Functional coupling of the alpha 2-adrenergic receptor-adenylate cyclase complex in the pregnant human myometrium.

We previously reported that in the pregnant human myometrium the binding sites labeled with [3H]idazoxan have the pharmacological characteristics of alpha 2-adrenergic receptors. Competitive experiments have also revealed that the stable guanine nucleotide analog guanyl-5'-imidodiphosphate decreases the apparent affinity of norepinephrine and clonidine for myometrial [3H]idazoxan-binding sites. In the present study, the alpha 2-adrenergic mechanism in this tissue was further approached by measuring adenylate cyclase responses and examining the different pertussis toxin-sensitive G-proteins. The two alpha 2-adrenergic agonists norepinephrine and clonidine inhibited adenylate cyclase activity in both the outer and inner layers of the pregnant human myometrium. The inhibitory effect of these agonists is completely reversed by alpha 2-adrenergic antagonists such as yohimbine and idazoxan. Pretreatment with pertussis toxin completely suppresses the inhibition of adenylate cyclase mediated by alpha 2-adrenergic receptors, suggesting that an inhibitory protein of the Gi type is involved. Pertussis toxin, known to catalyze the ADP ribosylation of the alpha-subunit of several G-proteins, labels three substrates at 39, 40, and 41 kDa. The more intense labeling occurring on the 40- to 41-kDa components are assigned to alpha-subunits of Gi-like proteins, whereas that at 39 kDa might correspond to a Go alpha-like substrate. These results indicate the presence of alpha 2-adrenergic receptors in the human myometrium at the end of pregnancy that are functionally linked to inhibition of adenylate cyclase activity via the Gi protein.

Sensitivity of the human myometrial adenylate cyclase to calcium and calmodulin.

The calcium-calmodulin-dependent regulation of adenylate cyclase was studied in membranes from pregnant human myometrium. In the absence or presence of exogenous calmodulin, free calcium concentrations greater than 50 nmol/l inhibited the adenylate cyclase activity. Activation of the enzyme by calmodulin (0.1-1 mumol/l) was calcium-dependent and maximal at 10 nmol/l free calcium. The myometrial adenylate cyclase activity was stimulated by the guanyl nucleotide, Gpp(NH)p. In the presence of the guanyl nucleotide, the activatory effect of the calcium-calmodulin complex disappeared. The activatory effect of exogenous calmodulin was dependent on endogenous calmodulin present in the myometrial membranes. Trifluoroperazine and calmidazolium were able to inhibit the adenylate cyclase activity.

Beta 2-adrenoceptor response in the rat uterus at the end of gestation and after induction of labor with RU 486.

To study the relationship between the progesterone environment and beta-adrenoceptors in the myometrium, rats were treated with the antiprogestin RU 486 (10 mg per rat) at 08:30 h on day 21 of gestation. Under these conditions, more than 60% of animals delivered within 24 h after this treatment, while none of the control animals delivered within the same time period. beta-Adrenoceptors were identified using the radiolabeled antagonist (-)-[125I] iodocyanopindolol. The density (Bmax approximately 33-45 fmol/mg protein) and the affinity (KD approximately 0.105-0.106 nM) were not changed (during the late stages of gestation) in RU 486 treated rats compared with control rats. These results were correlated with the relaxation of longitudinal and circular strips of myometrium placed in high KC1 medium and exposed to beta-adrenoceptor agonists. The adrenoceptors implicated in the relaxation of myometrial strips were mainly of the beta 2-subtype. There was no difference in their affinity between control and RU 486 treated rats. Mean pA2 values were 8.46 for propranolol and 8.27 for ICI 118-551 against salbutamol. Altogether these results indicate in the rat that the blockade of the action of progesterone at its receptor site by RU 486 did not modify either the affinity or the sensitivity of beta 2-adrenoceptors in the myometrium, although it induced parturition.

Disappearance of beta-adrenergic response of human myometrial adenylate cyclase at the end of pregnancy.

The density of beta-adrenergic receptors in both the outer and inner layers of the human myometrium decreases during the last weeks of pregnancy. Although in preterm myometrium (32-35 weeks of pregnancy) beta-adrenergic receptors are positively coupled to adenylate cyclase, we found that isoproterenol, epinephrine, and norepinephrine did not stimulate the enzyme in the inner or outer myometrial layer at term (39-40 weeks of pregnancy). At this stage, the addition of 10(-4) mol/L guanyl-5'-imidodiphosphate increased (from 10(-8) to 10(-4) mol/L) basal adenylate cyclase activity in a dose-dependent manner, indicating that the catalytic component of the enzyme remains linked to the stimulatory guanyl nucleotide binding protein (Gs). Compared to the preterm response, at term the myometrial adenylate cyclase response to 10(-4) mol/L guanyl-5'-imidodiphosphate was decreased, which may reflect a decrease in the amount of functional Gs. Altogether these changes are consistent with reduced Gs coupling to the catalytic component. However, the similarity of the responses of preterm and term myometrium to forskolin excluded the possibility of an alteration of the catalytic component of adenylate cyclase during the last weeks of pregnancy. The fact that a stimulatory effect of isoproterenol on adenylate cyclase was found after islet-activating protein pretreatment indicates that human term myometrium contains a functional inhibitory guanyl nucleotide binding protein which is involved in the modulation of the beta-adrenergic adenylate cyclase response. Our data suggest that modifications in the coupling mechanisms between receptors and the catalytic component are implicated in the loss of beta-adrenergic adenylate cyclase stimulation in the myometrium at the end of pregnancy.

Correlation between selective inhibition of the cyclic nucleotide phosphodiesterases and the contractile activity in human pregnant myometrium near term.

The present study was carried out to determine the ability of various pharmacological agents to selectively inhibit each cytosolic form of phosphodiesterase isolated from the longitudinal layer of human myometria near term. Among the drugs tested, zaprinast specifically inhibits the first form of PDE which hydrolyses both substrates (cAMP and cGMP) and is stimulated by the Ca2+-calmodulin complex. A second form of PDE specific for cAMP hydrolysis and Ca2+-calmodulin insensitive is only present during pregnancy. Rolipram is the most potent and selective inhibitor of this second form. It is also the most efficient compound to inhibit in vitro the spontaneous contractions of near term myometria. The double effect of rolipram suggests an important role of the second form of PDE in the mechanisms of contractility during the pregnancy. In addition rolipram or other derivatives might be of a therapeutic interest in the prevention of prematurity in so far as they are devoid of undesirable maternal and fetal side effects.

Beta-adrenergic receptors and stimulatory effects of (-) isoproterenol on testosterone production in fetal mouse Leydig cells.

Beta-adrenergic receptors were characterized in freshly excised fetal mouse testis using the radioiodinated antagonist iodocyanopindolol (ICYP). [125I]-CYP bound to a single class of high affinity sites with a KD value of 42.2 +/- 7.0 pM. Adrenergic agonists competed for ICYP binding sites with the following order of potency: (-)isoproterenol greater than (-)epinephrine much greater than (-)norepinephrine which is typical for a beta 2-adrenergic receptor. A selective beta 2-receptor antagonist ICI 118-551 showed an approximately 200 fold higher affinity than the beta 1-selective compound, betaxolol. The beta-adrenergic agonist (-)isoproterenol did not or slightly affect testosterone production by freshly isolated fetal Leydig cells. The ability of fetal Leydig cells to respond to (-)isoproterenol increased during culture. This change in responsiveness was not accompanied either by modification of the number of binding sites or by change in the binding affinity. Taken together these data suggest that i) the stimulatory effect of (-)isoproterenol on testosterone production by cultured fetal Leydig cells is mediated through beta 2-adrenergic receptors ii), the inability of freshly Leydig cells to respond to catecholamines is probably due to post receptor events.

Evolution of tissue levels of steroids: oestradiol-17 beta, oestrone and progesterone in the macaque myometrium during gestation.

In 15 cynomolgus monkeys between days 30-160 of gestation, tissue levels of oestrone (E1), oestradiol 17 beta (E2) and progesterone (P4) were assayed by RIA in the myometrium and the placenta. Myometrial samples were subdivided as follows: inner and outer layers adjacent to the placental area (IMP and OMP) and inner and outer layers from antiplacental areas (IMAP and OMAP). Steroid levels (ng/g wet wt) were in the range of known plasma values (ng/ml) and there was no asymmetric distribution of steroids between the various locations. When the results obtained in all the layers were pooled the gestational profiles indicated a decrease of E1 between days 80-130, whereas at the same time E2 and P4 increased. The ratio P4/E2 was 8 on day 40, 17 on day 80 and 9 on day 160. In the placenta, levels of E2 and P4 were 4 times higher, levels of E1 10 times higher than in the myometrium. Gestational profiles of the three steroids in the placenta increased from day 30 to day 160. Myometrium steroid content therefore does not appear to be a simple reflection of steroid diffusion from the site of production.

Different alpha 2-adrenoceptor densities in the circular and longitudinal layers of human near-term myometrium as revealed by [3H]-idazoxan binding studies.

[3H]-idazoxan, a specific alpha 2-adrenoceptor antagonist radioligand, has been used to characterize alpha 2-adrenoceptors in both circular and longitudinal layers of human near-term myometrium. [3H]-idazoxan binding is reversible, saturable and stereospecific. It labels with high affinity a single population of sites. Competition curves with different alpha 2-adrenoceptor agonists and antagonists show that the binding sites labelled with [3H]-idazoxan have the pharmacological characteristics of alpha 2-adrenoceptors. Though the KD value is similar in both layers (approximately 7 nM), the density of alpha 2-adrenoceptors in the circular layer (324 +/- 21 fmol/mg of protein) is significantly higher than in the longitudinal one (183 +/- 48 fmol/mg of protein).

Adrenergic receptors in inner and outer layers of human myometrium near term: characterization of beta-adrenergic receptor sites by [125I]-iodocyanopindolol binding.

The beta-adrenergic receptors present in inner and outer layers of human myometrium near term were characterized using the radioiodinated antagonist iodocyanopindolol (ICYP). In both layers ICYP binding is saturable, rapidly reversible, stereoselective, and appears to occur in a single class of sites with a KD of 30 pmol/l. Adrenergic agonists compete for ICYP binding sites with an order of potency consistent with beta 2-adrenergic potencies: isoproterenol greater than epinephrine much greater than norepinephrine. Studies in which CGP 20712 A, a beta 1-adrenergic antagonist, competes for ICYP binding sites in human myometrium reveal that at least 65% of the beta-receptors present are beta 2-subtype, whatever the layer considered. At the 35th week of pregnancy, the density of beta-adrenergic receptors in the inner layer (15.2 fmol/mg of protein) is about 50% higher than in the outer layer. At term, the densities of beta-adrenergic receptors are reduced and exhibit the same values for both layers (5 fmol/mg of protein). These results indicate that the beta-adrenergic receptors in the two layers diminish during pregnancy and reach, at term, an equal and low level of density.

Cyclic AMP metabolism in the inner and outer layers of human myometrium near term.

The metabolism of cAMP which appears to be the intracellular mediator of various relaxing agents was studied in biopsies obtained during elective caesarean section from inner and outer myometrial layers outside the placental insertion. In the inner layer, L-epinephrine, PGE1, PGE2, PGF2 alpha and PGI2 stimulated the cAMP formation process while 6-keto PGF1 alpha was ineffective. The fact that some of these prostaglandins are well-known to promote contraction, confirms that the effects of drugs on uterine motility are not necessarily related to changes in the cAMP level. On the other hand, L-epinephrine and prostaglandins did not strongly influence the cAMP formation process in the outer layer. Kinetic analysis and purification assays of phosphodiesterase (PDE) which catalyzes the degradation of cAMP revealed the presence of multiple molecular forms of the enzyme in human pregnant myometrium. Qualitative and quantitative differences between the two layers appeared in the two forms separated from the soluble fraction by DEAE-cellulose chromatography. An unequal distribution of calmodulin was also observed in the inner and outer layers. Our results support the concept of the regulatory heterogeneity of the pregnant human uterus and suggest that the myometrial inner layer plays an important role in the regulation of uterine motility at the end of pregnancy.

A longitudinal study of cyclic nucleotide phosphodiesterase activity and its relationship with nucleic acid content in the myometrium of cynomolgus monkeys (Macaca fascicularis) between days 39 and 162 of gestation.

Cyclic AMP and cGMP phosphodiesterase (PDE) activities and nucleic acid contents were assayed in crude homogenates prepared from biopsies excised between days 39 and 162 of gestation (normal full length of gestation: 165 days) in outer and inner layers of the macaque myometrium. In both layers, kinetic analysis of PDE indicated high (apparent Km 2 X 10(-6) M) and low (apparent Km 2 X 10(-5) M) affinity component for each substrate. When measured in high-affinity conditions, specific activities were elevated around day 40 and beyond day 130 of gestation. By contrast, low values were observed between days 50 and 100. They were related to the decrease of the Vmax values (expressed either per milligram protein or per microgram DNA). No significant differences were observed between outer and inner layers. Variations of tissue DNA and RNA were demonstrated throughout gestation indicating that both hyperplasia and hypertrophy were involved in uterine growth. Unlike DNA, RNA content (and consequently the RNA:DNA ratio) in the inner layer always remained above the values of the outer layer. In both layers, the RNA:DNA ratio, which presumably reflects the rate of protein synthesis and the PDE activity, reached maximum values at the same time.

Relationship between myometrial cyclic nucleotide phosphodiesterase (PDE) activity and the RNA/DNA ratio at various stages of gestation in primates.

Cyclic AMP and cGMP PDE activities were assayed in crude homogenates prepared from biopsies excised between day 39-162 of gestation (normal length of gestation, 165 days) in outer and inner layers of the macaque myometrium. In both layers, kinetic analysis of PDE indicated high (app Km approximately equal to 2 X 10(-6)M) and low (app Km approximately equal to 2 X 10(-5)M) affinity component for each substrate. Measured in high affinity conditions, specific activities were increased around day 40 and beyond day 130 of gestation. By contrast, low values were observed between days 50 and 100. No significant differences were observed between outer and inner layers. In both layers, the RNA/DNA ratio, which presumably reflects the rate of protein synthesis, culminated at the same time as the PDE activity. These variations were observed in the myometrium at specific stages of gestation. In late pregnancy, the human myometrium also displayed biphasic kinetics for cAMP and cGMP PDE activities. Non-human primates may be a partially representative model of what happens in the human myometrium.

Evolution of cAMP phosphodiesterase activity in cultured myometrial cells: effects of steroids and of successive subcultures.

Cyclic AMP phosphodiesterase (PDE) activity was characterized in culture of ewe myometrial cells and its sensitivity to steroid hormones was tested. Cultured myometrial cells were maintained from the first to the 20th subculture in the presence of 2% of serum in a medium supplemented with 1 microM of insulin. It was found that myometrial cells possess a PDE activity with atypical kinetics. The nonlinear responses in Lineweaver-Burke plots suggest the presence of high- and low-affinity PDE activities. In cell culture, apparent Km values were similar to those obtained from the original myometrium. Vmax values increased with successive subcultures, revealing an increase in the capacity of the cells to degrade cAMP; in parallel, the growth rate decreased. The PDE specific activity in cultured myometrial cells was inhibited by estradiol or progesterone. When added together, no synergistic effect was obtained. The rate of inhibition for both steroids was constant during successive passages for both low- and high-affinity conditions. Results obtained in myometrial cell long-term culture were compatible with reports in other species in vivo. Considering the role of cAMP in the regulation of uterine functions, subcultured myometrial cells provided us a useful experimental system with which to study the cAMP metabolism process.

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.

Effects of prostaglandins on cyclic nucleotide phosphodiesterase activity in the human term placenta.

Slices of human full-term placentas, obtained by elective cesarean section, were incubated in the absence or presence of prostaglandins (PGs) and the cyclic AMP phosphodiesterase (cAMP PDE) activity was measured. PGE1 and PGI2 were shown to stimulate cAMP PDE activity. The effect of PGE1 is related to an increase in the Vmax of the low Km activity without alteration of this apparent Km. Several findings suggest that the cAMP PDE is activated by its own substrate; PGE1 and PGI2, promote an increase of cAMP formation which is observed before the cAMP PDE activation. Dibutyryl cAMP or theophylline also activate cAMP PDE. In contrast, PGF2 alpha does not influence either adenylate cyclase or AMP PDE. In addition, we found that the ability of the placenta to degrade cAMP, increases after parturition. PG levels are higher in the foeto-placental unit during labor, and a causal relationship between these two phenomena is possible. Our data supporting the concept of hormonal control of cAMP PDE is consistent with the hypothesis that an accelerated cAMP metabolism in placenta contributes to the maintenance of a constant equilibrium of the cyclic nucleotide levels in the foeto-placental unit.

Steroid concentrations and delta 5, 3 beta-hydroxysteroid dehydrogenase activity in human placenta. Comparison between elective cesarean section and spontaneous vaginal delivery.

The level of delta 5, 3 beta-hydroxysteroid dehydrogenase (delta 5, 3 beta-HSDH) was measured in the mitochondrial and microsomal fractions of the human term placenta. The capability of the placenta to transform pregnenolone to progesterone was found to be significantly higher after spontaneous vaginal delivery than after elective cesarean section (p < 0.001). There was a significant rise in the placental concentration of estrone occurring at the time of parturition (p < 0.01). The placental concentration of estradiol-17 beta was also greater after than before parturition but the diffference was not statistically significant. Conversely to the estrogen concentration, no difference existed in the progesterone concentration between the two groups of placentas. The significance of these findings is discussed in relation to the parturition process.

[Comparative study of the 3 beta-hydroxy-delta 5-steroid dehydrogenase activity of the mitochondrial and microsomal fractions of human and cat placentas]. / Etude comparée de l'activité delta 5, 3 beta hydroxystéroïde deshydrogénase des fractions mitochondriales et microsomales de placentas humains et de chatte.

Delta 5, 3 beta hydroxysteroid dehydrogenase activity was studied on mitochondrial and microsomal fractions of human and cat placentas. This activity was assessed by measuring the rate of [3H] progesterone formation from [3H] pregnenolone and of [3H] delta 4-androstenedione from [3H] dehydroepiandrosterone (DHA), by a double isotope dilution method. Specific activities measured under initial velocity conditions were similar in both human and cat placentas. However, the kinetics of the reaction with DHA as substrate exhibit a zoological specificity.

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.