C the cellular uptake of UTI was relatively high.
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= internalized labelled UTI; Cross-linking of [125I]-labelled UTI to the cell surface of myometrial cell cultures
Cross-linking of labelled UTI to the cell surface of human myometrial cell monolayers revealed the presence of UTI–UTI-binding sites complexes with molecular weight of ~85 kDa (intense band) and 80 kDa (faint band) (Figure 8
). This indicates a molecular weight for the UTI-binding protein of ~40–45 kDa.
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) was carried out, followed by autoradiography of the gel. The data are representative of three independent experiments. Molecular weight markers (Bio-Rad) were analysed in adjacent lanes.Identification of UTI-binding protein on myometrial cells
We performed affinity matrix chromatography to determine the relative molecular size of the UTI-binding proteins on [125I]-labelled myometrial cells (Figure 9
). The supernatant from cell lysates was applied to a BSA-coupled Sepharose column and then reacted with UTI-activated Sepharose beads in the absence or presence of 8H11. The beads were washed and the UTI-Sepharose–reactive proteins eluted. Analysis by SDS–PAGE under non-reducing conditions revealed a specific band of relative molecular weight ~45 kDa whose interaction with the affinity beads was inhibited by 8H11. The ~45 kDa band was specific and reproducibly observed in three independent experiments.
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= 45 kDa UTI binding protein; and 
= the 40 kDa UTI as a control.Discussion
The present study is based upon previous studies suggesting that UTI may be important in control of uterine contractility during pregnancy (Maehara et al., 1995; Kanayama et al., 1995a,b, 1996; El Maradny et al., 1996; Kaga et al., 1996a,b; Kakinuma et al., 1997; Katsuki et al., 1997; Futamura et al., 1999). The main finding of this study was that human cultured myometrial cells obtained at biopsy during pregnancy at term express and contain specific binding sites for UTI. We report for the first time two classes of binding sites of differing affinities and the identification of a ~45 kDa binding protein. This protein is possibly the low affinity binding site for UTI and is internalized.
The present study gave the following results: the specific UTI-binding sites were identified functionally by their ability to bind [125I]-labelled UTI and competitive studies using unlabelled compounds. The binding of labelled UTI to the cell as a function of increasing concentrations of labelled UTI appeared saturable, approaching a maximum at 352 ± 71 nmol/l. This value agreed well with the maximal binding estimated visually from the binding curve obtained from earlier experiments using some types of neoplastic cell lines (Kobayashi et al., 1994, 1998a,b). The present quantitative binding experiments demonstrate two binding affinities for UTI on myometrial cells. We found 35 000 high affinity binding sites/cell with a Kd of 9.1x10–9 mol/l and 450 000 lower affinity sites/cell with a Kd of 3.5x10–7 mol/l. In previous studies, we observed only a single family of low affinity UTI binding sites with a Kd of ~2.0x10–7 mol/l in some types of tumour cells (Kobayashi et al., 1994, 1998a,,b). The reasons why we previously detected only a single class of binding sites in tumour cells are not understood, but again it may reflect differences in the cells used.
The UTI-binding sites were identified immunologically by the cross-linking of [125I]-UTI to its binding sites, demonstrating the presence of UTI–UTI-binding site complexes with a molecular weight of 80–85 kDa. Furthermore, the molecular weight of the UTI-binding sites was determined by the immunoaffinity precipitation method, demonstrating that the ~45 kDa UTI-binding protein is expressed in myometrial cells. Hence, we speculate that the ~45 kDa protein may correspond to the lower affinity binding site for UTI, although the reasons why only a single band is detected by such immunological analyses are not understood.
We found that the binding of [125I]-labelled UTI to myometrial cells was selective and domain specific. In the present study, we have demonstrated that mAb 8H11, which recognizes the NH2-terminal subdomain of UTI, very effectively inhibits UTI binding to the cells. mAb 8H11 may prevent ligand binding by steric hindrance. Recently, Xu et al. (Xu et al., 1998) reported that UTI has a hydrophobic domain close to the NH2-terminus, which is the most likely site for a cell-surface binding site. Taken together, our results indicate that the NH2-terminal domain of UTI contains the ligand for the UTI-binding sites on myometrial cells.
Furthermore, the binding of [125I]-labelled UTI to myometrial cells was displaceable. At 4°C cultured myometrial cells expressed [125I]-UTI mainly on their cell surface and in the extracellular matrix, while at 37°C, during dissociation a part of the radiolabelled material appeared in the incubation medium and labelled UTI was markedly deposited inside the cells. In addition, ~80% of the binding intensities were negated by the treatment with hyaluronidase, suggesting that the UTIbinding sites are bound to hyaluronic acid. The residual binding following hyaluronidase treatment may be other UTI-binding sites including the `UTI receptor'. Our previous report (Hirashima et al., 1997) showed that UTI failed to directly bind to hyaluronic acid. These results suggest that most of UTI binds to the cell surface possibly via UTI-binding sites bound to cell-associated hyaluronic acid.
We also showed that UTI is internalized by myometrial cells, possibly through an endocytotic pathway. It has been established that the uptake of UTI was observed by kidney epithelial cells (Yamasaki et al., 1996). The uptake of UTI by the proximal tubule is reported to be accomplished by adsorptive endocytosis on the luminal surface membrane. Our present results support the hypothesis that the cell-associated, hyaluronic acid-bound UTI-binding proteins may prevent UTI diffusing away from the cell surface and therefore keeping it in an environment rich in hyaluronic acid and mediating internalization of UTI. It is possible that UTI transduces a certain signal by endocytosis, although the mechanisms responsible for UTI uptake remain uncertain.
The present study adds much to the overall picture of the involvement of UTI in control of uterine contractility and the maintenance of pregnancy or initiation of labour. There have been many reports on the contractile effects of UTI in vitro (with animal and human tissues) and in vivo (in animal models) (Maehara et al., 1995; Kanayama et al., 1995a,b, 1996; El Maradny et al., 1996; Kaga et al., 1996a,b; Kakinuma et al., 1997; Futamura et al., 1999). UTI showed a strong inhibitory effect on oxytocin- and lipopolysaccharide- dependent myometrial contractility in vitro and in vivo. This study convincingly demonstrated that UTI has an important physiological role in the regulation of uterine contractility via an UTI-binding protein mechanism. UTI found in the myometrial cells may result from the transepithelial flux from UTI amniotic fluid. The UTI extravasated from the amniotic cavity is able to bind rapidly to UTI-binding sites on the surface of myometrial cells. These data strongly demonstrate that locally expressed UTI-binding sites accumulate amniotic fluid-UTI on uterine myometrial cells in pregnant women. When patients are close to parturition at term, amniotic fluid UTI concentrations decreased (Kobayashi et al., 1999), which may enhance the contractile activity of myometrium induced by oxytocin and/or prostaglandins, leading to a cascade of events critical for parturition.
Nothing is known so far about whether the expression of functional UTI-binding sites in myometrial tissues is quantitatively and qualitatively altered during labour. It is conceivable that qualitative and quantitative abnormalities in the myometrial UTI-binding sites and/or amniotic fluid UTI might be involved in the pathophysiology of both preterm labour and dysfunctional uterine activity at term. Further characterization of these proteins is needed to understand their physiological significance, as well as their involvement in preterm delivery.
Acknowledgments
We are grateful to Drs.T.Kobayashi, N.Kanayama, T.Nishiguchi, H.Ohi, N.Tokunaga, M.Yamashita, M.Yonezawa, T.Yamazaki, K.Suzuki, and H.Sadakata for the supply of tissue samples used in this study.The authors also thank M.Fujie and K.Shibata (Equipment Center, Hamamatsu University School of Medicine, Shizuoka) and Dr.T.Urano (2nd Physiology, Hamamatsu University School of Medicine, Shizuoka) for helping with the biochemical analysis and are thankful to Drs.E.Morishita and K.Kato (BioResearch Institute, Mochida Pharmaceutical Co, Tokyo), Drs.M.Ikeda and S.Miyauchi (Seikagaku Kogyo, Tokyo) and Drs.Y.Tanaka and T.Kondo (Chugai Pharmaceutical Co, Tokyo) for their continuous and generous support of our work.
Notes
1 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, Hamamatsu University School of Medicine, Handacho 3600, Hamamatsu, Shizuoka, 431-3192, Japan. E-mail: hirame{at}hama-med.ac.jp 
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Submitted on February 21, 2000; accepted on May 11, 2000.
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