F. Ferré

The status of the corticosteroid-binding globulin (CBG) at the fetomaternal interface, especially in the maternal intervillous blood space (I), was i

The 39-kDa Goalpha protein, the alpha subunit of a major heterotrimeric G protein of brain and neuroendocrine cells, was found to be present in human myometrium. Using three different antisera, we showed its strong expression in myometrium from pregnant patients as compared to nonpregnant ones. This is in agreement with the high expression level of its two isoforms (alphao1 and alphao2), previously described in late pregnancy. To better ascertain the nature of these immunoreactive isoforms, we investigated transcripts of the Goalpha gene in myometrium from pregnant and nonpregnant patients by reverse transcription-polymerase chain reaction (RT-PCR). In this tissue, the amplified cDNA product of a region common to both Go1alpha and Go2alpha mRNA variants was recognized as the Goalpha nucleotide sequence. Transcripts of Go1alpha and Go2alpha were identified by sequencing. A partial cDNA Go2alpha sequence was described, which differed from the Goalpha gene by two nucleotides in exon 8B. Levels of Go1alpha and Go2alpha transcripts analyzed by semi-quantitative RT-PCR were significantly higher in myometrium from pregnant than from nonpregnant patients. It is suggested that Goalpha gene expression in this tissue may contribute to modifications seen in the signaling pathways observed at the end of pregnancy.

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 (PKCα, PKCβ₁, PKCβ₂, PKCζ, and trace amounts of PKCε) 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.

Protein kinase C (PKC) activity in the muscular layer of stem villi vessels from the human term placenta was studied. Resting state PKC activity was distributed evenly between the cytosol and the particulate fractions. Upon stimulation by three different activators, phorbol 12‐myristate 13‐acetate, fluoride and endothelin‐1, a translocation of PKC activity from the cytosolic to the particulate fraction was observed. The expression and distribution of PKC isoforms were then examined by Western blot analysis using specific antibodies to PKC isoforms. At least four PKC isoforms, PKCα, PKCβ₁, PKCβ₂, PKCζ, and trace amounts of PKCϵ were detected in both fractions. Their relative responses to the different agonists were examined by quantifying their subcellular redistribution. No significant differential activation of the four mainly expressed PKC isoforms were observed in response to stimulation with any of the stimuli. Moreover, our results show that endothelin‐1 induced translocation/activation of PKC in this vascular smooth muscle.

The syncytiotrophoblast, which is delineated by two polar membranes (the microvillous and the basal plasma membranes), is the main placental structural element controlling maternal-fetal exchanges. These studies of the full-term placenta were undertaken in order to determine whether the microvillous membranes, which are bathed by the maternal intervillous circulation, and basal plasma membrane, which lines the fetal blood capillaries, have binding sites for insulin-like growth factor (IGF)-II. The microvillous and basal plasma membranes were purified and found to bind 125I-IGF-II with significantly different (p < 0.0001) Kd (0.51 and 1.02 nM, respectively). There were more available binding sites in the microvillous (4.4+/-0.3 pmol/mg protein) than in the basal (2.7+/-0.4 pmol/mg protein) plasma membranes (p < 0.0001). Both membranes contained three major (250, 135, and 130 kDa) 125I-IGF-II/binding-site protein complexes as determined by affinity cross-linking and PAGE. The 250-kDa band (type 2 IGF receptor) was the main band in the basal plasma membranes (46% total bound 125I-IGF-II). The 135-kDa band (insulin-receptor alpha subunit) was the main one in the microvillous membranes (48% total bound 125I-IGF-II). The amounts of 130-kDa band (type 1 IGF-receptor alpha subunit) in the two types of membranes were similar (30% total bound 125I-IGF-II). Only IGF-II displaced 125I-IGF-II from the 250-kDa band, while 125I-IGF-II bound to the 135-kDa band was displaced by insulin, and ligand bound to the 130-kDa band was displaced by IGF-I. Thus there are IGF receptors in both types of membranes of syncytiotrophoblast in the human full-term placenta, and the distributions of the IGF and insulin receptors are asymmetrical. This may reflect the fact that they face and interact with two independent, different media. Maternal IGF may influence the syncytiotrophoblast by binding to receptors on the microvillous membranes, while fetal IGF may also influence syncytiotrophoblast functions by activating receptors in the basal plasma membranes.

To determine if there are endothelin receptors on human uterine leiomyomas. 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. 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 endothelin_A receptor subtype. Binding of [¹²⁵I]endothelin-1 was displaced by an endothelin_A-selective antagonist, but not by sarafotoxin 6c, an endothelin_B-selective agonist. An endothelin_B-selective ligand was not specifically bound to leiomyoma. These results indicate that only endothelin_A receptors are present in human uterine leiomyomas. We speculate that endothelin-1 may act through these endothelin_A receptors to influence the development or regulation of hypertrophy and proliferation of the human myometrium during pregnancy and in uterine disorders like leiomyomas.

Endothelin-1 (ET-1) is a potent vasoactive peptide in stem villi vessels, which are considered to be the major sites of placental vascular resistance. To investigate the influence of pregnancy-specific hormonal environment on ET and ET receptor (ET-R) expression, we first developed and characterized a culture of vascular smooth muscle cells from stem villi vessels. Secondly, we investigated whether the muscular layer of stem villi vessels could be a site of the ET expression described in the placenta, and we examined this expression in placental vascular smooth muscle cells (PVSMCs). Prepro-ET-1 and prepro-ET-3 messenger ribonucleic acid (mRNA) were identified in stem villi vessels, whereas only prepro-ET-1 mRNA was observed in PVSMCs. Third, with the goal of using PVSMCs as ET target cells, we characterized the ET-R expressed by these cells in comparison with the muscular layer of stem villi vessels. Whereas both ETA-R and ETB-R are present in stem villi vessels, we found that PVSMCs express exclusively ETA-R. In addition to the previously reported ETA-R spliced transcripts, we described a new ETA-R transcript, ETA-R delta 3, generated by exclusion of exon 3 in stem villi vessels and PVSMCs. Alternative splicing mechanisms of ETA-R mRNA could constitute a control of the abundance of active ETA-R in terms of contractility. PVSMCs will be a useful model to study the environmental stimuli involved in the regulation of ET and ET-R expression in the muscular layer of feto-placental vasculature.

The initial signal for triggering human parturition might be fetal but of trophoblastic origin. Concomitantly, this placental signal would have as its target not only the uterus but also the fetus by activating its hypothalamo-pituitary-adrenocortical axis. The latter would represent a second fetal signal which, at the fetomaternal interface, would amplify and define in time the mechanisms responsible for the onset of labor, implying changes in the myometrial and cervical extracellular matrix associated with the accession of the contractile phenotype for myometrial cells. At each phase of these processes in the utero-feto-placental system, the nature of these signals remains to be identified. Is there a single substance, or rather, and more likely, a combination of several? We appear to be in the presence of dynamic systems of a neuro-immuno-hormonal type which are difficult to describe. Nevertheless, steroid hormones appear to coordinate their successive equilibriums until they become irreversible. Such irreversibility constitutes the essential sign of parturition.