In vitro effects of endothelin-1 on the contractility of myometrium obtained from pre- and postmenopausal women

This study was conducted to evaluate the responsiveness of human nonpregnant myometrium to endothelin 1 (ET1) (10 /p110 M-10/p16 M) and KCl (80 mM) in relation to the hormonal profile of the women, who were allocated into three groups: group 1, premenopausal follicular phase, n=14, group 2, premenopausal luteal phase, n=20, and group 3, postmenopausal women, n=12. At a concen- tration of 10 /p16 M, ET1 in both groups 1 and 2 induced very low ripples of high frequency (group 1: 80 /p514%, n=5, group 2: 314/p563%, n=11; P<0·05 compared with the pretreatment frequency) which lasted significantly longer in group 2 (29 /p52 min, n=10, P<0·05) than in group 1 (20 /p52 min, n=5), increasing the basal tone (group 1: 57·9 /p56%, n=5, group 2: 64·4 /p54%, n=6), the amplitude of myometrial contractility (group 1: 1·2/p50·07 g, n=5, group 2: 1·6 /p50·1 g, n=7, P<0·05) and the area under the contractility curve (AUC; group 1: 8·4/p51·1 g/p2min, n=6, group 2: 11·9 /p51·6 g/p2min, n=11). In group 3, ET1 (10 /p16 M) created a sustained long-lasting contraction (initial phase: 43 /p56 min, n=6) characterized by the complete obliteration of spontaneous contractility with no ripples at all, and increasing signifi- cantly (P<0·05) the amplitude of myometrial contractility (2·8/p50·5 g, n=6), the AUC (24·7 /p53·3 g/p2min, n=6), as well as the basal tone (183·6 /p521%, n=6) compared with the two premenopausal groups. In all three groups KCl exposure induced an initial rise (mean amplitude value: 1·1 g) followed by a relaxation phase to the primal baseline level (mean duration value: 12 min). Addition of ET1 (10 /p16 M) to KCl (80 mM) induced a similar pattern of contractility to that evoked by ET1 alone which, compared with KCl alone lasted significantly longer (P<0·05) in all three groups (group 1: 20 /p52 min, n=6; group 2: 23/p52 min, n=6; group 3: 35/p53 min, n=5). In group 3, the percentage change in basal tone was signifi- cantly smaller following KCl than after the combination of KCl plus ET1 (149 /p516%, n=5; P<0·01), indicating a different mechanism of contractility between KCl and ET1. These results demonstrate for the first time di ffer- ences in myometrial response to ET1 between pre- and postmenopausal women. It is suggested that KCl and ET1 affect uterine contractility through di fferent mechanisms and that ovarian steroids may play a regulatory role in human uterine responsiveness to ET1. Journal of Endocrinology (2001) 168, 153–162

Endothelins (ETs) consist of a family of three sarafotoxin- like peptides ET1, ET2 and ET3 originally isolated from the supernatant of cultured porcine endothelial cells (Yanagisawa et al. 1988). They are produced by di fferent cell types, endothelial and epithelial cells (Ohkubo et al. 1990, Sakurai et al. 1991, Kamada et al. 1992) and are primarily described for their potent vasoconstrictor actions (Davenport et al. 1990, Bodelsson et al. 1996, Elchalal & Schenker 1997). It has also been reported that ETs modulate the contractility in a variety of tissues. The effects of ETs are mediated through two cloned and sequenced subtypes of receptors, ETA and ETB, which are members of the G-protein-linked receptor superfamily (Masaki et al. 1994); ET1 is a selective ligand for ETA receptor (ET1 >ET2), whereas the three peptides, ET1, ET2 and ET3 have been reported to display equal affi nity for ETB receptor (ET1=ET2=ET3) (Arai et al. 1990, Sakurai et al. 1990, 1992, Bacon et al. 1995). In particular, the 21-amino acid peptide, ET1, is a potent, long-acting vasoconstrictor and proliferative agent produced by a wide range of human cell types (Sunnergen et al. 1990, Marciniak et al. 1992, Casey & MacDonald 1996) and plays a functional role in the female reproductive system (Kamada et al. 1993, Haq et al. 1996, Apa et al. 1998). Northern blot analysis demonstrated the release of ET1 by human decidual cells in early pregnancy 153 Journal of Endocrinology (2001) 168, 153–162 0022–0795/01/0168–153 /p302001 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology.org Downloaded from Bioscientifica.com at 06/13/2026 05:21:17AM via free access (Kubota et al. 1992) and the presence of immunoreactive prepro ET1 and prepro ET1 mRNA in human endo- metrial tissue (Economos et al. 1992, Salamonsen et al. 1992, Cameronet al. 1992, 1993, Marsh et al. 1994). It has been reported that the binding sites for ET1 are distributed in the human uterus throughout the menstrual cycle (O’Reilly et al. 1992) and that both subtypes of receptors are localized in human myometrium, where ETA binding sites represent the principal subtype (Schi ff et al. 1993, Breuiller-Fouche et al. 1994, Pekonen et al. 1994, Wolff et al. 1996). It has been demonstrated that in human nonpregnant myometrium, ET1 induces contractions (Word et al. 1990, Fried et al. 1993, Svane et al. 1993) activating exclusively the ETA receptors, and increasing the two phases of spontaneous myometrial contractility, the phasic and the tonic phase, despite the lesser sensitivity of nonpregnant compared with pregnant myometrium (Word et al. 1991, Osada et al. 1997). Although binding studies have shown that ET1 exhibits a ffi nity for ETB receptors, activation of ETB sites, using ETB selective ligands, has not been reported to mediate any contractile effect on human myometrial tissue; the lack of any contractile effect on human nonpregnant uterus has been attributed to the small population of ETB receptors (less than 25%) on human myometrium, or to their involve- ment in mechanisms of relaxation and proliferation; the precise role of ETB subtypes of receptors in human uterus needs to be further elucidated (Maggi et al. 1994, Bacon et al. 1995, Heluy et al. 1995, Wolff et al. 1996, Osada et al. 1997). However, in none of these studies has the effect of ET1 on myometrial contractility been examined specifically in relation to the hormonal pro file of the women. Knowing that human uterus is a target organ of sex steroids, the purpose of our study was to elucidate the effect of ET1 on human uterine contractility in relation to the sex steroid milieu of the women, and to determine possible alterations in premenopausal women (follicular or luteal phase of the normal menstrual cycle) and in postmenopausal women.

Specimens Myometrial tissue was collected from women undergoing hysterectomy for benign gynecological disorders. Informed consent was obtained. All women were operated on under the same conditions in terms of premedication and anes- thetic drugs. None of the patients had been taking any type of hormonal therapy for the previous three months. The samples were excised with a scalpel from the anterior and the posterior surface of the body of the uterus (macroscopically normal muscle), placed in ice-cold Krebs’ solution and taken immediately to the laboratory. The tissues collected from the women were allocated into three groups on the basis of the hormonal profile defined from the first day of their last menstrual period, and from serum progesterone and estradiol concentrations measured by enzyme linked fluorescent assay (ELFA) in peripheral blood samples obtained early in the morning of the day of the operation (means /p5...); group 1: premeno- pausal women in the follicular phase, mean estradiol value 222 ·4/p542 pmol/l, mean progesterone value 1·7/p50·1 nmol/l (age: 40 –45 years, n=14); group 2: premenopausal women in the luteal phase, mean estradiol value 653 ·5/p5141 pmol/l, mean progesterone value 57·1/p531 nmol/l (age: 40 –45 years, n=20); group 3: postmenopausal women, estradiol value <87·2 pmol/l, progesterone value <1·3 nmol/l (age: 65–70 years, n=12). Experiments The experiments were performed and completed within the first 10 h after the removal of the uterus from the abdomen, most usually within the first 6 –7 h. The viability of the tissue under investigation was con firmed by the responsiveness of human myometrium to KCl (80 mM) at the end of each experiment. The specimens were immediately dissected into longitudinal strips of 5/p22/p21 mm parallel to the muscle fiber orientation. Briefly, the strips were mounted horizontally in bathing chambers for isometric recording with one end fixed and the other attached to an isometric transducer connected to an amplifier. An initial resting tension of 1 g was applied to each strip. The tissues were continuously perfused with Krebs’ solution at 37 /p8C, gassed with 95% O 2 and 5% CO2. Tension generated by the muscle strips was recorded on a GRASS FTO3C. force displacement transducer and displayed on a universal oscillograph (Harvard) recorder. During the experiments, the strips were allowed to equilibrate for 1 to 2 h until the spontaneous contractility became regular in frequency and intensity. The tissues were then exposed to the various stimuli for 7 min, and washed out with Krebs’ solution. The duration of the drug application in our experimental procedure was dictated by two factors: first, the time required for the superfusing solution to reach steady-state concentration within the bath, and secondly the high cost of ET1. Therefore, the 7-min application period used in our experiments was considered a satisfactory period of time to ascertain the achievement of the full effect of the drug, and at the same time to limit the cost of the experiment. Two types of experiment were performed. In the first series of exper- iments five di fferent concentrations of ET1 (10 /p110 M, 10/p19 M, 10/p18 M, 10/p17 M, 10/p16 M) were applied sep- arately on each strip; the strips used during each exper- iment were from the same uterus (group 1: n=6 uteri, group 2: n=11 uteri, group 3: n=6 uteri). In the second series of experiments each strip was exposed to KCl (80 mM), allowed to re-equilibrate for 30 min at least and were then exposed to di fferent concentrations of the combination of KCl and ET1. The latter was used at E DOMALI and others · Endothelin in human nonpregnant uterus154 www.endocrinology.orgJournal of Endocrinology (2001) 168, 153–162 Downloaded from Bioscientifica.com at 06/13/2026 05:21:17AM via free access concentrations of 10/p110 M, 10/p19 M, 10/p18 M, 10/p17 M, and 10 /p16 M (group 1: n=8 uteri, group 2: n=9 uteri, group 3: n=6 uteri). To evaluate the contractile activity generated and the possible alterations before and after tissue treatment with the stimuli (namely KCl, ET1 or the combination of KCl and ET1), a number of parameters were studied. Change in basal tone The change in basal tone in the immediate 10-min period after tissue treatment with the stimulus was expressed as the percentage of the mean amplitude of the spontaneous contractions occurring in the 10-min period preceding the addition of the stimuli. Basal tone was de fined as the lowest point (baseline) of the spontaneous contractions before the application of the stimuli (Fig. 1). The amplitude of the spontaneous con- tractions was chosen because it expresses the contractile potential of each myometrial strip. The change in basal tone was calculated as shown in the examples in Fig, 1 using the formula x=/afii9826//afii9825/p2100 where x is the percentage change in the basal tone, /afii9825is the mean amplitude of the spontaneous myometrial contractions, and /afii9826is the mean values of the distance of the lower parts of the induced contractions from the initial baseline. In case I, /afii9825=1·94 g, /afii9826=0·842 g and x=43 ·4%, i.e. application of the stimulus raised the basal tone by 43 ·4% of the amplitude of the contractions preceding the stimulus. In case II, /afii9825=1·335 g, /afii9826=1·891 g and x=141 ·6%, i.e. tissue treatment with the stimulus elevated the basal tone by 141·6% of the amplitude of the contractions preceding the stimulus. Frequency of myometrial contractility Frequency of myometrial contractility in a 20-min period after tissue treatment with the stimulus was expressed as the percent- age change in the sum of spontaneous contractions occur- ring in a period of 20 min before the application of the stimulus. The area under the contractility curve The area under the contractility curve (AUC) was determined as the integrated force from the start of the induced contrac- tion up to 10 min from the application of the stimulus and was quantified by planimetry of the included area. Figure 1 The change in basal tone of our preparations after 10-min treatment with the stimuli ((a) premenopausal women; (b) postmenopausal women). The alterations in basal tone observed during the experiments were calculated using the formula: x =/afii9826//afii9825/p2100, where x is the percentage change in the basal tone, /afii9825is the mean amplitude values of spontaneous myometrial contractions, and /afii9826is the mean distance of the lower parts of the induced contractions from the initial baseline. Endothelin in human nonpregnant uterus · E DOMALI and others 155 www.endocrinology.org Journal of Endocrinology (2001) 168, 153–162 Downloaded from Bioscientifica.com at 06/13/2026 05:21:17AM via free access The amplitude of myometrial contractility The amplitude of myometrial contractility was de fined as the value of the distance between the highest point and the initial baseline of the evoked contractility in a period of 10 min after the application of each stimulus. Duration of alterations in myometrial contractility The duration of alterations in the myometrial contractility after the addition of each stimulus was determined as the period of time from the application of the stimulus until the reappearance of relatively regular spontaneous contractility. Reagents The stock solution consisted of a mixture of ET1 (0·1 mg) with 4 ml distilled water resulting in an ET1 concentration of 10 /p15 M. The concentrations of ET1 used in our experiments were prepared with sequential dilutions of the initial stock solution. ET1 (0 ·1 mg) was purchased from SIGMA-ALDRICH CHEMIE, GmbH P.O. 1120, 89552 Stenheim, Germany. The ionic composition of the modified Krebs’ solution was as follows: NaCl 110·9 mM, KCl 5 ·9 mM, MgCl 2 1·1 mM, CaCl 2 2 mM, NaH2PO4.H2O1 ·2 mM, glucose 9 ·6 mM, NaHCO 3 25 mM. Drugs were purchased from E. Merck, D-6100 Darmstadt, F.R. Germany, except for glucose and NaHCO 3 which were purchased from Mallinckrodt Chemical Works, St Louis, MO, USA and Mallinckrodt Baker B.V., Deventer, Holland respectively. Hormone assays Blood samples were centrifuged at 3000 cycles/min at least. The serum was extracted from the supernatant and assayed in an automated multiparametric immunoanalyzer; the analyzer functions on the basis of a technical method (ELFA), which combines enzyme immunoassay with fluorescent reading (450 nm). The immunoanalyzer and the reagents were purchased from bioMerieux sa 69280, Marcy-l’Etoile, Paris, France. Statistical analysis The responses of the human myometrium to di fferent stimuli (KCl, ET1, or KCl and ET1) were compared by one-way analysis of variance. Mean values and standard errors of the mean (means/p5...) were determined, and the statistical significance was confirmed by the use of the Student’s unpaired t-test, where appropriate.

During tissue equilibration, segments collected from pre- menopausal women (follicular phase, n=14; luteal phase, n=20) showed excessive spontaneous motility compared with those collected from postmenopausal women (n=12). In premenopausal women, spontaneous contrac- tions were revealed immediately (within 2 –3 min) after the application of 1 g tension and they increased progres- sively in amplitude and frequency (Fig. 2a). In postmeno- pausal women, myometrium contracted spontaneously much later (30 min), and the frequency of the contractions was lower than in the premenopausal groups (Fig. 2b) . During the first series of experiments, the effect of ET1 was evaluated on human uterine muscle strips. At concen- trations ranging from 10 /p110 Mt o1 0/p17 M, ET1 induced a dose-dependent increase in the frequency of the contrac- tions in tissues collected from premenopausal women without any statistically signi ficant di fference between groups 1 and 2. At a concentration of 10 /p16 M, ET1 in Figure 2 Representative traces of spontaneous myometrial contractility evoked after the application of 1 g resting tension in strips obtained from (a) premenopausal and (b) postmenopausal women. In post- menopausal preparations spontaneous contractions appeared later, after 30 min on average compared with 2–3 min in both groups 1 and 2 (premenopausal women). E DOMALI and others · Endothelin in human nonpregnant uterus156 www.endocrinology.orgJournal of Endocrinology (2001) 168, 153–162 Downloaded from Bioscientifica.com at 06/13/2026 05:21:17AM via free access both groups 1 and 2 caused a change in the pattern of myometrial contractility (Fig. 3), increasing the basal tone and inducing very low ripples of high frequency. Com- pared with the pretreatment frequency, an increase in group 1 of 80/p514% (n=5) and in group 2 of 314 /p563% (n=11, P<0·05) was found. The increase in basal tone was greater in group 2, in which the ripples were less discernible, than in group 1 (Fig. 3a and b). The height of the ripples increased gradually in both groups before regular contractility was re-established. The change in myometrial contractility up to the time of onset of regular contractions lasted signi ficantly longer in group 2 (29/p52 min, n=10) than in group 1 (20 /p52 min, n=5, P<0·05). During the period of re-establishment of regular contractions, the frequency of contractions was still higher than before the treatment with ET1, especially in group 2; in other words, the appearance of regular contractions was achieved quicker in group 1 than in group 2 (Fig. 3a,b). In postmenopausal women (group 3), the application of the lower concentrations (10 /p110 M, 10 /p19 M, 10 /p18 M) of ET1 showed no significant effect on the frequency of the evoked contractions. At concentrations of 10 /p17 M and particularly at 10 /p16 M, as in groups 1 and 2, ET1 caused a remarkable change in the pattern of myometrial contrac- tility which, however, differed from that in the other two groups in that the action of ET1 resulted in a sustained long-lasting contraction, the initial part of which lasted 43/p56 min (n=6) and was characterized by the complete obliteration of spontaneous contractility with no ripples at all (Fig. 3c). Following this, ripples appeared without any sign of onset of regular contractility for a period of at least 2 h. This pattern was closer to that in group 2, but in group 3 the basal tone increased further and the change lasted much longer. Application of KCl (80 mM) alone to myometrial strips (Fig. 4) evoked a contraction characterized by an initial rise followed by a slow relaxation phase to the initial baseline level which lasted on average 12 min with no significant difference between the three groups. Addition of ET1 (10 /p16 M) and KCl (80 mM) induced a pattern of contractility which was similar to that induced by ET1 alone with no signi ficant di fference between the corre- sponding groups. Compared, however, with the contrac- tile pattern evoked by KCl alone, the combination of KCl and ET1 signi ficantly increased the duration of change in myometrial contractility ( P<0·05) in groups 1 (20 /p5 2 min, n=6), and 2 (23 /p52 min, n=6), while in group 3 (35 /p53 min, n=5) regular myometrial motility was re-established much later ( P<0·01) than in the two premenopausal groups (Fig. 4a,b,c). Table 1 compares the amplitude of myometrial contrac- tility, the AUC, and the percentage increase in basal tone during application of KCl alone, ET1 alone, and the combination of KCl and ET1 in the three groups. A dose–response effect of ET1 on AUC was found (Fig. 5). Treatment with ET1 alone (10 /p16 M) induced amplitude and AUC values that did not di ffer significantly between groups 1 and 2, but were significantly greater (P<0·01) in group 3 compared with the two premenopausal groups (Table 1). Addition of ET1 to KCl significantly restricted (P<0·05) the myometrial responsiveness induced by ET1 alone only in group 3. Normalization of the data to KCl confirmed the statistical di fferences between the groups (Fig. 6). This signi ficant attenuation should be attributed Figure 3 Representative traces showing alterations in human spontaneous myometrial contractility after treatment with ET1 at a concentration of 10 /p16 M in (a) group 1, premenopausal women, follicular phase (n=6), (b) group 2, premenopausal women, luteal phase ( n=11) and (c) group 3, postmenopausal women (n=6). In the premenopausal groups, ET1 altered the pattern of spontaneous myometrial contractility, inducing very low ripples of high frequency, which lasted longer in group 2 than in group 1; in group 3, ET1 induced a sustained long-lasting contraction with no ripples at all which lasted much longer than those in both groups 1 and 2. Endothelin in human nonpregnant uterus · E DOMALI and others 157 www.endocrinology.org Journal of Endocrinology (2001) 168, 153–162 Downloaded from Bioscientifica.com at 06/13/2026 05:21:17AM via free access to the smaller amplitude of spontaneous contractions that occurred for technical reasons during the period preceding the application of the stimulus in strips obtained from postmenopausal women as compared with premenopausal women (Table 2). In fact, when the data were analyzed on the basis of basal tone, expressed as the percentage change in myometrial contractility induced by the stimuli, this difference was eliminated. In particular, we found that ET1 in both premenopausal groups elevated the initial baseline, without signi ficant di fferences between them, while in postmenopausal women (group 3) the percentage increase in basal tone was significantly greater than in the other two groups. Addition of ET1 to KCl did not a ffect the elevation in basal tone induced by ET1 alone in any of the three groups, which remained signi ficantly (P<0·05) greater in group 3 compared with groups 1 and 2 (Table 1). Similarly, although the amplitude and the AUC induced by KCl alone were significantly smaller in group 3 than in groups 1 and 2, the percentage increase in basal tone induced by KCl did not di ffer significantly between the three groups (Table 1). In group 3, the percentage change in basal tone was signi ficantly smaller following KCl than following ET1 or the combination of KCl plus ET1. No signi ficant correlations were found between estradiol or progesterone concentrations and the contractile parameters used on an individual basis.

This study demonstrates signi ficant changes in the spon- taneous contractility of human nonpregnant myometrium evoked by ET1 in postmenopausal and premenopausal women. The main finding of the study was the long- lasting e ffectiveness of ET1 in strips collected from postmenopausal women compared with premenopausal Figure 4 Representative traces showing the effects of KCl alone (80 mM) and the combination of KCl and ET1 at a concentration 10 /p16 M on human myometrial contractility in (a) group 1, premenopausal women, follicular phase (n=8), (b) group 2, premenopausal women, luteal phase ( n=9) and (c) group 3, post- menopausal women (n=6). Addition of ET1 extended significantly the contractile pattern induced by KCl in group 2 and especially in group 3. Table 1 Alterations in the amplitude of myometrial contractility, the area under the contractility curve (AUC), and the basal tone induced by KCl, ET1, and the combination of KCl plus ET1 at a concentration 10 /p16 M in premenopausal (group 1, follicular phase; group 2, luteal phase) and postmenopausal (group 3) women Group 1 Group 2 Group 3 KCl ET1 KCl +ET1 KCl ET1 KCl +ET1 KCl ET1 KCl +ET1 AUC (g /p2min) 5·5 /p50·2 8·4 /p51·1a 7·1/p50·2 5·0 /p50·6 11·9 /p51·6b 9·6/p51·5 4·9 /p50·8 24·7 /p53·3 12·3 /p53·2+ Amplitude (g) 1·2 /p50·1 1·2 /p50·07c 1·2/p50·1 1·2 /p50·1 1·6 /p50·1d 1·1/p50·1 1·0 /p50·2 2·8 /p50·5 1·3 /p50·3++ Basal tone (%) 43·0 /p53 57·9 /p56e 53·6/p57* 52·0 /p59 64·4 /p54e 61·0/p55* 53·0 /p55 183·6 /p521 149·0 /p516 aP<0·001, bP<0·01, cP<0·001, dP<0·05, eP<0·001 compared with group 3 (ET1); +P<0·05, ++P<0·05 compared with ET1 of group 3; * P<0·05 compared with group 3 (KCl and ET1). E DOMALI and others · Endothelin in human nonpregnant uterus158 www.endocrinology.orgJournal of Endocrinology (2001) 168, 153–162 Downloaded from Bioscientifica.com at 06/13/2026 05:21:17AM via free access women, but signi ficant di fferences were also found between the two premenopausal groups. In particular, the pattern of changes in myometrial contractility induced by ET1 in the myometrium from women in the luteal phase was between the patterns of the other two groups. These differences are di ffi cult to explain. It is known that the contractile effects of ET1 on human nonpregnant myometrium are mediated exclusively by ETA receptors (Baconet al. 1995, Heluy et al. 1995). The discrepancies in the myometrial responsiveness to ET1 in the three groups observed in the present study could not be attributed to the density or the affi nity of the ligand for its receptor as it has already been reported that the binding capacity of ET1 (affi nity) does not alter between pre- and postmenopausal women (Schi ff et al. 1993, Maggi et al. 1994). Therefore, other mechanisms possibly related to the differentiated hormonal profile of the women may be important. Although individual hormonal values did not correlate significantly with the changes in the contractility induced by the stimuli, it is possible that myometrial contractility to ET1 is enhanced by the estrogen deficiency after menopause, thus explaining the long- lasting effect of ET1 seen in group 3. That the pattern of contractility in group 2 was closer to that of group 3 suggests that in terms of responsiveness to the contractile agent, ET1, the myometrium during the luteal phase behaves in a more or less similar manner to that after the menopause. It could be postulated that the counteractive effect of progesterone to estrogen during the luteal phase creates conditions of contractility in the myometrium similar to those in postmenopausal women, but this requires investigation. The intracellular mechanism that could mediate the enhanced responsiveness of estrogen- deprived human uterus may be associated with alterations in the receptor ’s functions leading to changes in the post-receptor biochemical events (Osada et al. 1997). In the present study, application of KCl (80 mM) provoked an initial rise in the spontaneous myometrial Figure 5 Effect of KCl, ET1 and a combination of KCl and ET1 on myometrial contractility (expressed as the AUC) in a period of 10 min after treatment with the stimulus in premenopausal ((a) follicular and (b) luteal) and postmenopausal (c) women; a dose –response effect of ET1 on AUC was found. /p12, KCl, /p11, ET1, /p20, KCl +ET1. Endothelin in human nonpregnant uterus · E DOMALI and others 159 www.endocrinology.org Journal of Endocrinology (2001) 168, 153–162 Downloaded from Bioscientifica.com at 06/13/2026 05:21:17AM via free access contractility followed by a relaxation phase to the original baseline without significant differences between the three groups. This means that the responsiveness of the human nonpregnant uterus to KCl did not alter, despite the change in the hormonal pro file of the women, while in pregnant myometrium increased sensitivity to KCl during the progress of gestation has already been reported (Izumi et al. 1990). The nonchanging responsiveness of nonpregnant human myometrium to KCl is in contrast to the e ffect of ET1 that was enhanced in the luteal phase and particularly in the postmenopausal women, thus demonstrating a di fferential response of human nonpregnant myometrium to ET1 and KCl. Although the addition of ET1 extended signi ficantly the relaxation phase of the KCl-induced contractions in the three groups, basically the pattern of contractility induced by ET1 alone was una ffected by the presence of KCl. The differential response of human uterus to ET1 and KCl might re flect the involvement of a variety of bio- chemical mechanisms in the evoked myometrial contrac- tility. A series of studies has already suggested that KCl induces contractions through voltage-dependent Ca ++ influx, which is completely abolished by the presence of voltage-dependent Ca ++ channel blockers, such as nifedipine and verapamil (Izumi et al. 1995). In the case of ET1, the dominant requirement and the final event leading to uterine contractility is the increase in the intracellular level of calcium (Word et al. 1990) which is realized, however, not only through voltage-dependent Ca ++ influx, but also through release of Ca++ from intra- cellular pools and sustained entry of Ca ++ from receptor- operated Ca++ channels (Word et al. 1990, Fried et al. 1993). The latter involves a cascade of events including activation of phospholipase C and A and some isoforms of protein kinase C (Xuan et al. 1994, Tertrin-Clary et al. 1999). Continuance of the Ca ++ supply to the cell could maintain the strength and the duration of the contraction induced by ET1 and could explain the di fference in regard to the pattern of myometrial contractility seen with KCl alone. Additionally, it has been reported that the intact smooth muscle cells show increased sensitivity in terms of contractions to ET1 than KCl (Himpens & Casteels 1987). The physiological importance of the present findings remains to be elucidated. One can postulate that ET1, released by the vascular endothelium or the adjacent endometrium, reaches the myometrium and its vasculature and modifies the spontaneous contractility in a hormonally dependent manner, thus providing an important regulator of uterine function especially in pathological situations such as dysmenorrhea, involved in appearance of ischaemic pain. In conclusion, the present study demonstrates for the first time di fferences in the in vitro responsiveness of human nonpregnant myometrium to ET1 between the two phases of the cycle (follicular and luteal) as well as between pre- and postmenopausal women. It is suggested, Figure 6 Myometrial responsiveness to ET1 and the combination of KCl +ET1 at a concentration 10 /p16 M as shown by (a) the AUC and (b) the amplitude of myometrial contractility. Data were normalized to data obtained after tissue treatment with KCl (80 mM). FE, follicular phase, ET1; LE, luteal phase, ET1; PE, postmenopausal, ET1; FKE, follicular phase, KCl +ET1; LKE, luteal phase, KCl +ET1; PKE, postmenopausal, KCl +ET1. Table 2 Mean amplitude values of spontaneous contractions in the three groups before the application of ET1 ( first series of experiments) and KCl plus ET1 (second series of experiments) ET1 (g) KCl and ET1 (g) Group 1 (n=6) 1 ·3/p50·11 ·4/p50·1 Group 2 (n=7) 1 ·7/p50·21 ·4/p50·1 Group 3 (n=5) 1 ·4/p50·10 ·3/p50·1 E DOMALI and others · Endothelin in human nonpregnant uterus160 www.endocrinology.orgJournal of Endocrinology (2001) 168, 153–162 Downloaded from Bioscientifica.com at 06/13/2026 05:21:17AM via free access first, that the hormonal milieu may regulate the respon- siveness of the myometrium to ET1 at least in in vitro conditions and, secondly, that ET1 and KCl, two utero- tonic agents, may regulate myometrial contractility through different mechanisms.

We wish to thank I Makantasis for his technical assistance. This work was supported by a research scholarship to E Domali by the University of Thessaly.

Apa R, MiceliF&d eF e oD 1998 Endothelin-1 inhibits basal and human chorionic gonadotropin-stimulated progesterone production. Human Reproduction 13 2425–2429. A r a iH ,H o r iS ,A r a m o r iI ,O h k u b oH&Nakanishi S 1990 Cloning and expression of a cDNA encoding an endothelin receptor. Nature 348 730–732. Bacon CR, Morrison JJ, O’Reilly G, Cameron IT & Davenport 1995 ETA and ETB endothelin receptors in human myometrium characterized by the subtype selective ligands BQ123, BQ3020, FR139317 and PD151242. Journal of Endocrinology 144 127–134. Bodelsson G, Laursen M & Stjernquist M 1996 Oxygen promotes contraction by endothelin-1 in human umbilical artery. Human Reproduction 11 2767–2768. Breuiller-Fouche M, HeluyV&F o u r n i e rT1994 Endothelin receptors: binding and phosphoinositide breakdown in human myometrium. Journal of Pharmacology and Experimental Therapeutics 270 973–978. Cameron IT, Davenport AP, van Papendorp C, Barker PJ, Huskisson NS, Gilmour RS, Brown MJ & Smith SK 1992 Endothelin-like immunoreactivity in human endometrium. Journal of Reproduction and Fertility 95 623–628. Cameron IT, Plumpton C, Champency R, van Papendorf C, Ashby MJ & Davenport AP 1993 Identi fication of endothelin-1, endothelin-2 and endothelin-3 in human endometrium. Journal of Reproduction and Fertility 98 251–255. Casey ML & MacDonald PC 1996 The endothelin –parathyroid hormone-related protein vasoactive peptide system in human endometrium: modulation by transforming growth factor-beta. Human Reproduction 11 (Suppl 2) 62–82. Davenport AP, Ashby MJ, Easton P, Ella S, Bedford J, Dickerson C, Numez DJ, Capper SJ & Brown MJ 1990 A sensitive radioimmunoassay measuring endothelin-like immunoreactivity in human plasma: comparison of levels in patients with essential hypertension and normotensive control subjects. Clinical Science 78 261–264. E c o n o m o sK ,M a c D o n a l dP&Casey L 1992 Endothelin-1 gene expression and protein biosynthesis in human endometrium: potential modulator of endometrial blood flow. Journal of Clinical Endocrinology and Metabolism 74 14–19. Elchalal U & Schenker JG 1997 The pathophysiology of ovarian hyperstimulation syndrome - views and ideas. Human Reproduction 12 1129–1137. Fried G, Liu YA & Andersson E 1993 Endothelin contracts human uterine myometrium by a partly dihydropyridine-sensitive mechanism. Acta Physiologica Scandinavica 147 131–136. Haq A, Kayali M, Hammami MM, Jaroud i K & al-Sedairy ST 1996 Immunoreactive endothelin-1, endothelin-2 and big endothelin-1 in follicular fluids of women undergoing ovulation induction for in vitro fertilization. Human Reproduction 11 269–273. Heluy V, Germain G, Fournier T, Ferr eF&B r e u iller-Fouche M 1995 Endothelin ETA receptors mediate human uterine smooth muscle contraction. European Journal of Pharmacology 285 89–94. H i m p e n sB&Casteels R 1987 Measurement by Quin2 of changes of the intracellular calcium concentration in strips of the rabbit ear artery and of the guinea-pig ileum. Pflügers Archiv 408 32–37. Izumi H, Ichihara J, Uchium iY&S h irakawa K 1990 Gestational changes in mechanical properties of skinned muscle tissues of human myometrium. American Journal of Obstetrics and Gynecology 163 638–647. Izumi H, Byam-Smith M & Gar field RE 1995 Gestational changes in oxytocin- and endothelin-1-induced contractility of pregnant rat myometrium. European Journal of Pharmacology 278 187–194. Kamada S, Kubota T, Hirata Y, Tagushi M, Egushi S, Marumo F & Aso T 1992 Direct e ffect of endothelin-1 on the granuloma cells of the porcine ovary. Journal of Endocrinology 134 59–66. Kamada S, Kubota T & Taguchi M 1993 High levels of immuno- reactive endothelin-1 in human follicularfluids. Human Reproduction 8 674–677. Kubota T, Kamada S, Hirata Y, Eguchi S, Imai T, Marumo F & Aso T 1992 Synthesis and release of endothelin-1 by human decidual cells. Journal of Clinical Endocrinology and Metabolism 75 1230–1234. Maggi M, Vanelli GB, Fantoni G, Baldi E, Magini A, Peri A, Giannini S, Gloria L, Del Carlo P, Gasparis D et al. 1994 Endothelin in the human uterus during pregnancy. Journal of Endocrinology 142 385–396. Marciniak SJ, Plumpton C, Barker PJ, Huskisson NS & Davenport AP 1992 Localization of immunoreactive endothelin and proendothelin in the human lung. Pulmonary Pharmacology 5 175–182. Marsh MM, Hampton AL, Riley SC, Findlay JK & Salamonsen LA 1994 Production and characterization of endothelin released by human endometrial epithelial cells in culture. Journal of Clinical Endocrinology and Metabolism 79 1625–1631. Masaki T, Vane JR & Vanhoutte PM 1994 International Union of Pharmacology nomenclature of endothelin receptors. Pharmacological Reviews 46 137–142. Ohkubo S, Ogi K, Hosoya M, Suzuki N, Kimura C, Ondo H & Fujino M 1990 Speci fic expression of human endothelin-2 (ET-2) gene in a renal adenocarcinoma cell line. Molecular cloning of cDNA encoding the precursor of ET-2 and its characterization. FEBS Letters 274 136–140. O’Reilly G, Charnock-Jones DS, Davenport AP, Cameron IT & Smith SK 1992 Presence of messenger ribonucleic acid for endothelin-1, endothelin-2 and endothelin-3 in human endometrium and a change in the ratio of ETA and ETB receptor subtype across the menstrual cycle. Journal of Clinical Endocrinology and Metabolism 75 1545–1549. Osada K, Tsunoda H, Miyauchi T, Sughisita Y, Kub oT&G o t oK 1997 Pregnancy increases ET-1-induced contraction and changes receptor subtypes in uterine smooth muscle in humans. American Journal of Physiology 272 R541–R548. P e k o n e nF ,N y m a nT&Rutanen E-M 1994 Di fferential expression of mRNAs for endothelin-related proteins in human endometrium, myometrium and leiomyoma. Molecular and Cellular Endocrinology 103 165–170. Sakurai T, Yanagisawa M, Takuwa Y, Miyazaki H, Kimura S, Goto K & Masaki T 1990 Cloning of a cDNA encoding a non-isopeptide- selective subtype of the endothelin receptor.Nature 348 732–735. Sakurai T, Yanagisawa M, Inoue A, Ryan US, Kimura S, Mitsui Y, G o t oK&Masaki T 1991 cDNA cloning, sequence analysis and tissue distribution of rat preproendothelin-1 mRNA. Biochemical and Biophysical Research Communications 175 44–47. Sakurai T, YanagisawaM&M asaki T 1992 Molecular characterization of endothelin receptors.Trends in Pharmacological Sciences 13 103–108. Endothelin in human nonpregnant uterus · E DOMALI and others 161 www.endocrinology.org Journal of Endocrinology (2001) 168, 153–162 Downloaded from Bioscientifica.com at 06/13/2026 05:21:17AM via free access Salamonsen LA, Butt AR, Macpherson AM, Roger sP&F i n dlay JK 1992 Immunolocalization of the vasoconstrictor endothelin in human endometrium during the menstrual cycle and in umbilical cord at birth. American Journal of Obstetrics and Gynecology 167 163–167. Schiff E, Ben-Baruch G, Galron R, Mashiac hS&S o k o l ovsky M 1993 Endothelin-1 receptors in the human myometrium: evidence for different binding properties in post-menopausal as compared with premenopausal and pregnant women. Clinical Endocrinology 38 321–324. Sunnergen KP, Word RA, Sambrook JF, MacDonald PC & Casey ML 1990 Expression and regulation of endothelin precursor mRNA in avascular human amnion. Molecular and Cellular Endocrinology 68 R7–R14. Svane D, Larsson B, Alm P, Andersson KE & Forman A 1993 Endothelin-1: immunocytochemistry, localization of binding sites, and contractile effects in human uteroplacental smooth muscle. American Journal of Obstetrics and Gynecology 168 233–241. Tertrin-Clary C, Eude L, Fournier T, Paris B, Breuiller-Fouche M & Ferre F 1999 Contribution of protein kinase C to ET1-induced proliferation in human myometrial cells. American Journal of Physiology 276 E503–E511. Wolff K, Faxen M, Lunell NO, Nisel lH&L i n d b l o mB1996 Endothelin receptor type A and B gene expression in human nonpregnant, term pregnant, and preeclamptic uterus. American Journal of Obstetrics and Gynecology 175 1295–1300. Word RA, Kamm KE, Stull JT & Casey ML 1990 Endothelin increases cytoplasmic calcium and myosin phosphorylation in human myometrium. American Journal of Obstetrics and Gynecology 162 1103–1108. Word RA, Kamm KE & Casey L 1991 Contractile e ffects of prostaglandins, oxytocin and endothelin-1 in human myometrium in vitro: refractoriness of myometrial tissue of pregnant women to prostaglandins E 2 and F2/afii9825. Journal of Clinical Endocrinology and Metabolism 75 1027–1032. Xuan Y-T, Wang O-L & Whorton AR 1994 Regulation of endothelin-induced Ca2+ mobilization in smooth muscle cells by protein kinase C. American Journal of Physiology 266 c1560–c1567. Yanagisawa M, Kurihara H, Kimura S, Got oK&M asaki T 1988 A novel peptide vasoconstrictor, endothelin 1, is produced by vascular endothelium and modulates smooth muscle Ca 2+ channels. Journal of Hypertension Supplement 6 188–191. Received 19 May 2000 Revised manuscript received 10 August 2000 Accepted 20 September 2000 E DOMALI and others · Endothelin in human nonpregnant uterus162 www.endocrinology.orgJournal of Endocrinology (2001) 168, 153–162 Downloaded from Bioscientifica.com at 06/13/2026 05:21:17AM via free access