Receptors In Human Myometrium Research Articles

Expression of Prothrombin and Protease Activated Receptors in Human Myometrium During Pregnancy and Labor1

As thrombin is proposed to be involved in stimulating myometrial contractility during labor and preterm labor, we aimed to investigate the expression of prothrombin (F7), the precursor of thrombin, its receptors, the protease-activated receptor (PAR) family (F2R, F2RL1, F2RL2, and F2RL3), and prothrombinase FGL2 in human myometrium during pregnancy and labor. Messenger RNA and protein were isolated from human pregnant laboring and nonlaboring myometrial tissue and from human primary myometrial smooth muscle cells. Semiquantitative RT-PCR, real-time fluorescence RT-PCR, Western blotting, and fluorescence microscopy were performed to determine the expression levels of F7, FGL2, F2R, F2RL1, F2RL2, and F2RL3 in the myometrial tissues and cells. The expression of mRNA and protein for these molecules is reported for the first time in human myometrium at term pregnancy, at labor, and in the nonpregnant state. Importantly, an increase in F2R and a significant increase in F2RL3 mRNA expression at labor were demonstrated. Statistically significant increases in F2R and F2RL3 protein expression was also detected in human myometrium at labor. Furthermore, FGL2 mRNA expression at labor, and FGL2 protein expression at term pregnancy and at labor was observed in this tissue for the first time. The expression of F7, FGL2, F2R, F2RL1, F2RL2, and F2RL3 in human myometrium reveals that all the machinery necessary for thrombin activation and cellular activity is present in the myometrium during pregnancy and labor. These data, in conjunction with the demonstrated increase in F2R and F2RL3 expression at labor, suggest a principal role for these molecules in the regulation of myometrial function at labor, including preterm labor.

Expression of the Oxytocin and V1a Vasopressin Receptors in Human Myometrium in Differing Physiologic States and following Misoprostol Administration

Objective: To assess relative expression levels of mRNA for the oxytocin receptor (OTR) and the V1a vasopressin receptor (V1aR) in human myometrium in various physiologic states and to determine if misoprostol exposure affects the expression of mRNA for these receptors. Study Design: Using semiquantitative reverse transcription-polymerase chain reaction, we analyzed myometrial samples from 37 women (10 menopausal, 10 nongravid premenopausal, 10 gravidae, 7 premenopausal misoprostol-treated nongravid women) for the specific expression of mRNA messages for OTR and V1aR. The presence of the OTR and V1aR protein was confirmed by immunohistochemistry. Results: We found that the expression of OTR was significantly higher in gravid compared to premenopausal nongravid (p < 0.0001), misoprostol-treated (p < 0.001) and menopausal myometrium (p < 0.0001). Differences in V1aR expression were not statistically significant among the four study groups. Conclusion: The expression of OTR mRNA in myometrium is markedly increased during late gestation. V1aR expression is present in all physiologic states, and does not appear to be altered by the presence or absence of labor. Misoprostol administration does not appear to have an effect on OTR and V1aR mRNA receptor expression.

Regional Expression of Prostaglandin E2 and F2alpha Receptors in Human Myometrium, Amnion, and Choriodecidua with Advancing Gestation and Labor1

The change from uterine quiescence to enhanced contractile activity may be due to the differential expression of prostaglandin receptors within the myometrium and fetal membranes, in a temporal and topographically distinct manner. To address this question, we determined the localization and expression of the PGE2 receptor subtypes (PTGER1-4) and the PGF2alpha receptor (PTGFR) in paired upper and lower segment myometrium, amnion, and choriodecidual samples throughout human pregnancy, with and without labor. All receptor subtypes were found throughout the muscle layers in both the upper and lower uterine segments, colocalizing with alpha smooth muscle actin. A change in intracellular localization was observed at term labor, where PTGER1 and PTGER4 were predominately associated with the nucleus. Minimal changes in the expression of the PGE2 and PGF2alpha receptor subtypes were observed with gestational age, labor, or between the upper and lower myometrial segments. Receptor expression in maternal and fetal tissues differed between the receptor subtypes; PTGER1 and PTGER4 were predominately expressed in the fetal membranes, PTGER2 was greatest in the myometrium, whereas PTGER3 and PTGFR were similarly expressed in the myometrium and fetal membranes. Myometrial activation through the prostaglandin receptors is perhaps more subtle and may be mediated by a balance between one or several of the prostaglandin receptor subtypes together with other known contraction associated proteins. Lack of coordination in receptor expression between the myometrium and fetal membranes may indicate different regulatory mechanisms between these tissues, or it may suggest a function for these receptors in the amnion and choriodecidua that is independent of that seen in the myometrium.

Activin betaA-subunit and activin receptors in human myometrium at term and during labour.

To measure activin A content and to localise and semi-quantitate activin receptors in human myometrium at term and during labour. Myometrium was collected from non-pregnant women (n = 6), pregnant women at term not in labour (n = 6) and at term in labour (n = 6). Monash Medical Centre, Melbourne, Australia. Tissue lysates of myometrium were analysed for activin A content using an enzyme-linked immunosorbent assay and activin receptor proteins IA, IIA and IIB using Western hybridisation. Activin betaA-subunit and activin receptors were localised in myometrium by immunohistochemistry. Activin A was detected by ELISA in non-pregnant, pregnant and labouring myometrium. Levels were significantly higher in labouring myometrium. The three activin receptors IA, IIA and IIB were detected in all myometrial samples by Western hybridisation. Receptor IA was expressed in significantly higher levels in pregnant myometrium. Receptor IIA was very weakly expressed throughout. The expression of receptor IIB was similar in all three groups. Activin betaA-subunit and all three receptors were localised to the endothelial cells of myometrial blood vessels. Neither activin betaA-subunit nor any of the three activin receptors were immunolocalised to myometrial smooth muscle cells in the three groups. This result was confirmed by Western blotting for expression of activin receptors in isolated myometrial smooth muscle and microvascular endothelial cells. The myometrium is not a target for activin A during late pregnancy or labour. However, activin A may have a role in the regulation of microvascular endothelial cell function in the myometrium.

Activin β A-subunit and activin receptors in human myometrium at term and during labour

Objective To measure activin A content and to localise and semi-quantitate activin receptors in human myometrium at term and during labour. Design Myometrium was collected from non-pregnant women ( n = 6), pregnant women at term not in labour ( n = 6) and at term in labour ( n = 6). Setting Monash Medical Centre, Melbourne, Australia. Main outcome measures Tissue lysates of myometrium were analysed for activin A content using an enzyme-linked immunosorbent assay and activin receptor proteins IA, IIA and IIB using Western hybridisation. Activin β A-subunit and activin receptors were localised in myometrium by immunohistochemistry. Results Activin A was detected by ELISA in non-pregnant, pregnant and labouring myometrium. Levels were significantly higher in labouring myometrium. The three activin receptors IA, IIA and IIB were detected in all myometrial samples by Western hybridisation. Receptor IA was expressed in significantly higher levels in pregnant myometrium. Receptor IIA was very weakly expressed throughout. The expression of receptor IIB was similar in all three groups. Activin β A-subunit and all three receptors were localised to the endothelial cells of myometrial blood vessels. Neither activin β A-subunit nor any of the three activin receptors were immunolocalised to myometrial smooth muscle cells in the three groups. This result was confirmed by Western blotting for expression of activin receptors in isolated myometrial smooth muscle and microvascular endothelial cells. Conclusion The myometrium is not a target for activin A during late pregnancy or labour. However, activin A may have a role in the regulation of microvascular endothelial cell function in the myometrium.

Loss of myometrial oxytocin receptors during oxytocin-induced and oxytocin-augmented labour

Oxytocin is used widely for the induction and augmentation of labour, but there is little information about the dynamics of oxytocin receptors in human myometrium during parturition, and the possible effect of oxytocin infusion. This information is important because G protein-coupled receptors, such as the oxytocin receptor, undergo desensitization after prolonged or repeated stimulation. The concentration of myometrial oxytocin receptors and the steady state of its mRNA were measured in patients undergoing Caesarean sections before or during spontaneous or induced labour. The concentration of receptors before labour was 477 (175-641) fmol mg(-1) protein (median, quartile range), and decreased to 140 (72-206; P &lt; 0.05) and 118 (69-75; P &lt; 0.01) fmol mg(-1) protein during prolonged oxytocin-augmented and oxytocin-induced labour, respectively. The corresponding oxytocin receptor mRNA concentrations decreased by 60- and 300-fold, respectively. The decrease in receptor binding and mRNA in women receiving oxytocin infusion indicates that homologous receptor desensitization occurs in vivo.

Loss of myometrial oxytocin receptors during oxytocin-induced and oxytocin-augmented labour.

Oxytocin is used widely for the induction and augmentation of labour, but there is little information about the dynamics of oxytocin receptors in human myometrium during parturition, and the possible effect of oxytocin infusion. This information is important because G protein-coupled receptors, such as the oxytocin receptor, undergo desensitization after prolonged or repeated stimulation. The concentration of myometrial oxytocin receptors and the steady state of its mRNA were measured in patients undergoing Caesarean sections before or during spontaneous or induced labour. The concentration of receptors before labour was 477 (175-641) fmol mg(-1) protein (median, quartile range), and decreased to 140 (72-206; P < 0.05) and 118 (69-75; P < 0.01) fmol mg(-1) protein during prolonged oxytocin-augmented and oxytocin-induced labour, respectively. The corresponding oxytocin receptor mRNA concentrations decreased by 60- and 300-fold, respectively. The decrease in receptor binding and mRNA in women receiving oxytocin infusion indicates that homologous receptor desensitization occurs in vivo.

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.

Expression of Endothelin Receptors in Human Myometrium During Pregnancy and in Uterine Leiomyomas

Expression of Endothelin Receptors in Human Myometrium During Pregnancy and in Uterine Leiomyomas

Focal sarcoplasmic reticulum calcium stores and diffuse inositol 1,4,5-trisphosphate and ryanodine receptors in human myometrium

Intracellular calcium stores of human uterine myocytes in primary and second passage cell culture were visualized using the low-affinity calcium-sensitive fluorescent dye, fluo-3FF. The calcium stores appeared as numerous small (0.2–0.5 μm diameter) focal fluorescences. The stores were not depleted by exposing the cells to oxytocin or ryanodine under standard conditions. The stores were rapidly depleted by oxytocin or ryanodine exposure when sarcoplasmic reticulum (SR) calcium re-uptake was inhibited by pretreatment with thapsigargin. Immunofluorescence experiments indicated that both ryanodine and inositol 1,4,5-trisphosphate (IP3) receptors were smoothly distributed throughout the SR, and neither receptor co-localized with the calcium stores. Since IP3and ryanodine calcium channels are tightly associated with their receptor, these results suggest that SR calcium release occurs via second messenger channels that are remote from the SR calcium stores. These observations are consistent only with a mechanism for release of calcium stores where the SR serves three functions: (1) as site of calcium storage, (2) as the structure that contains the IP3- and ryanodine receptors and their associated release channels, and (3) as a conduit between the calcium stores and the release channels.

Roles of endothelin-l(ET-1) and receptors in human myometrium and leiomyoma.

Roles of endothelin-l(ET-1) and receptors in human myometrium and leiomyoma.

Sex Steroid Receptors in Human Myometrium and Fibroids: Changes during the Menstrual Cycle and Gonadotropin-Releasing Hormone Treatment

Sex Steroid Receptors in Human Myometrium and Fibroids: Changes during the Menstrual Cycle and Gonadotropin-Releasing Hormone Treatment

Desensitization of oxytocin receptors in human myometrium.

In the present study, we investigated the possible mechanisms by which oxytocin might regulate oxytocin receptor (OTR) density. Exposure of cultured myometrial cells to oxytocin for a prolonged period caused desensitization: the steady-state level of oxytocin binding was 210 x 10(3) binding sites/cell, but this was time-dependently reduced to 20.1 x 10(3) sites/cell by exposing the cells to oxytocin for up to 20 h. In contrast, Western blotting data showed that the total amount of OTR protein was not affected by oxytocin treatment for up to 24 h. Flow cytometry experiments demonstrated that OTRs were not internalized during this treatment. However, RNase protection assays and Northern analysis showed that in cultured myometrial cells OTR mRNA was reduced by oxytocin treatment to reach a new low steady-state concentration. Analysis of this mRNA in myometrial biopsies from 17 patients undergoing emergency Caesarean section showed how it decreased with advancing labour. Samples obtained after 12 h of labour contained approximately 50 times less OTR mRNA than samples obtained from patients in labour for less than 12 h. We speculate that this decrease in OTR mRNA represents in-vivo OTR desensitization.

Expression of contractile FP and relaxatory EP receptors in human myometrium during gestation and with labor

Expression of contractile FP and relaxatory EP receptors in human myometrium during gestation and with labor

Tocolytic therapy with fenoterol induces selective down-regulation of beta-adrenergic receptors in human myometrium.

Tocolytic therapy with beta-adrenergic receptor agonists is a standard regimen to prevent preterm birth. Agonists exposure of beta-adrenergic receptors causes receptor desensitization in other organs, and this may limit the therapeutic value of beta-adrenergic receptor agonists. To study the effects of prolonged beta-adrenergic agonist treatment in human myometrium, we obtained biopsies during Caesarean section of 14 pregnant patients who had received fenoterol for at least 5 days and 14 untreated pregnant controls. The densities of total beta-adrenergic receptors, which are mainly of the beta 2-subtype as assessed by [125I]iodo-cyanopindolol binding in crude membrane fractions, were more than 50% smaller in women receiving fenoterol, whereas alpha 2-adrenergic receptor densities were similar. Gs and Gi G-protein alpha-subunit densities were unaltered as assessed by Western blotting and pertussis toxin-catalyzed [32P]ADP-ribosylation. beta-Adrenergic receptor kinase (beta ARK) activity, as determined using bovine rhodopsin as the substrate, was the same in the two groups. Adenylyl cyclase activities in the presence of guanine nucleotides, NaF, forskolin, or Mn+2 were also not altered by fenoterol treatment. The messenger RNA (mRNA) concentrations of beta 2-adrenergic receptors, beta ARK-I and glyceraldehyde-3-phosphate dehydrogenase (as a reference), as determined by quantitative PCR, were unaffected by fenoterol treatment. We conclude that tocolysis with fenoterol results in a selective down-regulation of myometrial beta-adrenergic receptors, which is not associated with a reduction in the respective mRNA concentrations or alterations of alpha 2-adrenergic receptors, Gs and Gi alpha-subunits, or beta ARK activity or mRNA.

Tocolytic Therapy with Fenoterol Induces Selective Down-Regulation of -Adrenergic Receptors in Human Myometrium

Tocolytic Therapy with Fenoterol Induces Selective Down-Regulation of -Adrenergic Receptors in Human Myometrium

Epidermal growth factor receptors in uteroplacental tissues in term pregnancy before and after the onset of labor.

Using saturation binding assays and Scatchard analyses, we determined the concentrations and binding affinities of epidermal growth factor (EGF) receptors in human myometrium (n = 13) and decidua (n = 10) before and during labor and in placenta (n = 15), chorion (n = 17), and amnion (n = 17) before labor, during labor, and after vaginal delivery. Each tissue was individually assayed. In myometrium and chorion, EGF receptors increased significantly from 5.6 +/- 0.8 and 13.5 +/- 1.7 fmol/mg protein (mean +/- SEM) before labor to 11.1 +/- 2.8 and 26.7 +/- 3.0 fmol/mg protein, respectively, after the onset of labor (P < 0.05). In amnion, EGF receptors increased from 12.8 +/- 2.7 fmol/mg protein before labor to 33.0 +/- 2.3 fmol/mg protein during labor, but decreased significantly (5.9 +/- 1.2 fmol/mg protein) with vaginal delivery (P < 0.05). Decidual and placental concentrations of EGF receptors did not change significantly with labor. The binding affinity of EGF receptors in all tissues studied did not change significantly with labor, as reflected by their respective association and dissociation constants. Up-regulation of EGF receptors in myometrium, chorion, and amnion with spontaneous labor may enhance stimulation of prostanoid production and stimulate uterine activity.

Sex steroid modulation of AT2 receptors in human myometrium.

In contrast to the abundant expression of the AT2 subtype of angiotensin II (AII) receptors during fetal development, AT2 receptor in adult life is expressed in few tissues. We now report studies on the presence and hormonal regulation of AT2 receptor in human pregnant and nonpregnant myometrium obtained from a large study population (n = 50). AT2 receptor subtypes have been characterized using self- and cross-competition curves among [125I]CGP42112A (a selective AT2 ligand), [125I](Sar1,Ile8)AII (a unselective antagonist), the corresponding unlabeled ligands, and several peptidic and nonpeptidic analogs with different affinities for the AT1 and AT2 receptor subtypes. We found that the human nonpregnant uterus expresses almost exclusively the AT2 subtype, and that [125I]CGP42112A is a selective probe to study human AT2 receptor. By using [125I]CGP42112A, we demonstrated that the density of AT2 receptor in human myometrium is dramatically affected by the hormonal milieu. Indeed, in the estrogen-dominant uterus of normal cycling women in the proliferative phase and that of perimenopausal women with anovulatory cycles, the density of binding sites was very high [1565 +/- 246 fmol/mg protein (n = 11) and 2176 +/- 429 (n = 7), respectively]. The concomitant presence of progestogens blunted the estrogen effect [term pregnancy, 61 +/- 12 fmol/mg protein (n = 5); secretive phase of the cycle, 453 +/- 154 (n = 10); combined oral contraceptive, 243 +/- 74 fmol/mg protein (n = 6)]. Very low concentrations of binding sites are also present in the sex steroid-deprived uterus of postmenopausal women (100 +/- 12 fmol/mg protein; n = 8) and the uterus of fertile women chronically treated with GnRH agonists (199 +/- 100 fmol/mg protein; n = 3). Hence, these data confirm the presence of AT2 receptors in human uterus and indicate their regulation by sex steroids.

Sex steroid modulation of AT2 receptors in human myometrium

Sex steroid modulation of AT2 receptors in human myometrium

Restoration of human chorionic gonadotropin response in human myometrial smooth muscle cells by treatment with follicle-stimulating hormone (FSH): evidence for the presence of FSH receptors in human myometrium.

Human myometrial smooth muscle cells contain receptors for human chorionic gonadotropin (hCG)/luteinizing hormone (LH). Exogenous hCG and LH can cause a modest hyperplasia in myometrial smooth muscle cells in culture. This response is lost after about the third subculture of the cells. The present study investigated whether the loss of hCG response could be restored by co-culturing with human follicle stimulating hormone (FSH). The results showed that co-culturing with FSH can indeed restore a modest mitogenic response of hCG. However, FSH alone was not mitogenic. The FSH restoration of hCG response can be blocked by antibodies to FSH or hCG but not by non-specific rabbit IgG. The FSH treatment resulted in an increase of steady state levels of hCG/LH receptor mRNA and protein in myometrial smooth muscle cells. Since the FSH actions could be receptor mediated, we investigated the presence of FSH receptor mRNA transcripts and protein in freshly dispersed myometrial smooth muscle cells. Northern blotting demonstrated that myometrial smooth muscle cells, just as rat ovary, a classical target of FSH action, contain multiple FSH receptor mRNA transcripts. Western immunoblotting demonstrated that myometrial smooth muscle cells also contain a 60 kDA FSH receptor protein just as rat ovary and human granulosa cells used as positive control tissues. The immunocytochemistry also demonstrated that myometrial smooth muscle cells, as rat ovary and human granulosa cells, contain FSH receptor immunostaining. In summary, it is novel that FSH could restore the mitogenic response of hCG in human myometrial smooth muscle cells and these cells contain FSH receptors. These findings may have functional implications for direct regulation of human myometrium not only by hCG/LH but also by FSH.