[Biochemistry of fetal membranes rupture]. / Biochimie de la rupture des membranes fœtales.

Fetal membranes, amnion and chorion, line up the amniotic cavity and are essential for its integrity towards normal term of pregnancy. They consist of a pluristratified structure whose composition assures their cohesion and elasticity. They firstly function in retaining the fluctuant amniotic fluid in a half-rigid cavity. Their elastic limit depends on the organization of the extracellular matrix and firstly on the collagen type it contains. The compact layer of the amnion, responsible for the elastic limit, contains mainly type I collagen, organized in lattice; this allows elongation or spreading. Underneath, the spongy layer, principally of collagen III, is organized in a loose mesh, enriched in hydrated proteoglycans, which allows the absorption of the shocks and the sliding of the amnion on the chorion. The cascade of events leading to the membrane rupture displays: (i) membranes distension with elasticity loss, (ii) separation of the chorion from the amnion, (iii) chorion fracture, (iv) amnion distension which produces an hernia, (v) amnion rupture. The rupture mechanism was long thought to be a consequence of uterine contractions. However, the observation before labour of a zone of altered morphology, with biochemical variations (modifications of metalloprotease activity and of proteoglycans, apoptosis...) associated with focal physical weakness in the region overlying the cervix suggests programming of the rupture before parturition. A better understanding of the biochemical mechanisms of membranes rupture will provide new insights into how to anticipate and to intervene in the case of risk of premature rupture.

Stimulation of the ADRB3 adrenergic receptor induces relaxation of human placental arteries: influence of preeclampsia.

Preeclampsia, which complicates 3-8% of pregnancies, is one of the leading causes of neonatal morbidity and mortality. Its pathophysiology remains unclear. The aim of the present study was to investigate the presence and the role of beta2- and beta2-adrenergic receptors (ADRB2 and ADRB3, respectively) in human placental arteries and to assess the influence of preeclampsia on ADRB responsiveness. SR 59119A, salbutamol, and isoproterenol (ADRB3, ADRB2, and nonselective ADRB agonists, respectively) induced a concentration-dependent relaxation of placental artery rings obtained from women with uncomplicated or preeclamptic pregnancies. SR 59119A-induced relaxation was unaffected by the blockade of ADRB1 and ADRB2 by 0.1 microM propranolol but was significantly decreased by the blockade of ADRB1, ADRB2, and ADRB3 by 10 microM propranolol. Both SR 59119A and salbutamol were associated with a significant increase in cAMP production that was significantly inhibited by pretreatment with 0.1 microM propranolol only for salbutamol. SR 59119A-induced relaxation (E(max) = 28% +/- 5% vs. 45% +/- 4%, respectively) and cAMP production (2.7 +/- 0.5 vs. 4.9 +/- 0.4 pmol/mg of protein, respectively; P < 0.01) were decreased in arteries obtained from preeclamptic compared to normotensive women. Both ADRB2 and ADRB3 transcripts were expressed at the same level between arteries from normotensive and preeclamptic women. Western blot analysis, however, revealed a decreased expression of the ADRB3 immunoreactive protein in arteries from preeclamptic compared to normotensive women. We suggest the presence of functional ADRB2 and ADRB3 in human placental arteries. Even if preeclampsia is associated with an impairment of the ADRB3 responsiveness, ADRB3 agonists may have future pharmaceutical implications in the management of pregnancy-related disorders.

Beta3-adrenoceptor is the predominant beta-adrenoceptor subtype in human myometrium and its expression is up-regulated in pregnancy.

To assess whether pregnancy might influence the functionality and expression of human myometrial beta(2)- and beta(3)-adrenoceptors (beta(2)- and beta(3)-AR), we performed functional, binding, Western blot, and molecular biology experiments in human nonpregnant and near-term pregnant myometrium. Inhibition of spontaneous contractions induced by a beta(3)-AR agonist, SR 59119A, was significantly greater in pregnant, compared with nonpregnant, myometrial strips (E'(max) = 61 +/- 5% vs. 44 +/- 5% for pregnant and nonpregnant myometrium, respectively), whereas salbutamol, a beta(2)-AR agonist, was significantly less efficient in pregnant, compared with nonpregnant, myometrium (E(max) = 29 +/- 4 vs. 54 +/- 8%). Although two populations of binding sites corresponding to beta(2)- and beta(3)-AR were identified in both nonpregnant and pregnant myometrium, we found a clear predominance of the beta(3)-AR subtype. Moreover, beta(3)-AR binding sites were up-regulated 2-fold in myometrium at the end of pregnancy. Both beta(2)- and beta(3)-AR mRNA were expressed in human nonpregnant and pregnant myometrium. Contrary to beta(2)-AR, the expression of the beta(3)-AR transcripts and immunoreactive proteins was increased in pregnant, compared with nonpregnant, myometrium. Such compelling data suggest a predominant role for beta(3)-AR in the regulation of human myometrium contractility, especially at the end of pregnancy, which might have important consequences for the clinical management of preterm labor.

The human near-term myometrial beta 3-adrenoceptor but not the beta 2-adrenoceptor is resistant to desensitisation after sustained agonist stimulation.

1. In order to compare the beta(2)- and beta(3)-adrenoceptor (beta-AR) desensitisation process in human near-term myometrium, we examined the influence of a pretreatment of myometrial strips with either a beta(2)- or a beta(3)-AR agonist (salbutamol or SR 59119A, respectively, both at 10 microm, for 5 and 15 h) on the relaxation and the cyclic adenosine monophosphate (cAMP) production induced by these agonists. 2. To assess some of the mechanisms potentially implicated in the beta-AR desensitisation process, we studied the influence of such treatment on the number of beta(2)- and beta(3)-AR binding sites, the beta(2)- and beta(3)-AR transcripts expression and the phosphodiesterase 4 (PDE4) activity. 3. Salbutamol, but not SR 59119A, concentration-response curve (CRC) was shifted by a 15 h salbutamol preincubation, with a significant difference in -log EC(20) values (6.31+/-0.13 vs 5.58+/-0.24, for control and 15 h salbutamol pretreatment, respectively, P<0.05). Neither salbutamol nor SR 59119A CRCs were modified after a 15 h preincubation with SR 59119A. 4. A 15 h exposure of myometrial strips to salbutamol significantly reduced the salbutamol-induced (0.60+/-0.26 vs 1.54+/-0.24 pmol mg(-1) protein, P<0.05), but not the SR 59119A-induced, cAMP production. No decrease in cAMP production was observed after a 15 h SR 59119A exposure. 5. A 15 h salbutamol exposure of myometrial strips significantly reduced the beta(2)- but not the beta(3)-AR binding site density, whereas no decrease in the number of beta(2)- and beta(3)-AR binding sites was observed after a 15 h SR 59119A treatment. 6. Neither PDE4 activity nor the beta(2)- and beta(3)-AR mRNA expression levels were affected by salbutamol or SR 59119A treatments. 7. Our results indicate that beta(3)-AR, but not beta(2)-AR, are resistant to the agonist-induced desensitisation. In our model, beta(2)-AR desensitisation is mediated by a decreased number of beta(2)-AR that was not explained by transcriptional regulation of the receptor.

A critical role for PKC zeta in endothelin-1-induced uterine contractions at the end of pregnancy.

We have previously shown that protein kinase C (PKC) zeta and/or PKC delta are necessary for endothelin-1 (ET-1)-induced human myometrial contraction at the end of pregnancy (Eude I, Paris P, Cabrol D, Ferré F, and Breuiller-Fouché M. Biol Reprod 63: 1567-1573, 2000). Here, we report that the selective inhibitor of PKC delta isoform, Rottlerin, does not prevent ET-1-induced contractions, whereas LY-294002, a phosphatidylinositol (PI) 3-kinase inhibitor, affects the contractile response. This study characterized the in vitro contractile response of cultured human pregnant myometrial cells to ET-1 known to induce in vitro contractions of intact uterine smooth muscle strips. Cultured myometrial cells incorporated into collagen lattices have the capacity to reduce the size of these lattices, referred to as lattice contraction. Neither the selective conventional PKC isoform inhibitor, Gö-6976, or rottlerin affected myometrial cell-mediated gel contraction by ET-1, whereas this effect was blocked by LY-294002. We found that treatment of myometrial cell lattices with an inhibitory peptide specific for PKC zeta or with an antisense against PKC zeta resulted in a significant loss of ET-1-induced contraction. Evidence is also presented by using confocal microscopy that ET-1 induced translocation of PKC zeta to a structure coincident with the actin-rich microfilaments of the cytoskeleton. We have shown that PKC zeta has a role in the actin organization in ET-1-stimulated cells. Accordingly, our results suggest that PKC zeta plays a role in myometrial contraction in pregnant women.

A functional genomic study to identify differential gene expression in the preterm and term human myometrium.

The mechanisms that lead to the onset of human parturition are still unknown, although selected critical factors have been identified. To investigate the changes in myometrial gene expression associated with parturition, we used two macroarrays each containing 1176 different complementary human cDNA clones. Methods involving hierarchical clustering and conventional statistical analysis allowed us to generate a profile of genes expression at three stages of late pregnancy: preterm (29 wk amenorrhea); full term, not in labor (38 wk amenorrhea); and full term in labor (39 wk amenorrhea). Only 4% of the genes investigated were differentially expressed between the preterm and term groups (P < 0.05). These genes could be clustered as groups of either down-regulated or up-regulated transcripts. The changes in transcript abundance were particularly marked between the preterm and term stages of gestation, whereas the differences between term not in labor and term in labor were less pronounced. The parturition was characterized by a massive down-regulation of a large panel of developmental, cell adhesion molecule and proliferation-related genes, along with the up-regulation of inflammatory, contraction and apoptosis associated genes. We propose that the mechanisms of parturition consist primarily in the arrest of the processes of myometrial development, a step that might be essential to allow the uterus to recover appropriate contractile function before delivery.

Interleukin-1beta induces glycosaminoglycan synthesis via the prostaglandin E2 pathway in cultured human cervical fibroblasts.

The aim of this study was to identify, in cultured human cervical fibroblasts, the mechanisms by which interleukin (IL)-1beta induces the synthesis of glycosaminoglycans (GAG) and to explore the putative role of prostaglandin E(2) (PGE(2)) in this process. Exposure of the cells for 24 h to IL-1beta induced a significant (P < 0.05) dose-dependent increase in GAG synthesis. IL-1beta (1 ng/ml) induced the expression of cyclooxygenase-2 (COX-2) protein 6 h after treatment, accompanied by a 7.5-fold increase in PGE(2) production. We confirmed that NS398, a selective COX-2 inhibitor, dose-dependently blocked PGE(2) augmentation following IL-1beta treatment. AH23848, the selective EP(4) receptor antagonist, completely abolished IL-1beta-induced GAG synthesis, whereas AH6809, an EP(2) receptor antagonist, had no effect on the stimulatory effects of IL-1beta. Furthermore, we demonstrated that 6 h exposure to IL-1beta induced a notable increase in EP(4) receptor mRNA expression and a decrease in EP(1) receptor mRNA but had no effect on the expression of EP(2) and EP(3) receptor transcripts. In conclusion, these findings indicate that IL-1beta not only induced GAG synthesis by increasing COX-2 protein expression and the subsequent PGE(2) production but also enhanced the responsiveness of cervical fibroblasts to PGE(2) by selectively up-regulating EP(4) receptor mRNA expression. These results suggest that PGE(2) may regulate human cervical ripening in an autocrine/paracrine manner via EP(4) receptors.

Potentiation response of cultured human uterine leiomyoma cells to various growth factors by endothelin-1: role of protein kinase C.

OBJECTIVE: Factors responsible for the abnormal proliferation of myometrial cells that accompanies leiomyoma formation are unknown, although steroid hormones and peptide growth factors have been implicated. We hypothesized that endothelin-1 (ET-1) is a physiological regulator of tumor growth. DESIGN: In this study, we investigated the role of ET-1 on growth of human leiomyoma cells and its synergistic effect with growth factors, as well as the signaling pathway involved in this interaction. METHODS: Leiomyoma cell proliferation was assayed by [H]thymidine incorporation and cell number. Protein kinase C (PKC) isoforms were analyzed by Western blot using specific antibodies. RESULTS: ET-1 on its own was unable to stimulate DNA synthesis but potentiated the leiomyoma cell growth effects of basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), IGF-I and IGF-II. The failure of a protein tyrosine kinase (PTK) inhibitor, tyrphostin 51, to affect the potentiating effect of ET-1, supports the hypothesis of non-involvement of PTK in this process. The inhibition of PKC by calphostin C or its down-regulation by phorbol 12,13-dibutyrate (PDB) eliminated the potentiating effect of ET-1, but did not block cell proliferation induced by the growth factors alone. Five PKC isoforms (alpha, beta1, epsilon, delta and zeta) were detected in leiomyoma cells, but only phorbol ester-sensitive PKC isoforms (PKCalpha, epsilon and delta) contribute to the potentiating effect of leiomyoma cell growth by ET-1. CONCLUSIONS: We have demonstrated that ET-1 potentiates leiomyoma cell proliferation to growth factors through a PKC-dependent pathway. These findings suggest a possible involvement of ET-1 in the pathogenesis of leiomyomas.

Differential regulation of protein kinase C isoforms in human uterine leiomyoma.

The protein kinase C (PKC) isoenzyme expression pattern in human uterine leiomyoma was compared with that obtained in homologous myometrium distal from the tumor. The six PKC isoforms (PKCalpha, PKCbeta1, PKCbeta2, PKCdelta, PKCepsilon and PKCzeta) evidenced in the myometrium were found to be similarly expressed in leiomyoma. Quantitative immunoblotting revealed that all PKC isoforms were preferentially localized in the particulate fraction. To gain insight into the possible functional consequences of PKC expression patterns, subcellular redistribution in response to the mitogenic peptide endothelin-1 (ET-1) was studied. After stimulation with ET-1, differential redistribution occurred in leiomyoma and myometrium, suggesting a selective role of PKC isoforms in the myometrial growth process.

EP(4) receptors mediate prostaglandin E(2)-stimulated glycosaminoglycan synthesis in human cervical fibroblasts in culture.

The aim of this study was to determine the prostaglandin E (EP) receptors and second messengers implicated in glycosaminoglycan (GAG) synthesis by human cervical fibroblasts in culture. Human cervical fibroblasts were obtained from cervical biopsies in pre-menopausal, cycling women. Cultured cells were incubated with prostaglandin E(2) (PGE(2)) and an array of agonists and antagonists. Glycosaminoglycan synthesis was assayed after extraction by measuring the [(3)H]glucosamine and [(35)S]sulphate incorporated into GAG and cAMP production was determined by radioimmunoassay. PGE(2) significantly stimulated GAG synthesis. Neither 17-phenyl-trinor-PGE(2), the EP(1) selective agonist, nor sulprostone, an EP(3) agonist, had any effect on GAG production. Butaprost, the EP(2) selective agonist, also failed to increase GAG synthesis. AH6809, an EP(2) antagonist, had no effect on PGE(2)-stimulated GAG production. AH23848, an EP(4) antagonist, inhibited the GAG synthesis provoked by PGE(2). PGE(2) and butaprost significantly increased cAMP production. Both AH6809 and AH23848 inhibited the PGE(2)-stimulated cAMP production. H89, a cAMP-dependent protein kinase (PKA) inhibitor, did not inhibit PGE(2)-stimulated GAG synthesis and Sp-cAMPS, a selective PKA activator, failed to increase GAG production. In conclusion, both EP(4) and EP(2) receptors are present and functional in human cervical fibroblasts. Only EP(4) receptors mediate PGE(2) stimulated GAG synthesis in a PKA-independent pathway.

Expression of endothelin receptors in human myometrium during pregnancy and in uterine leiomyomas.

The distribution of mRNAs for endothelinA and B (ET(A) and ET(B)) receptors and their binding properties was studied in human nonpregnant and pregnant term myometrium and in uterine leiomyomas. ET(A)- and ET(B)-receptors functionally coupled to phospholipase C (PLC) coexisted in myometrial tissues, but only the functional ET(A)-receptor subtype was detected in leiomyomas. ET(A)-receptor mRNA and three other spliced variants were distributed in all tissue studied. We reported an increase in the proportion of ET(A)-receptors coupled to PLC in term pregnant myometrium when compared to nonpregnant tissue. These results suggest that upregulation of the myometrial ET(A)-receptors may account for or contribute to the control of normal development and growth of human myometrium during pregnancy. They also support a pathological role for the endothelin-1 (ET-1)/ET(A)-receptor system in leiomyoma development.

Selective protein kinase C isoforms are involved in endothelin-1-induced human uterine contraction at the end of pregnancy.

The role of protein kinase C (PKC) in contraction of the human myometrium induced by endothelin-1 (ET-1) was investigated at the end of pregnancy. The expression and subcellular distribution of PKC isoforms were examined by Western blot analysis using isoform-specific antibodies. At least three conventional PKC isoforms (cPKC; alpha, beta1, and beta2), two novel PKC isoforms (epsilon and delta), and an atypical PKC isoform (zeta) were detected in pregnant myometrium. Quantitative immunoblotting revealed that all these isoforms were mainly distributed in the particulate fraction. The lack of a calcium chelator to modify the particulate sequestration of cPKC suggests an interaction with an anchoring protein such as receptor-activated C kinase-1, which is evidenced in the particulate fraction of the pregnant myometrium. Of the six isoforms, only PKCbeta1, PKCbeta2, PKCdelta, and PKCzeta were translocated to the particulate fraction, and PKCepsilon to the cytoskeletal fraction, after stimulation with ET-1. Involvement of PKC in the ET-1-induced contractile response is supported by the inhibition caused by the PKC inhibitor calphostin C. However, we demonstrated that the selective cPKC isoform inhibitor, Gö 6976, as well as the substantial depletion of PKCbeta1 and PKCepsilon and the partial depletion of PKCalpha and PKCdelta by a long-term treatment with phorbol 12,13-dibutyrate did not prevent ET-1-induced contraction. Accordingly, our results suggest that PKCdelta and PKCzeta activation mediated ET-1-induced contraction, whereas cPKC isoforms were not implicated in the human pregnant myometrium.

[Endothelin receptors in benign human tumours of uterine muscle]. / Récepteurs des endothélines dans les tumeurs bénignes du muscle utérin humain.

The endothelins (ET1, ET2, ET3) are a family of peptides that exert vasoactive and mitogenic effects. ETs bind to at least two subtypes of receptors: the ETA subtype is ET1 selective whereas the ETB subtype binds ET1, ET2 and ET3. By RT-PCR, we detected ETA receptor mRNA and ETB receptor mRNA in leiomyoma and in homologous myometrium distal from the tumor. Despite the presence of four spliced variants of ETA receptors, we identified a single class of ETA-binding sites. The level of ETB receptor mRNA was found to be higher in myometrium versus leiomyomas. Using complementary pharmacologic approach, we demonstrated the predominance of ETA receptors in normal myometrium (75% of total receptors). Both ETA and ETB transcripts coexist in leiomyomas, but we have reported only ETA binding sites. Because of growth properties of ET1, we suggest a role for this peptide in the tumoral development of human uterine smooth muscle.

Contribution of protein kinase C to ET-1-induced proliferation in human myometrial cells.

The role of protein kinase C (PKC) in endothelin-1 (ET-1)-induced proliferation of human myometrial cells was investigated. ET-1 dose dependently stimulated DNA synthesis and the number of cultured myometrial cells. Inhibition of PKC by calphostin C or Ro-31-8220 or downregulation of PKC eliminated the proliferative effects of ET-1. The failure of two protein tyrosine kinase (PTK) inhibitors (tyrphostin 51 and tyrphostin 23) to affect ET-1-induced proliferation supports the hypothesis of noninvolvement of the tyrosine kinase signaling pathway in this process. The expression and distribution of PKC isoforms were examined by Western blot analysis. The five PKC isoforms (PKC-alpha, -beta1, -beta2, -zeta, -epsilon) evidenced in human myometrial tissue were found to be differentially expressed in myometrial cells, with a predominant expression of PKC-alpha and PKC-zeta. Treatment with phorbol 12, 13-dibutyrate (PDBu) resulted in the translocation of all five isoforms to the particulate fraction, whereas ET-1 induced a selective increase in particulate PKC-beta1, PKC-beta2, and PKC-epsilon. Our findings that multiple PKC isoforms are differentially responsive to ET-1 or PDBu suggest that they play distinct roles in the myometrial growth process.

Role of protein kinase C in endothelin-1-induced contraction of human myometrium.

The role of protein kinase C (PKC) in the contraction of human myometrium induced by endothelin-1 was investigated. The PKC inhibitor, calphostin C, reduced the sustained phase of endothelin-1-induced contraction. The expression and subcellular distribution of PKC isoforms were determined in unstimulated myometrium by Western blotting using isoform-specific antisera. At least five PKC isoforms (PKCalpha, PKCbeta1, PKCbeta2, PKCzeta, and trace amounts of PKCepsilon) were detected. Quantitative immunoblotting revealed that all these isoforms were diversely distributed between the cytosolic and particulate fractions. After stimulation with phorbol 12,13-dibutyrate (PDB) and endothelin-1, differential redistribution occurred, suggesting a selective role of these isoforms in the physiological function of the myometrium. Biochemical assay confirmed that PDB as well as endothelin-1 evoked a decrease in cytosolic PKC activity. Taken together, these results suggest that PKC may play a role in endothelin-1-induced contraction of human uterine smooth muscle.

Role of endothelin-1 in regulating proliferation of cultured human uterine smooth muscle cells.

Endothelin-1 (ET-1) exhibits vasoconstricting and growth-promoting properties in vascular smooth muscle. Whether ET-1 has mitogenic properties in uterine smooth muscle cells, and which ET receptor subtype mediates this response, is unknown. The present study was undertaken to examine the proliferative potential of the ET family on human myometrial cells in culture. ET-1 stimulated DNA synthesis and proliferation of myometrial cells. The absence of a stimulating effect of endothelin-3 (ET-3) or the ETB agonist sarafotoxin 6c (S6c) was observed. The proliferative effect of 100nM ET-1 was blocked by the two ETA antagonists (BQ 123 and FR 139317), whereas the ETB antagonist IRL 1038 was ineffective. These data indicated that ET-1-induced DNA synthesis was mediated only by the ETA receptor subtype. Pertussis toxin (PTX) pretreatment completely abolished this effect, indicating that this pathway was coupled to the ETA receptor via the Gi protein family. PTX treatment partially decreased serum-induced DNA synthesis. This suggests that some factors from serum may operate via the G-protein in initiation of mitogenesis. Insulin-like growth factors (IGFs), epidermal growth factor (EGF) and insulin were found to be mitogens in the absence of serum, and they had no potentiating effect on ET-1-induced DNA synthesis. In the presence of 0.5% serum, EGF alone caused a weak increase in DNA synthesis, while all the growth factors were able to reduce the proliferative effect of ET-1. These findings on human myometrial cells in culture raise the possibility that, under certain conditions, ET-1 may function as a positive or as a negative modulator of smooth muscle proliferation.

EndothelinA receptors in human uterine leiomyomas.

OBJECTIVE: To determine if there are endothelin receptors on human uterine leiomyomas. METHODS: Samples of leiomyomas from eight patients were analyzed for [iodine (I)-125]endothelin-1 binding. Several subtype-selective ligands were used to determine the endothelin receptor population. RESULTS: Binding of [125I]endothelin-1 to uterine leiomyoma membranes was specific and saturable, with a mean +/- dissociation constant 85.5 +/- 8.4 pM. Competition binding studies showed that the order of potency was endothelin-1 > endothelin-3, which was consistent with the presence of the endothelinA receptor subtype. Binding of [125I]endothelin-1 was displaced by an endothelinA-selective antagonist, but not by sarafotoxin 6c, an endothelinB-selective agonist. An endothelins-selective ligand was not specifically bound to leiomyoma. CONCLUSION: These results indicate that only endothelinA receptors are present in human uterine leiomyomas. We speculate that endothelin-1 may act through these endothelinA receptors to influence the development or regulation of hypertrophy and proliferation of the human myometrium during pregnancy and in uterine disorders like leiomyomas.

Endothelin ETA receptors mediate human uterine smooth muscle contraction.

Receptors mediating endothelin-induced contraction of myometrium were investigated in the human uterus. Endothelin-1 and endothelin-3 (10 pM to 0.3 microM) caused concentration-dependent contraction of myometrial strips. Endothelin-1 was approximately ten times more potent than endothelin-3, with pD2 values of 8.24 and 7.20, respectively. By contrast, two endothelin ETB receptor selective agonist, BQ 3020 (N-acetyl-[Ala11,15]endothelin-1-(6-21) and sarafotoxin 6c (up to 0.3 microM), did not induce contraction of human myometrium. The endothelin ETA receptor selective antagonist, FR139317 (1-hexahydroazepino-CO-Leu-D-Trp(CH3)-D-(2-pyridyl)alanine) (0.1, 0.3 and 1 microM), competitively antagonized the endothelin-1-elicited contraction, with a pA2 value of 7.10, whereas another endothelin ETA receptor-selective blocking drug, BQ 123 [cyclo(-D-Trp-D-Asp-Pro-D-Val-Leu)], behaved as a non-competitive antagonist. Pretreatment of myometrial strips with an endothelin ETB receptor selective antagonist, IRL 1038 ([Cys11-Cys15]endothelin-1-(11-21)), had no effect on contractions induced by endothelin-1. All these data indicate that only endothelin ETA receptors mediate endothelin-1-induced contractions of human myometrium.

Characterization of type A endothelin receptors in cultured human myometrial cells.

The aim of the present study was to characterize endothelin (ET)-receptors in human myometrial cells in culture. 125I-labeled ET-1 binding to myometrial cells was specific and saturable, with a dissociation constant of 64.2 +/- 12.8 pM. Competition binding studies showed the following order of potency: ET-1 > ET-3, which is consistent with the presence of the ETA receptor subtype. FR-139317 and BQ-123, two ETA antagonists, both inhibited 125I-ET-1 binding. BQ-123 only elicited a partial inhibition. The fraction resistant to BQ-123 did not represent the ETB receptor subtype, since no specific 125I-ET-3 binding could be detected. ET-1 and ET-3 were found to stimulate [3H]inositol phosphate (IP) accumulation in cultured myometrial cells, with corresponding half-maximal effective concentration values of 0.26 +/- 0.04 and 87 +/- 17 nM, respectively. Both ETA antagonists inhibited ET-1-induced accumulation of [3H]IP. BQ-123 was only a partial inhibitor, whereas FR-139317 totally suppressed ET-1-stimulated production of [3H]IP. We conclude that human myometrial cells in culture exclusively possess ETA receptor subtypes coupled to phospholipase C.