G. Tanguy

Selective Up-Regulation of Phosphodiesterase-4 Cyclic Adenosine 3′,5′-Monophosphate (cAMP)-Specific Phosphodiesterase Variants by Elevated cAMP Content in Human Myometrial Cells in Culture

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.

Steroid concentrations in human myometrial and placental tissues at week 39 of pregnancy.

Endogenous levels of P, E1, and E2 were determined by radioimmunoassay in human myometrium and placenta at week 39 of pregnancy. In the myometrium, P and estrogens were higher in the inner layer near the placenta than in other zones. Higher E2/P ratios were found in placental sites. A large difference in the E2/P ratios was observed between placenta and corresponding adjacent myometrial area, indicating an easier diffusion of E2 from the site of its production.

Isolation and characterization of the rolipram-sensitive cyclic AMP-specific phosphodiesterase (type IV PDE) in human term myometrium

On the basis of the potencies of classical selective modulators of cyclic nucleotide phosphodiesterase (PDE) activities, five cyclic nucleotide PDE isoforms have been isolated and characterized in the cytosolic fraction of human term myometrium. By means of successive ion-exchange chromatographies, a calcium-calmodulin sensitive isoform, a cyclic GMP-stimulated isoform, a cyclic GMP-inhibited isoform, a rolipram-sensitive cyclic AMP-specific isoform and a cyclic GMP-specific isoform, corresponding to PDE I, PDE II, PDE III, PDE IV and PDE V, respectively, have been identified. We found that near term, human myometrium contains a higher proportion of the rolipram-sensitive type IV PDE isoform (about 50% of total cyclic AMP hydrolytic activity) than the type III cyclic GMP-inhibited PDE isoform (only 10%). Type IV PDE displays simple Michaelis-Menten kinetics with a high affinity for cyclic AMP (Km approximately 4.4 microM) and is selectively and competitively inhibited by rolipram (K(i) approximately 0.9 microM) and Ro 20-1724 (K(i) approximately 2.6 microM). The predominance of type IV PDE at the end of pregnancy suggests that this isoform contributes, via a modulation of the intracellular cyclic AMP level, to local control of uterine motility and thus could help the myometrium prepare for pronounced contractile activity at the time of parturition.

The "dysharmonic luteal phase" syndrome: endometrial progesterone receptor and estradiol dehydrogenase.

The dysharmonic luteal phase (DLP) syndrome is defined by delayed endometrial maturation despite normal plasma progesterone (P) values. In ten patients with DLP the actual date of the endometrial biopsy, dated retrospectively, was 24.7 +/- 2.3 days, whereas the histologic date was 20.0 +/- 2.6 days. The concentration of cytosolic P receptor in DLP endometrium tended to be lower, whereas the concentration of nuclear receptor was significantly higher in DLP than in seven matched patients with normal luteal phases. Endometrial estradiol-dehydrogenase activities were identical in both groups. The DLP syndrome cannot be explained by a decreased sensitivity of the endometrium to P and is probably merely functional in nature.

Radioimmunoassay of plasma and salivary progesterone during the menstrual cycle

We have developed specific and sensitive radioimmunoassays for progesterone in saliva (SP4) easily applicable in routine practice to assess ovarian function. One of them uses [3H]progesterone, the other 3-CMO-[125I]histamine as tracer. The reliability criteria of both techniques have been controlled and the results obtained with the two methods in 50 saliva samples are correlated well. Our clinical study has been chiefly done with [3H]progesterone. In the present study we reported the values obtained daily during the greatest part of the menstrual cycle in 14 normal cases and they are in agreement with previous reports. The use of [125I]tracer, by increasing the sensitivity of the RIA and shortening the duration of the assay, improves the use of SP4 as a practical non-invasive sampling alternative for many clinical situations involving evaluation of the ovarian function.

Oral administration of micronized natural progesterone in late human pregnancy: Effects on progesterone and estrogen concentrations in the plasma, placenta, and myometrium

A single dose of micronized oral progesterone was administered to 15 pregnant women immediately prior to elective cesarean section. Levels of progesterone, 17 beta-estradiol, and estrone were measured in the plasma, in the placenta, and at different sites in myometrium obtained during the surgical procedure. Results were compared to those observed in a control group of women who did not receive progesterone. Progesterone levels demonstrated a marked increase in plasma and in the whole myometrium 150 minutes after administration. The levels then decreased rapidly to control values in 1 hour. The concentrations of progesterone in the placenta did not show any changes. No difference appeared in 17 beta-estradiol levels in the plasma or the myometrium, whereas an increase was observed in the placenta. Estrone levels did not change in the plasma, but they decreased in the myometrium and in the placenta.

Effect of pregnancy on PDE4 cAMP-specific phosphodiesterase messenger ribonucleic acid expression in human myometrium.

In light of the important role of the second messengers cAMP and cGMP in the mechanism of relaxation in the human myometrium, specific regulation of the phosphodiesterase (PDE) enzymatic system responsible for cyclic nucleotide inactivation is essential. We previously identified in the human myometrium PDE4 cAMP-specific PDE as by far the most abundant isoform. Here we have studied the expression patterns of mRNAs for the four cloned human PDE4 genes in the myometria of pregnant and non-pregnant women. Concurrent expression of the PDE4A, 4B, 4C and 4D genes is demonstrated. We found that the PDE4D transcripts are the most prominently expressed. PDE4A and PDE4B mRNAs also are markedly abundant, whereas lower expression is observed for PDE4C mRNAs. Interestingly, we showed that transcripts of PDE4B2 are more abundant in the myometria of pregnant women than in non-pregnant women, whereas no difference between the two tissues was detected for PDE4A, 4C and 4D mRNAs. Cultured human myometrial cells, which present a high level of PDE4 activity and express the four PDE4 mRNA subtypes, provide us with an appropriate model to further evaluate whether the level of expression of the PDE 4B2 mRNA subtype is under hormonal regulation.

Placental sulfatase deficiency: Clinical and biochemical study of 16 cases

Clinical and biochemical data of 16 typical cases of placental sulfatase deficiency have been observed. In vivo loading tests with DHA-S allowed us to make a prenatal diagnosis. In vitro experiments gave confirmation, showing zero or virtually zero placental sulfatase activity towards delta 5P or DHA sulfates Aromatase activities, when tested, were normal or more often less than standard values, the latter showing themselves rather large individual variations. All pregnancies were associated with the delivery of male neonates in good health but 3. The 15 living babies have been developing normally since then. These results, together with those reported in the literature, suggest that placental sulfatase deficiency is under control of an X-linked recessive character, this being supported by the recent observation of such a disorder in two sisters simultaneously pregnant. As to the high frequency problem of cesarian section, pointed out by several authors, we cannot conclude, from our own observations, that the defect has an obvious influence on the good outcome of labor, as 10 out of the 16 women delivered vaginally near term.

The effect of vasoactive intestinal peptide (VIP) on the contractile activity of human uterine smooth muscle.

1. In the present study we examined the in vitro effect of vasoactive intestinal peptide (VIP) on spontaneous contractions in both inner and outer layers of non‐pregnant human myometrium. A dose‐dependent relaxation was observed, but with a marked difference in sensitivity to VIP between the two layers, with an IC50 value of 1 X 10−8 and 1 X 10−5 mol/L in the outer and inner layers, respectively.

DHEA-S-loading test in cases of intrauterine growth retardation: relationship between the pattern of the maternal plasma metabolites and the fetoplacental dysfunction.

Dehydroepiandrosterone sulfate loading tests (DLT) have been performed in 17 cases of clinically suspected intrauterine fetal growth retardation. At birth, 9 babies had adequate birth weights for gestational age (AGA) and 8 infants were small for gestational age (SGA). In both groups, plasma levels of dehydroepiandrosterone sulfate (DHEA-S), unconjugated DHEA, delta4-adrostenedione (delta4-A), testosterone (T), unconjugated estrone (E1) and estradiol (E2) were measured seven times from 15 min to 4 h after the intravenous injection of 50 mg DHEA-S. Several differences appear between the AGA group and the SGA group: (1) in the latter group, the rate of disappearance of the injected DHEA-S is decreased; (2) plasma levels of delta4-A and T are significantly increased and remain elevated for 2-3 h, whereas in the AGA group these values are increased only for a short period of time just after the injection. E1 and E2 patterns are similar in both groups. It has previously been demonstrated that an impaired response to DLT reflects a reduced uteroplacental blood flow. In addition, our results suggests a concomitant impairment of the aromatizing activity which reduces the placental metabolization of neutral steroids to estrogens.