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Proinflammatory cytokines produced at the fetomaternal interface, such as IL-1beta, have been implicated in preterm and term labor. The present study was performed to evaluate the influence of IL-1beta on the endothelin (ET)/ET receptor system in human myometrial cells. We report that myometrial cells under basal conditions not only respond to but also secrete ET-1, one of the main regulators of uterine contractions. Prolonged exposure of the cells to IL-1beta led to a decrease in prepro-ET-1 and ET-3 mRNA correlated with a decrease in immunoreactive ET-1 and ET-3 levels in the culture medium. Whereas ETA receptor expression at both protein and mRNA levels was not affected by IL-1beta treatment, we demonstrated an unexpected predominance of the ETB receptor subtype under this inflammatory condition. Whereas the physiological function of ETB remains unclear, we confirmed that only ETA receptors mediate ET-1-induced myometrial cell contractions under basal conditions. By contrast, prolonged exposure of the cells to IL-1beta abolished the contractile effect induced by ET-1. Such a regulation of IL-1beta on the ET release and the balance of ETA to ETB receptors leading to a loss of ET-1-induced myometrial cell contractions suggest that complex regulatory mechanisms take place to constraint the onset of infection-induced premature contractions.

The status of the corticosteroid-binding globulin (CBG) at the fetomaternal interface, especially in the maternal intervillous blood space (I), was investigated and compared to that of CBG in the maternal (M) and fetal (umbilical arteries [A] and vein [V]) peripheral circulations at term. Immunoquantitation of plasma CBG showed that the CBG concentration in I was 30% less than that in M (P < 0.001) and threefold higher than that in umbilical cord blood (P < 0.001). The microheterogeneity of CBG studied by immunoaffinoelectrophoresis in the presence of concanavalin A and Western blotting indicated that the CBG in I was mainly of maternal origin and different from fetal CBG. A CBG mRNA, but no classic 50- to 59-kDa CBG, was found in isolated term trophoblastic cells. The steroid environment of the CBG in I differed greatly from that in the peripheral maternal and fetal circulations, because the progesterone:cortisol molar ratio in I was 75-fold higher than that in M and 7- to 10-fold higher than that in the fetal circulation. Binding studies revealed that the affinity constants of CBG for cortisol in I, A, and V were significantly lower than that in M plasma (P < 0.02) in their respective hormonal contexts. The binding parameters for I-CBG stripped of endogenous steroids and lipids were close to those for M-CBG but different from those of fetal CBG (P < 0.001). These data reflect the physiological relevance of the CBG-steroid interaction, especially with very CBG-loaded progesterone at the fetomaternal interface during late pregnancy.

The presence of progesterone receptors (PR) throughout the human term fetoplacental vascular tree was investigated. By reverse transcription-polymerase chain reaction (RT-PCR), we showed expression of PR mRNAs in stem villi vessels, chorionic arteries and veins, and umbilical arteries and veins. Binding studies and Scatchard analysis revealed a single class of high-affinity binding sites for (3)H-R5020 (promegestone) in cytosolic extracts of all placental vessels, with K(d) values in the range of 2.5-4 nM. High levels of PR were detected in placental vessels compared to other vascular tissues. Thus, maximum binding capacities of stem villi vessels, chorionic arteries and veins, and umbilical arteries and veins were 247 +/- 25, 377 +/- 58, 295 +/- 40, 371 +/- 118, and 672 +/- 144 fmol/mg protein, respectively. Endothelial cell elimination in chorionic arteries did not significantly modify the number of PR. RT-PCR and binding studies also assessed PR expression in cultured placental vascular smooth muscle cells isolated from stem villi vessels. All these data suggested that most of the PR of fetoplacental vessels were from the media. In conclusion, we report here the first evidence of the presence of PR in the muscular layer of human term fetoplacental vessels. This finding, together with the high progesterone concentrations in cord blood, suggests that the interactions between the PR and its ligand may play a role in the physiology and physiopathology of human fetoplacental vascularization.

In human myometrium, the modulation of intracellular cAMP content resulting from agonist-mediated stimulation of the receptor-adenylyl cyclase complex is largely influenced by the rate of cAMP hydrolysis by phosphodiesterase (PDE) isoenzymes. We have previously shown that the PDE4 family contributes to the predominant cAMP-hydrolyzing activity in human myometrium and that elevation of the PDE4B2 messenger RNA steady state level occurs in pregnant myometrial tissue. In the present study, we used a model of human myometrial cells in culture to determine whether an elevated cAMP concentration could influence PDE expression. As in myometrial tissue, high levels of PDE4 activity were detected in these smooth muscle cells. Long term treatment with 8-bromo-cAMP or forskolin resulted in a selective induction of PDE4B and of PDE4D short form messenger RNA variants. Concurrently, an increased immunoreactive signal for the PDE4B- and PDE4D-related isoenzymes was detected. This induction was consistent with an observed significant up-regulation of PDE4 activity. Accordingly, our results demonstrate that in human cultured myometrial cells, cAMP-elevating agents manipulate PDE4 activity through selective induction of synthesis of PDE4B and PDE4D short forms. Such a mechanism might have physiological importance during pregnancy by dampening hormonal stimulation and could thereby be involved in tolerance to the tocolytic effect of beta-adrenoceptor agonists.

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.

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 primary function of the placenta is to ensure an optimal environment for fetal growth and development. In normal pregnancy, placental vascular tone regulation assures fetus well-being and normal development by maintaining adequate blood flow so as to ensure materno-fetal exchanges. In human placenta, synthesis of insulin-like growth factor (IGF)-II and specific binding sites have been previously characterized in the trophoblast; in contrast, no studies have dealt with this subject in the fetoplacental vascular system, particularly in stem villi vessels. We thus investigated whether membranes of the muscular layer of stem villi vessels contained 125I-IGF-II binding sites. Two complementary approaches were used: 125I-IGF-II binding and affinity cross-linking studies. 125I-IGF-II labeled, in a saturable and noncooperative manner, a single class of high-affinity binding sites characterized by a Kd of 1.24 +/- 0.26 nM (n = 6), a maximum binding capacity (Bmax) of 3.02 +/- 0.45 pmol/mg protein, and a Hill coefficient of 1.00 +/- 0.15. Competitors for 125I-IGF-II binding to membranes were in the order of potency IGF-II > IGF-I. Insulin was not a competitor. Affinity cross-linking of membranes with 125I-IGF-II, followed by SDS-PAGE and autoradiography, revealed two labeled bands: a protein complex of 250 kDa, which corresponds to the type II IGF receptor, and another of 135 kDa, corresponding to the type I IGF receptor. Only IGF-II could displace 125I-IGF-II binding from the major 250-kDa band, while 125I-IGF-II bound to the minor 135-kDa band was displaced by either IGF-I, IGF-II, or insulin. In conclusion, high levels of specific binding sites for 125I-IGF-II are present in the muscular layer of stem villi vessels, which are considered placenta resistance vessels. The involvement of both type I and type II IGF receptors in the growth-promoting action of IGF-II remains to be determined in the fetoplacental vascular system.

Serum concentrations of total cholesterol, triglycerides, and apolipoproteins (apo) A-I, B, CII, CIII, and E in 36 hemodialysis patients and nine anephric patients were compared with the concentrations in 34 normolipidemic subjects. The dialysis patients displayed a moderate hypertriglyceridemia (1.94 +/- 0.12 vs 1.09 +/- 0.11 mmol/L in controls, mean +/- SEM; P less than 0.001), apo CIII concentrations were also increased (130.2 +/- 2.1 vs 108.4 +/- 0.7 mg/L; P less than 0.001), whereas apo CII (34.5 +/- 0.5 vs 36 +/- 0.5 mg/L; P less than 0.05), apo E (22.7 +/- 0.3 vs 27.9 +/- 0.2 mg/L; P less than 0.001), and apo A-I (1.18 +/- 0.05 vs 1.31 +/- 0.04 g/L; P less than 0.05) were decreased. Concentrations of serum apo B were normal (0.86 +/- 0.03 vs 0.97 +/- 0.07 g/L). In the hemodialysis patients, apo CIII concentrations were increased in apo B-containing lipoproteins (30.1 +/- 0.5 vs 25.0 +/- 0.1 mg/L; P less than 0.001), whereas CII and E were decreased below control values (14.4 +/- 0.2 vs 16.8 +/- 0.1, and 8.2 +/- 0.2 vs 11.4 +/- 0.1 mg/L, respectively; P less than 0.001 each). By calculation, non-B-containing lipoproteins in the hemodialysis group had increased concentrations of apo CIII (100.1 +/- 2.1 vs 83.3 +/- 0.7 mg/L; P less than 0.001) and decreased amounts of apo E (14.5 +/- 0.4 vs 16.4 +/- 0.3 mg/L; P less than 0.001); apo CII content was unchanged (20.1 +/- 0.5 vs 19.3 +/- 0.5 mg/L). Results for apo CII, CIII, and E among apo A-I-containing lipoproteins in both normolipidemic and hemodialysis groups were similar to those in non-B-containing lipoproteins. Finally, the sole significant (P less than 0.01) difference between the anephric and hemodialysis groups was the lower apo E concentrations in the former group. Accumulation of triglyceride-rich lipoproteins in hemodialysis patients may thus be related to the enrichment of apo CIII in apo B-containing lipoproteins and to a marked decrease in the apo CII and E contents.

We describe a new enzyme-linked immunosorbent assay (ELISA) that permits direct determination of apoprotein (apo) CII, CIII, and E in total serum as well as in apo B-containing lipoprotein particles. To validate this ELISA technique, we studied several aspects of the assay: its specificity, the influence of the conditions of conservation of plasma and of lipoprotein fractions, the effect of delipidation, and its reproducibility. We measured the concentrations of apo CII, CIII, and E in total serum and in apo B-containing lipoproteins from a pool of normal sera and in sera from 75 healthy subjects. After sequential ultracentrifugation, the content of apo CII, CIII, and E in the major lipoprotein fractions was also determined. Total serum or plasma could be stored at -20 or -50 degrees C for at least six weeks and the isolated lipoprotein fractions for as long as four weeks, which suggests a protective effect of total serum on lipoprotein particle structure. Advantages of this ELISA include (a) its specificity, sensitivity, and reliability; (b) better discrimination than determination of total serum apoprotein; (c) easier application and greater rapidity; and (d) the possibility of application to population screening.