{"paper_id":"f9b1d82a-812e-4264-ae17-8e157ee8f636","body_text":"In vitro effects of endothelin-1 on the contractility of myometrium\nobtained from pre- and postmenopausal women\nE Domali1,2, E Asprodini3, P A Molyvdas 2 and I E Messinis1\n1Department of Obstetrics and Gynecology, University of Thessalia, 22 Papakiriazi Street, 41222 Larissa, Greece\n2Department of Physiology, University of Thessalia, 22 Papakiriazi Street, 41222 Larissa, Greece\n3Department of Pharmacology, University of Thessalia, 22 Papakiriazi Street, 41222 Larissa, Greece\n(Requests for offprints should be addressed to I E Messinis)\nAbstract\nThis study was conducted to evaluate the responsiveness of\nhuman nonpregnant myometrium to endothelin 1 (ET1)\n(10\n/p110 M-10/p16 M) and KCl (80 mM) in relation to the\nhormonal profile of the women, who were allocated into\nthree groups: group 1, premenopausal follicular phase,\nn=14, group 2, premenopausal luteal phase, n=20, and\ngroup 3, postmenopausal women, n=12. At a concen-\ntration of 10\n/p16 M, ET1 in both groups 1 and 2 induced\nvery low ripples of high frequency (group 1: 80 /p514%,\nn=5, group 2: 314/p563%, n=11; P<0·05 compared with\nthe pretreatment frequency) which lasted significantly\nlonger in group 2 (29 /p52 min, n=10, P<0·05) than\nin group 1 (20 /p52 min, n=5), increasing the basal tone\n(group 1: 57·9 /p56%, n=5, group 2: 64·4 /p54%, n=6),\nthe amplitude of myometrial contractility (group 1:\n1·2/p50·07 g, n=5, group 2: 1·6 /p50·1 g, n=7, P<0·05)\nand the area under the contractility curve (AUC; group 1:\n8·4/p51·1 g/p2min, n=6, group 2: 11·9 /p51·6 g/p2min,\nn=11). In group 3, ET1 (10\n/p16 M) created a sustained\nlong-lasting contraction (initial phase: 43 /p56 min, n=6)\ncharacterized by the complete obliteration of spontaneous\ncontractility with no ripples at all, and increasing signifi-\ncantly (P<0·05) the amplitude of myometrial contractility\n(2·8/p50·5 g, n=6), the AUC (24·7 /p53·3 g/p2min, n=6),\nas well as the basal tone (183·6 /p521%, n=6) compared\nwith the two premenopausal groups. In all three groups\nKCl exposure induced an initial rise (mean amplitude\nvalue: 1·1 g) followed by a relaxation phase to the primal\nbaseline level (mean duration value: 12 min). Addition of\nET1 (10\n/p16 M) to KCl (80 mM) induced a similar pattern\nof contractility to that evoked by ET1 alone which,\ncompared with KCl alone lasted significantly longer\n(P<0·05) in all three groups (group 1: 20 /p52 min, n=6;\ngroup 2: 23/p52 min, n=6; group 3: 35/p53 min, n=5). In\ngroup 3, the percentage change in basal tone was signifi-\ncantly smaller following KCl than after the combination of\nKCl plus ET1 (149 /p516%, n=5; P<0·01), indicating a\ndifferent mechanism of contractility between KCl and\nET1. These results demonstrate for the first time di ffer-\nences in myometrial response to ET1 between pre- and\npostmenopausal women. It is suggested that KCl and ET1\naffect uterine contractility through di fferent mechanisms\nand that ovarian steroids may play a regulatory role in\nhuman uterine responsiveness to ET1.\nJournal of Endocrinology (2001) 168, 153–162\nIntroduction\nEndothelins (ETs) consist of a family of three sarafotoxin-\nlike peptides ET1, ET2 and ET3 originally isolated from\nthe supernatant of cultured porcine endothelial cells\n(Yanagisawa et al. 1988). They are produced by di fferent\ncell types, endothelial and epithelial cells (Ohkubo et al.\n1990, Sakurai et al. 1991, Kamada et al. 1992) and are\nprimarily described for their potent vasoconstrictor actions\n(Davenport et al. 1990, Bodelsson et al. 1996, Elchalal &\nSchenker 1997). It has also been reported that ETs\nmodulate the contractility in a variety of tissues. The\neffects of ETs are mediated through two cloned and\nsequenced subtypes of receptors, ETA and ETB, which\nare members of the G-protein-linked receptor superfamily\n(Masaki et al. 1994); ET1 is a selective ligand for ETA\nreceptor (ET1 >ET2), whereas the three peptides, ET1,\nET2 and ET3 have been reported to display equal affi nity\nfor ETB receptor (ET1=ET2=ET3) (Arai et al. 1990,\nSakurai et al. 1990, 1992, Bacon et al. 1995).\nIn particular, the 21-amino acid peptide, ET1, is a\npotent, long-acting vasoconstrictor and proliferative\nagent produced by a wide range of human cell types\n(Sunnergen et al. 1990, Marciniak et al. 1992, Casey &\nMacDonald 1996) and plays a functional role in the female\nreproductive system (Kamada et al. 1993, Haq et al. 1996,\nApa et al. 1998). Northern blot analysis demonstrated the\nrelease of ET1 by human decidual cells in early pregnancy\n153\nJournal of Endocrinology (2001) 168, 153–162\n0022–0795/01/0168–153 /p302001 Society for Endocrinology Printed in Great Britain\nOnline version via http://www.endocrinology.org\nDownloaded from Bioscientifica.com at 06/13/2026 05:21:17AM\nvia free access\n\n\n(Kubota et al. 1992) and the presence of immunoreactive\nprepro ET1 and prepro ET1 mRNA in human endo-\nmetrial tissue (Economos et al. 1992, Salamonsen et al.\n1992, Cameronet al. 1992, 1993, Marsh et al. 1994). It has\nbeen reported that the binding sites for ET1 are distributed\nin the human uterus throughout the menstrual cycle\n(O’Reilly et al. 1992) and that both subtypes of receptors\nare localized in human myometrium, where ETA binding\nsites represent the principal subtype (Schi ff et al. 1993,\nBreuiller-Fouche et al. 1994, Pekonen et al. 1994, Wolff\net al. 1996). It has been demonstrated that in human\nnonpregnant myometrium, ET1 induces contractions\n(Word et al. 1990, Fried et al. 1993, Svane et al. 1993)\nactivating exclusively the ETA receptors, and increasing\nthe two phases of spontaneous myometrial contractility,\nthe phasic and the tonic phase, despite the lesser sensitivity\nof nonpregnant compared with pregnant myometrium\n(Word et al. 1991, Osada et al. 1997). Although binding\nstudies have shown that ET1 exhibits a ffi nity for ETB\nreceptors, activation of ETB sites, using ETB selective\nligands, has not been reported to mediate any contractile\neffect on human myometrial tissue; the lack of any\ncontractile effect on human nonpregnant uterus has been\nattributed to the small population of ETB receptors (less\nthan 25%) on human myometrium, or to their involve-\nment in mechanisms of relaxation and proliferation; the\nprecise role of ETB subtypes of receptors in human uterus\nneeds to be further elucidated (Maggi et al. 1994, Bacon\net al. 1995, Heluy et al. 1995, Wolff et al. 1996, Osada\net al. 1997). However, in none of these studies has the\neffect of ET1 on myometrial contractility been examined\nspecifically in relation to the hormonal pro file of the\nwomen. Knowing that human uterus is a target organ of\nsex steroids, the purpose of our study was to elucidate the\neffect of ET1 on human uterine contractility in relation to\nthe sex steroid milieu of the women, and to determine\npossible alterations in premenopausal women (follicular\nor luteal phase of the normal menstrual cycle) and in\npostmenopausal women.\nMaterials and Methods\nSpecimens\nMyometrial tissue was collected from women undergoing\nhysterectomy for benign gynecological disorders. Informed\nconsent was obtained. All women were operated on under\nthe same conditions in terms of premedication and anes-\nthetic drugs. None of the patients had been taking any\ntype of hormonal therapy for the previous three months.\nThe samples were excised with a scalpel from the anterior\nand the posterior surface of the body of the uterus\n(macroscopically normal muscle), placed in ice-cold\nKrebs’ solution and taken immediately to the laboratory.\nThe tissues collected from the women were allocated into\nthree groups on the basis of the hormonal profile defined\nfrom the first day of their last menstrual period, and from\nserum progesterone and estradiol concentrations measured\nby enzyme linked fluorescent assay (ELFA) in peripheral\nblood samples obtained early in the morning of the day\nof the operation (means /p5...); group 1: premeno-\npausal women in the follicular phase, mean estradiol\nvalue 222 ·4/p542 pmol/l, mean progesterone value\n1·7/p50·1 nmol/l (age: 40 –45 years, n=14); group 2:\npremenopausal women in the luteal phase, mean estradiol\nvalue 653 ·5/p5141 pmol/l, mean progesterone value\n57·1/p531 nmol/l (age: 40 –45 years, n=20); group 3:\npostmenopausal women, estradiol value <87·2 pmol/l,\nprogesterone value <1·3 nmol/l (age: 65–70 years, n=12).\nExperiments\nThe experiments were performed and completed within\nthe first 10 h after the removal of the uterus from the\nabdomen, most usually within the first 6 –7 h. The\nviability of the tissue under investigation was con firmed\nby the responsiveness of human myometrium to KCl\n(80 mM) at the end of each experiment. The specimens\nwere immediately dissected into longitudinal strips of\n5/p22/p21 mm parallel to the muscle fiber orientation.\nBriefly, the strips were mounted horizontally in bathing\nchambers for isometric recording with one end fixed and\nthe other attached to an isometric transducer connected to\nan amplifier. An initial resting tension of 1 g was applied to\neach strip. The tissues were continuously perfused with\nKrebs’ solution at 37 /p8C, gassed with 95% O\n2 and 5%\nCO2. Tension generated by the muscle strips was recorded\non a GRASS FTO3C. force displacement transducer and\ndisplayed on a universal oscillograph (Harvard) recorder.\nDuring the experiments, the strips were allowed to\nequilibrate for 1 to 2 h until the spontaneous contractility\nbecame regular in frequency and intensity. The tissues\nwere then exposed to the various stimuli for 7 min, and\nwashed out with Krebs’ solution. The duration of the drug\napplication in our experimental procedure was dictated by\ntwo factors: first, the time required for the superfusing\nsolution to reach steady-state concentration within the\nbath, and secondly the high cost of ET1. Therefore, the\n7-min application period used in our experiments was\nconsidered a satisfactory period of time to ascertain the\nachievement of the full effect of the drug, and at the same\ntime to limit the cost of the experiment. Two types of\nexperiment were performed. In the first series of exper-\niments five di fferent concentrations of ET1 (10\n/p110 M,\n10/p19 M, 10/p18 M, 10/p17 M, 10/p16 M) were applied sep-\narately on each strip; the strips used during each exper-\niment were from the same uterus (group 1: n=6 uteri,\ngroup 2: n=11 uteri, group 3: n=6 uteri). In the second\nseries of experiments each strip was exposed to KCl\n(80 mM), allowed to re-equilibrate for 30 min at least and\nwere then exposed to di fferent concentrations of the\ncombination of KCl and ET1. The latter was used at\nE DOMALI and others · Endothelin in human nonpregnant uterus154\nwww.endocrinology.orgJournal of Endocrinology (2001) 168, 153–162\nDownloaded from Bioscientifica.com at 06/13/2026 05:21:17AM\nvia free access\n\n\nconcentrations of 10/p110 M, 10/p19 M, 10/p18 M, 10/p17 M,\nand 10 /p16 M (group 1: n=8 uteri, group 2: n=9 uteri,\ngroup 3: n=6 uteri). To evaluate the contractile activity\ngenerated and the possible alterations before and after\ntissue treatment with the stimuli (namely KCl, ET1 or the\ncombination of KCl and ET1), a number of parameters\nwere studied.\nChange in basal tone The change in basal tone in the\nimmediate 10-min period after tissue treatment with the\nstimulus was expressed as the percentage of the mean\namplitude of the spontaneous contractions occurring in the\n10-min period preceding the addition of the stimuli. Basal\ntone was de fined as the lowest point (baseline) of the\nspontaneous contractions before the application of the\nstimuli (Fig. 1). The amplitude of the spontaneous con-\ntractions was chosen because it expresses the contractile\npotential of each myometrial strip. The change in basal\ntone was calculated as shown in the examples in Fig, 1\nusing the formula x=/afii9826//afii9825/p2100 where x is the percentage\nchange in the basal tone, /afii9825is the mean amplitude of\nthe spontaneous myometrial contractions, and /afii9826is the\nmean values of the distance of the lower parts of the\ninduced contractions from the initial baseline. In case I,\n/afii9825=1·94 g, /afii9826=0·842 g and x=43 ·4%, i.e. application of\nthe stimulus raised the basal tone by 43 ·4% of the\namplitude of the contractions preceding the stimulus. In\ncase II, /afii9825=1·335 g, /afii9826=1·891 g and x=141 ·6%, i.e. tissue\ntreatment with the stimulus elevated the basal tone by\n141·6% of the amplitude of the contractions preceding the\nstimulus.\nFrequency of myometrial contractility Frequency of\nmyometrial contractility in a 20-min period after tissue\ntreatment with the stimulus was expressed as the percent-\nage change in the sum of spontaneous contractions occur-\nring in a period of 20 min before the application of the\nstimulus.\nThe area under the contractility curve The area\nunder the contractility curve (AUC) was determined as\nthe integrated force from the start of the induced contrac-\ntion up to 10 min from the application of the stimulus and\nwas quantified by planimetry of the included area.\nFigure 1 The change in basal tone of our preparations after 10-min treatment with the\nstimuli ((a) premenopausal women; (b) postmenopausal women). The alterations in basal\ntone observed during the experiments were calculated using the formula: x =/afii9826//afii9825/p2100,\nwhere x is the percentage change in the basal tone, /afii9825is the mean amplitude values of\nspontaneous myometrial contractions, and /afii9826is the mean distance of the lower parts of the\ninduced contractions from the initial baseline.\nEndothelin in human nonpregnant uterus · E DOMALI and others 155\nwww.endocrinology.org Journal of Endocrinology (2001) 168, 153–162\nDownloaded from Bioscientifica.com at 06/13/2026 05:21:17AM\nvia free access\n\n\nThe amplitude of myometrial contractility The\namplitude of myometrial contractility was de fined as the\nvalue of the distance between the highest point and\nthe initial baseline of the evoked contractility in a period of\n10 min after the application of each stimulus.\nDuration of alterations in myometrial contractility\nThe duration of alterations in the myometrial contractility\nafter the addition of each stimulus was determined as\nthe period of time from the application of the stimulus\nuntil the reappearance of relatively regular spontaneous\ncontractility.\nReagents\nThe stock solution consisted of a mixture of ET1 (0·1 mg)\nwith 4 ml distilled water resulting in an ET1 concentration\nof 10\n/p15 M. The concentrations of ET1 used in our\nexperiments were prepared with sequential dilutions of\nthe initial stock solution. ET1 (0 ·1 mg) was purchased\nfrom SIGMA-ALDRICH CHEMIE, GmbH P.O. 1120,\n89552 Stenheim, Germany. The ionic composition of the\nmodified Krebs’ solution was as follows: NaCl 110·9 mM,\nKCl 5 ·9 mM, MgCl\n2 1·1 mM, CaCl 2 2 mM,\nNaH2PO4.H2O1 ·2 mM, glucose 9 ·6 mM, NaHCO 3\n25 mM. Drugs were purchased from E. Merck, D-6100\nDarmstadt, F.R. Germany, except for glucose and\nNaHCO\n3 which were purchased from Mallinckrodt\nChemical Works, St Louis, MO, USA and Mallinckrodt\nBaker B.V., Deventer, Holland respectively.\nHormone assays\nBlood samples were centrifuged at 3000 cycles/min at\nleast. The serum was extracted from the supernatant and\nassayed in an automated multiparametric immunoanalyzer;\nthe analyzer functions on the basis of a technical method\n(ELFA), which combines enzyme immunoassay with\nfluorescent reading (450 nm). The immunoanalyzer and\nthe reagents were purchased from bioMerieux sa 69280,\nMarcy-l’Etoile, Paris, France.\nStatistical analysis\nThe responses of the human myometrium to di fferent\nstimuli (KCl, ET1, or KCl and ET1) were compared by\none-way analysis of variance. Mean values and standard\nerrors of the mean (means/p5...) were determined, and\nthe statistical significance was confirmed by the use of the\nStudent’s unpaired t-test, where appropriate.\nResults\nDuring tissue equilibration, segments collected from pre-\nmenopausal women (follicular phase, n=14; luteal phase,\nn=20) showed excessive spontaneous motility compared\nwith those collected from postmenopausal women\n(n=12). In premenopausal women, spontaneous contrac-\ntions were revealed immediately (within 2 –3 min) after\nthe application of 1 g tension and they increased progres-\nsively in amplitude and frequency (Fig. 2a). In postmeno-\npausal women, myometrium contracted spontaneously\nmuch later (30 min), and the frequency of the contractions\nwas lower than in the premenopausal groups (Fig. 2b) .\nDuring the first series of experiments, the effect of ET1\nwas evaluated on human uterine muscle strips. At concen-\ntrations ranging from 10\n/p110 Mt o1 0/p17 M, ET1 induced\na dose-dependent increase in the frequency of the contrac-\ntions in tissues collected from premenopausal women\nwithout any statistically signi ficant di fference between\ngroups 1 and 2. At a concentration of 10\n/p16 M, ET1 in\nFigure 2 Representative traces of spontaneous myometrial contractility evoked after the application of 1 g\nresting tension in strips obtained from (a) premenopausal and (b) postmenopausal women. In post-\nmenopausal preparations spontaneous contractions appeared later, after 30 min on average compared\nwith 2–3 min in both groups 1 and 2 (premenopausal women).\nE DOMALI and others · Endothelin in human nonpregnant uterus156\nwww.endocrinology.orgJournal of Endocrinology (2001) 168, 153–162\nDownloaded from Bioscientifica.com at 06/13/2026 05:21:17AM\nvia free access\n\n\nboth groups 1 and 2 caused a change in the pattern of\nmyometrial contractility (Fig. 3), increasing the basal tone\nand inducing very low ripples of high frequency. Com-\npared with the pretreatment frequency, an increase in\ngroup 1 of 80/p514% (n=5) and in group 2 of 314 /p563%\n(n=11, P<0·05) was found. The increase in basal tone\nwas greater in group 2, in which the ripples were less\ndiscernible, than in group 1 (Fig. 3a and b). The height of\nthe ripples increased gradually in both groups before\nregular contractility was re-established. The change in\nmyometrial contractility up to the time of onset of regular\ncontractions lasted signi ficantly longer in group 2\n(29/p52 min, n=10) than in group 1 (20 /p52 min, n=5,\nP<0·05). During the period of re-establishment of regular\ncontractions, the frequency of contractions was still higher\nthan before the treatment with ET1, especially in group 2;\nin other words, the appearance of regular contractions was\nachieved quicker in group 1 than in group 2 (Fig. 3a,b). In\npostmenopausal women (group 3), the application of the\nlower concentrations (10\n/p110 M, 10 /p19 M, 10 /p18 M) of\nET1 showed no significant effect on the frequency of the\nevoked contractions. At concentrations of 10 /p17 M and\nparticularly at 10 /p16 M, as in groups 1 and 2, ET1 caused\na remarkable change in the pattern of myometrial contrac-\ntility which, however, differed from that in the other two\ngroups in that the action of ET1 resulted in a sustained\nlong-lasting contraction, the initial part of which lasted\n43/p56 min (n=6) and was characterized by the complete\nobliteration of spontaneous contractility with no ripples at\nall (Fig. 3c). Following this, ripples appeared without any\nsign of onset of regular contractility for a period of at least\n2 h. This pattern was closer to that in group 2, but in\ngroup 3 the basal tone increased further and the change\nlasted much longer.\nApplication of KCl (80 mM) alone to myometrial strips\n(Fig. 4) evoked a contraction characterized by an initial\nrise followed by a slow relaxation phase to the initial\nbaseline level which lasted on average 12 min with no\nsignificant difference between the three groups. Addition\nof ET1 (10\n/p16 M) and KCl (80 mM) induced a pattern of\ncontractility which was similar to that induced by ET1\nalone with no signi ficant di fference between the corre-\nsponding groups. Compared, however, with the contrac-\ntile pattern evoked by KCl alone, the combination of KCl\nand ET1 signi ficantly increased the duration of change\nin myometrial contractility ( P<0·05) in groups 1 (20 /p5\n2 min, n=6), and 2 (23 /p52 min, n=6), while in group\n3 (35 /p53 min, n=5) regular myometrial motility was\nre-established much later ( P<0·01) than in the two\npremenopausal groups (Fig. 4a,b,c).\nTable 1 compares the amplitude of myometrial contrac-\ntility, the AUC, and the percentage increase in basal tone\nduring application of KCl alone, ET1 alone, and the\ncombination of KCl and ET1 in the three groups. A\ndose–response effect of ET1 on AUC was found (Fig. 5).\nTreatment with ET1 alone (10\n/p16 M) induced amplitude\nand AUC values that did not di ffer significantly between\ngroups 1 and 2, but were significantly greater (P<0·01) in\ngroup 3 compared with the two premenopausal groups\n(Table 1). Addition of ET1 to KCl significantly restricted\n(P<0·05) the myometrial responsiveness induced by ET1\nalone only in group 3. Normalization of the data to KCl\nconfirmed the statistical di fferences between the groups\n(Fig. 6). This signi ficant attenuation should be attributed\nFigure 3 Representative traces showing alterations in human spontaneous myometrial contractility after\ntreatment with ET1 at a concentration of 10 /p16 M in (a) group 1, premenopausal women, follicular phase\n(n=6), (b) group 2, premenopausal women, luteal phase ( n=11) and (c) group 3, postmenopausal women\n(n=6). In the premenopausal groups, ET1 altered the pattern of spontaneous myometrial contractility,\ninducing very low ripples of high frequency, which lasted longer in group 2 than in group 1; in group 3, ET1\ninduced a sustained long-lasting contraction with no ripples at all which lasted much longer than those in\nboth groups 1 and 2.\nEndothelin in human nonpregnant uterus · E DOMALI and others 157\nwww.endocrinology.org Journal of Endocrinology (2001) 168, 153–162\nDownloaded from Bioscientifica.com at 06/13/2026 05:21:17AM\nvia free access\n\n\nto the smaller amplitude of spontaneous contractions that\noccurred for technical reasons during the period preceding\nthe application of the stimulus in strips obtained from\npostmenopausal women as compared with premenopausal\nwomen (Table 2). In fact, when the data were analyzed on\nthe basis of basal tone, expressed as the percentage change\nin myometrial contractility induced by the stimuli, this\ndifference was eliminated. In particular, we found that\nET1 in both premenopausal groups elevated the initial\nbaseline, without signi ficant di fferences between them,\nwhile in postmenopausal women (group 3) the percentage\nincrease in basal tone was significantly greater than in the\nother two groups. Addition of ET1 to KCl did not a ffect\nthe elevation in basal tone induced by ET1 alone in any of\nthe three groups, which remained signi ficantly (P<0·05)\ngreater in group 3 compared with groups 1 and 2 (Table\n1). Similarly, although the amplitude and the AUC\ninduced by KCl alone were significantly smaller in group\n3 than in groups 1 and 2, the percentage increase in basal\ntone induced by KCl did not di ffer significantly between\nthe three groups (Table 1). In group 3, the percentage\nchange in basal tone was signi ficantly smaller following\nKCl than following ET1 or the combination of KCl plus\nET1. No signi ficant correlations were found between\nestradiol or progesterone concentrations and the contractile\nparameters used on an individual basis.\nDiscussion\nThis study demonstrates signi ficant changes in the spon-\ntaneous contractility of human nonpregnant myometrium\nevoked by ET1 in postmenopausal and premenopausal\nwomen. The main finding of the study was the long-\nlasting e ffectiveness of ET1 in strips collected from\npostmenopausal women compared with premenopausal\nFigure 4 Representative traces showing the effects of KCl alone (80 mM) and the combination of KCl and\nET1 at a concentration 10 /p16 M on human myometrial contractility in (a) group 1, premenopausal women,\nfollicular phase (n=8), (b) group 2, premenopausal women, luteal phase ( n=9) and (c) group 3, post-\nmenopausal women (n=6). Addition of ET1 extended significantly the contractile pattern induced by KCl in\ngroup 2 and especially in group 3.\nTable 1 Alterations in the amplitude of myometrial contractility, the area under the contractility curve (AUC), and the basal tone induced\nby KCl, ET1, and the combination of KCl plus ET1 at a concentration 10 /p16 M in premenopausal (group 1, follicular phase; group 2, luteal\nphase) and postmenopausal (group 3) women\nGroup 1 Group 2 Group 3\nKCl ET1 KCl +ET1 KCl ET1 KCl +ET1 KCl ET1 KCl +ET1\nAUC (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+\nAmplitude (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++\nBasal 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\naP<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\ngroup 3 (KCl and ET1).\nE DOMALI and others · Endothelin in human nonpregnant uterus158\nwww.endocrinology.orgJournal of Endocrinology (2001) 168, 153–162\nDownloaded from Bioscientifica.com at 06/13/2026 05:21:17AM\nvia free access\n\n\nwomen, but signi ficant di fferences were also found\nbetween the two premenopausal groups. In particular, the\npattern of changes in myometrial contractility induced by\nET1 in the myometrium from women in the luteal phase\nwas between the patterns of the other two groups. These\ndifferences are di ffi cult to explain.\nIt is known that the contractile effects of ET1 on human\nnonpregnant myometrium are mediated exclusively by\nETA receptors (Baconet al. 1995, Heluy et al. 1995). The\ndiscrepancies in the myometrial responsiveness to ET1 in\nthe three groups observed in the present study could not\nbe attributed to the density or the affi nity of the ligand for\nits receptor as it has already been reported that the binding\ncapacity of ET1 (affi nity) does not alter between pre- and\npostmenopausal women (Schi ff et al. 1993, Maggi et al.\n1994). Therefore, other mechanisms possibly related to\nthe differentiated hormonal profile of the women may be\nimportant. Although individual hormonal values did not\ncorrelate significantly with the changes in the contractility\ninduced by the stimuli, it is possible that myometrial\ncontractility to ET1 is enhanced by the estrogen\ndeficiency after menopause, thus explaining the long-\nlasting effect of ET1 seen in group 3. That the pattern of\ncontractility in group 2 was closer to that of group 3\nsuggests that in terms of responsiveness to the contractile\nagent, ET1, the myometrium during the luteal phase\nbehaves in a more or less similar manner to that after the\nmenopause. It could be postulated that the counteractive\neffect of progesterone to estrogen during the luteal phase\ncreates conditions of contractility in the myometrium\nsimilar to those in postmenopausal women, but this\nrequires investigation. The intracellular mechanism that\ncould mediate the enhanced responsiveness of estrogen-\ndeprived human uterus may be associated with alterations\nin the receptor ’s functions leading to changes in the\npost-receptor biochemical events (Osada et al. 1997).\nIn the present study, application of KCl (80 mM)\nprovoked an initial rise in the spontaneous myometrial\nFigure 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\nafter treatment with the stimulus in premenopausal ((a) follicular and (b) luteal) and postmenopausal (c) women; a dose –response effect\nof ET1 on AUC was found. /p12, KCl, /p11, ET1, /p20, KCl +ET1.\nEndothelin in human nonpregnant uterus · E DOMALI and others 159\nwww.endocrinology.org Journal of Endocrinology (2001) 168, 153–162\nDownloaded from Bioscientifica.com at 06/13/2026 05:21:17AM\nvia free access\n\n\ncontractility followed by a relaxation phase to the original\nbaseline without significant differences between the three\ngroups. This means that the responsiveness of the human\nnonpregnant uterus to KCl did not alter, despite the\nchange in the hormonal pro file of the women, while in\npregnant myometrium increased sensitivity to KCl\nduring the progress of gestation has already been reported\n(Izumi et al. 1990). The nonchanging responsiveness\nof nonpregnant human myometrium to KCl is in\ncontrast to the e ffect of ET1 that was enhanced in the\nluteal phase and particularly in the postmenopausal\nwomen, thus demonstrating a di fferential response of\nhuman nonpregnant myometrium to ET1 and KCl.\nAlthough the addition of ET1 extended signi ficantly\nthe relaxation phase of the KCl-induced contractions in\nthe three groups, basically the pattern of contractility\ninduced by ET1 alone was una ffected by the presence\nof KCl.\nThe differential response of human uterus to ET1 and\nKCl might re flect the involvement of a variety of bio-\nchemical mechanisms in the evoked myometrial contrac-\ntility. A series of studies has already suggested that KCl\ninduces contractions through voltage-dependent Ca\n++\ninflux, which is completely abolished by the presence\nof voltage-dependent Ca ++ channel blockers, such as\nnifedipine and verapamil (Izumi et al. 1995). In the case of\nET1, the dominant requirement and the final event\nleading to uterine contractility is the increase in the\nintracellular level of calcium (Word et al. 1990) which is\nrealized, however, not only through voltage-dependent\nCa\n++ influx, but also through release of Ca++ from intra-\ncellular pools and sustained entry of Ca ++ from receptor-\noperated Ca++ channels (Word et al. 1990, Fried et al.\n1993). The latter involves a cascade of events including\nactivation of phospholipase C and A and some isoforms of\nprotein kinase C (Xuan et al. 1994, Tertrin-Clary et al.\n1999). Continuance of the Ca\n++ supply to the cell could\nmaintain the strength and the duration of the contraction\ninduced by ET1 and could explain the di fference in\nregard to the pattern of myometrial contractility seen with\nKCl alone. Additionally, it has been reported that the\nintact smooth muscle cells show increased sensitivity in\nterms of contractions to ET1 than KCl (Himpens &\nCasteels 1987).\nThe physiological importance of the present findings\nremains to be elucidated. One can postulate that ET1,\nreleased by the vascular endothelium or the adjacent\nendometrium, reaches the myometrium and its vasculature\nand modifies the spontaneous contractility in a hormonally\ndependent manner, thus providing an important regulator\nof uterine function especially in pathological situations\nsuch as dysmenorrhea, involved in appearance of\nischaemic pain.\nIn conclusion, the present study demonstrates for the\nfirst time di fferences in the in vitro responsiveness of\nhuman nonpregnant myometrium to ET1 between the\ntwo phases of the cycle (follicular and luteal) as well as\nbetween pre- and postmenopausal women. It is suggested,\nFigure 6 Myometrial responsiveness to ET1 and the combination of KCl +ET1 at a concentration 10 /p16 M as shown by (a) the AUC and\n(b) the amplitude of myometrial contractility. Data were normalized to data obtained after tissue treatment with KCl (80 mM). FE, follicular\nphase, ET1; LE, luteal phase, ET1; PE, postmenopausal, ET1; FKE, follicular phase, KCl +ET1; LKE, luteal phase, KCl +ET1; PKE,\npostmenopausal, KCl +ET1.\nTable 2 Mean amplitude values of spontaneous contractions in\nthe three groups before the application of ET1 ( first series of\nexperiments) and KCl plus ET1 (second series of experiments)\nET1 (g) KCl and ET1 (g)\nGroup 1 (n=6) 1 ·3/p50·11 ·4/p50·1\nGroup 2 (n=7) 1 ·7/p50·21 ·4/p50·1\nGroup 3 (n=5) 1 ·4/p50·10 ·3/p50·1\nE DOMALI and others · Endothelin in human nonpregnant uterus160\nwww.endocrinology.orgJournal of Endocrinology (2001) 168, 153–162\nDownloaded from Bioscientifica.com at 06/13/2026 05:21:17AM\nvia free access\n\n\nfirst, that the hormonal milieu may regulate the respon-\nsiveness of the myometrium to ET1 at least in in vitro\nconditions and, secondly, that ET1 and KCl, two utero-\ntonic agents, may regulate myometrial contractility\nthrough different mechanisms.\nAcknowledgements\nWe wish to thank I Makantasis for his technical assistance.\nThis work was supported by a research scholarship to\nE Domali by the University of Thessaly.\nReferences\nApa R, MiceliF&d eF e oD 1998 Endothelin-1 inhibits basal and\nhuman chorionic gonadotropin-stimulated progesterone production.\nHuman Reproduction 13 2425–2429.\nA r a iH ,H o r iS ,A r a m o r iI ,O h k u b oH&Nakanishi S 1990 Cloning\nand expression of a cDNA encoding an endothelin receptor. 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Journal of\nHypertension Supplement 6 188–191.\nReceived 19 May 2000\nRevised manuscript received 10 August 2000\nAccepted 20 September 2000\nE DOMALI and others · Endothelin in human nonpregnant uterus162\nwww.endocrinology.orgJournal of Endocrinology (2001) 168, 153–162\nDownloaded from Bioscientifica.com at 06/13/2026 05:21:17AM\nvia free access","source_license":"CC0","license_restricted":false}