The human near-term myometrial beta 3-adrenoceptor but not the beta 2-adrenoceptor is resistant to desensitisation after sustained agonist stimulation.

1. In order to compare the beta(2)- and beta(3)-adrenoceptor (beta-AR) desensitisation process in human near-term myometrium, we examined the influence of a pretreatment of myometrial strips with either a beta(2)- or a beta(3)-AR agonist (salbutamol or SR 59119A, respectively, both at 10 microm, for 5 and 15 h) on the relaxation and the cyclic adenosine monophosphate (cAMP) production induced by these agonists. 2. To assess some of the mechanisms potentially implicated in the beta-AR desensitisation process, we studied the influence of such treatment on the number of beta(2)- and beta(3)-AR binding sites, the beta(2)- and beta(3)-AR transcripts expression and the phosphodiesterase 4 (PDE4) activity. 3. Salbutamol, but not SR 59119A, concentration-response curve (CRC) was shifted by a 15 h salbutamol preincubation, with a significant difference in -log EC(20) values (6.31+/-0.13 vs 5.58+/-0.24, for control and 15 h salbutamol pretreatment, respectively, P<0.05). Neither salbutamol nor SR 59119A CRCs were modified after a 15 h preincubation with SR 59119A. 4. A 15 h exposure of myometrial strips to salbutamol significantly reduced the salbutamol-induced (0.60+/-0.26 vs 1.54+/-0.24 pmol mg(-1) protein, P<0.05), but not the SR 59119A-induced, cAMP production. No decrease in cAMP production was observed after a 15 h SR 59119A exposure. 5. A 15 h salbutamol exposure of myometrial strips significantly reduced the beta(2)- but not the beta(3)-AR binding site density, whereas no decrease in the number of beta(2)- and beta(3)-AR binding sites was observed after a 15 h SR 59119A treatment. 6. Neither PDE4 activity nor the beta(2)- and beta(3)-AR mRNA expression levels were affected by salbutamol or SR 59119A treatments. 7. Our results indicate that beta(3)-AR, but not beta(2)-AR, are resistant to the agonist-induced desensitisation. In our model, beta(2)-AR desensitisation is mediated by a decreased number of beta(2)-AR that was not explained by transcriptional regulation of the receptor.

Is up-regulation of phosphodiesterase 4 activity by PGE2 involved in the desensitization of beta-mimetics in late pregnancy human myometrium?

Elevation of cAMP content resulting from stimulation of the receptor-adenylyl cyclase complex is involved in maintaining the quiescence of the human myometrium during pregnancy. The magnitude of this elevation is critically influenced by the rate of cAMP hydrolysis by phosphodiesterase (PDE) isoenzymes. In the present study we report that in term myometrium, enhanced cAMP-specific PDE4 activity takes part in the heterologous desensitization to the beta-mimetic, salbutamol. Indeed, pretreatment with a PDE4-selective inhibitor potentiates the relaxant effect of salbutamol on myometrial strips of women at term. Furthermore, the reduced relaxant effect of salbutamol after long-term treatment of myometrial strips with PGE2, a potent myometrial effector, can be reversed by PDE4 inhibition. Using a model of cultured myometrial cells, we also demonstrated that PGE2 is able to up-regulate PDE4 activity, at least through the induction of synthesis of PDE4B and PDE4D short forms, which, in turn, dampen the cAMP accumulation provoked by the stimulation of adenylyl cyclase. Such data suggest that in late pregnancy endogenous PGE2 might up-regulate PDE4 activity and lessen the responsiveness of myometrium to beta-mimetic activation. Accordingly, coapplication of a selective PDE4 inhibitor might greatly improve the usefulness of beta-mimetic in tocolysis.

EP(4) receptors mediate prostaglandin E(2)-stimulated glycosaminoglycan synthesis in human cervical fibroblasts in culture.

The aim of this study was to determine the prostaglandin E (EP) receptors and second messengers implicated in glycosaminoglycan (GAG) synthesis by human cervical fibroblasts in culture. Human cervical fibroblasts were obtained from cervical biopsies in pre-menopausal, cycling women. Cultured cells were incubated with prostaglandin E(2) (PGE(2)) and an array of agonists and antagonists. Glycosaminoglycan synthesis was assayed after extraction by measuring the [(3)H]glucosamine and [(35)S]sulphate incorporated into GAG and cAMP production was determined by radioimmunoassay. PGE(2) significantly stimulated GAG synthesis. Neither 17-phenyl-trinor-PGE(2), the EP(1) selective agonist, nor sulprostone, an EP(3) agonist, had any effect on GAG production. Butaprost, the EP(2) selective agonist, also failed to increase GAG synthesis. AH6809, an EP(2) antagonist, had no effect on PGE(2)-stimulated GAG production. AH23848, an EP(4) antagonist, inhibited the GAG synthesis provoked by PGE(2). PGE(2) and butaprost significantly increased cAMP production. Both AH6809 and AH23848 inhibited the PGE(2)-stimulated cAMP production. H89, a cAMP-dependent protein kinase (PKA) inhibitor, did not inhibit PGE(2)-stimulated GAG synthesis and Sp-cAMPS, a selective PKA activator, failed to increase GAG production. In conclusion, both EP(4) and EP(2) receptors are present and functional in human cervical fibroblasts. Only EP(4) receptors mediate PGE(2) stimulated GAG synthesis in a PKA-independent pathway.

Pregnancy induces a modulation of the cAMP phosphodiesterase 4-conformers ratio in human myometrium: consequences for the utero-relaxant effect of PDE4-selective inhibitors.

The inhibitory impacts of RP 73401, a phosphodiesterase type 4 (PDE4) selective inhibitor of the second generation, versus rolipram, the prototypal PDE4 inhibitor, were evaluated and compared on cAMP phosphodiesterase (PDE) activity and contractility of the myometrium in nonpregnant and pregnant women. In enzymatic studies, RP 73401 and rolipram inhibited the cAMP PDE activity with significantly greater maximal efficiency in the myometrium of pregnant compared with nonpregnant women (75 versus 55%; P <.05). Although myometrial PDE4 presented a single class of interaction with RP 73401 [pD(2) (-log [IC(50)]) = -8.2], it exhibited at least two classes of interaction with rolipram (pD(2) = -8.2 and -5.6). In the myometrium of pregnant versus nonpregnant women, rolipram is significantly more efficacious in the concentration range >0.01 to 100 microM (P <.01), whereas no difference was observed for the concentration range <0.01 microM. In contractility studies, RP 73401 was equally effective in relaxing myometrial strips from both nonpregnant and pregnant women (pD(2) = -8.8). Conversely, the ability of rolipram to inhibit contractions of the myometrium in pregnant women was significantly lower (pD(2) = -7.2) compared with that in nonpregnant women (pD(2) = -8.2; P <.01). Concomitantly, in the myometrium of pregnant women, a rise in immunoreactive PDE4B2 signal was detected, whereas the PDE4D3 signal was less intense. These results demonstrate that parallel to an accumulation of PDE4B2 isoform, a modification in the ratio of PDE4 conformers HPDE4 and LPDE4 (conformer that binds rolipram with high and low affinity, respectively) occurs in the myometrium of near-term pregnant women with an increase of LPDE4 functionally implicated in the contractile process. Such modifications provide a strong rationale to propose LPDE4 as potential pharmacologic targets for the design of new tocolytic treatments.

Does high polyunsaturated free fatty acid level at the feto-maternal interface alter steroid hormone message during pregnancy?

Polyunsaturated fatty acids (PUFA) are important in pregnancy, fetal development and parturition. We measured free fatty acids (FFA), albumin and alpha-fetoprotein (AFP) in the maternal and fetal circulations of women undergoing elective Caesarean section at term. We also studied the impact of PUFAs on estrogen (ER) and progesterone receptors (PR) binding properties in vitro in the myometria of pregnant women and ex vivo in human myometrial cells in culture. FFA in intervillous blood (I) (feto-maternal interface) and maternal peripheral blood (M) were similar, while those in the umbilical vein (V) and arteries (A) were 2-4 fold lower (P<0.001). PUFA levels were low in M and 3 fold higher in I, A and V (P< 0.001); consequently C20:4 and C22:6 were most abundant in intervillous space. Albumin was uniformly distributed throughout the maternal-fetal unit, but there was a transplacental gradient in AFP. The AFP in the intervillous space had a special conformation (less immuno-reactive, more anionic), suggesting loading with PUFA. Physiological concentrations of C20:4 stimulated estradiol binding, but inhibited progestin binding. C20:4 inhibited progesterone binding by decreasing the number of binding sites, with no change in apparent affinity, in vitro in myometrial tissue and ex vivo in myometrial cells. Thus PUFA may modulate the steroid hormone message, so that the high C20:4 concentration at the maternal-fetal interface at term may help amplify the estrogen signal and inhibit the progesterone signal.

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.

Estrogen receptors (ERalpha/ERbeta) in normal and pathological growth of the human myometrium: pregnancy and leiomyoma.

The distributions of the mRNAs for estrogen receptors (ERalpha and ERbeta) and their binding properties in myometria of pregnant and nonpregnant women and in leiomyoma were studied. RT-PCR analysis indicated that the term pregnancy myometria had little ERalpha mRNA, whereas the amounts of ERbeta mRNAs in pregnant or nonpregnant myometria appeared to be similar. Both ERalpha and ERbeta mRNA were greater in certain leiomyoma than in normal nonpregnant myometria. The binding kinetics revealed that two specific binding sites (with high or low affinity) for 17beta-estradiol were present in the nonpregnant myometrium. Only the low-affinity binding sites were detectable in late-pregnancy myometria and in leiomyoma, and their capacities were increased two- to threefold (P < 0.001) in leiomyoma. The pregnancy- and leiomyoma-related changes in myometrial ER status, especially the low concentration of ERalpha mRNA and the lack of high-affinity ER in pregnant women, plus the increased ERalpha and ERbeta mRNAs and the increased low-affinity ER in some leiomyoma, suggest that the redistribution of ER subtypes is associated with the pathological and/or normal growth of the myometrium.

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.

Identification of the site in the cGMP-inhibited phosphodiesterase phosphorylated in adipocytes in response to insulin and isoproterenol.

Stimulation of rat adipocytes with insulin and isoproterenol results in serine phosphorylation and activation of the adipocyte cGMP-inhibited phosphodiesterase (cGI PDE), events believed to be important in the antilipolytic action of insulin (Degerman, E., Smith, C.J., Tornqvist, H., Vasta, V., Manganiello, V.C., and Belfrage, P. (1990) Proc. Natl. Acad. Sci. U.S.A. 87,533-537). Here we demonstrate, by two-dimensional phosphopeptide mapping, that the major phosphopeptide generated by trypsin, or trypsin followed by Asp-N protease digestion of [32P]cGI PDE phosphorylated in adipocytes in response to isoproterenol and/or insulin, in each case co-migrates with the phosphopeptide released by the same treatment of M297FRRPS(P)LPCISREQ310. This peptide was synthesized based on the deduced sequence of the cloned rat adipocyte cGI PDE and phosphorylated by cAMP-dependent protein kinase (protein kinase A). Radiosequencing of authentic and synthetic tryptic 32P-peptides showed that a single site in cGI PDE (Ser302) was phosphorylated in adipocytes incubated with isoproterenol and/or insulin. The more than additive phosphorylation and activation of cGI PDE in response to the two hormones found in this report and previously (Smith, C.J., Vasta, V., Degerman, E., Belfrage, P., and Manganiello, V.C. (1991) J. Biol. Chem. 266, 13385-13390) is proposed to reflect cross-talk between their respective signal transduction pathways at the level of the cGI PDE serine protein kinase or upstream regulatory component(s).

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.

Electrogenic malate uptake and improved growth energetics of the malolactic bacterium Leuconostoc oenos grown on glucose-malate mixtures.

Growth of the malolactic bacterium Leuconostoc oenos was improved with respect to both the specific growth rate and the biomass yield during the fermentation of glucose-malate mixtures as compared with those in media lacking malate. Such a finding indicates that the malolactic reaction contributed to the energy budget of the bacterium, suggesting that growth is energy limited in the absence of malate. An energetic yield (YATP) of 9.5 g of biomass.mol ATP-1 was found during growth on glucose with an ATP production by substrate-level phosphorylation of 1.2 mol of ATP.mol of glucose-1. During the period of mixed-substrate catabolism, an apparent YATP of 17.7 was observed, indicating a mixotrophy-associated ATP production of 2.2 mol of ATP.mol of glucose-1, or more correctly an energy gain of 0.28 mol of ATP.mol of malate-1, representing proton translocation flux from the cytoplasm to the exterior of 0.56 or 0.84 H+.mol of malate-1(depending on the H+/ATP stoichiometry). The growth-stimulating effect of malate was attributed to chemiosmotic transport mechanisms rather than proton consumption by the malolactic enzyme. Lactate efflux was by electroneutral lactate -/H+ symport having a constant stoichiometry, while malate uptake was predominantly by a malate -/H+ symport, though a low-affinity malate- uniport was also implicated. The measured electrical component (delta psi) of the proton motive force was altered, passing from -30 to -60 mV because of this translocation of dissociated organic acids when malolactic fermentation occurred.

A new peptide (1150Da) selectively activates the calcium-calmodulin sensitive isoform of cyclic nucleotide phosphodiesterase from human myometrium.

The cyclic nucleotide phosphodiesterase enzymatic system is examined in extracts of human myometrium and four individual phosphodiesterase isoforms have been isolated and characterized. A new thermostable peptide, recently purified in rat and calf myometrium, is able to stimulate up to 55-fold, the calcium-calmodulin dependent phosphodiesterase isoform. Activation of cAMP hydrolysis is by far the most marked with a 55-fold maximal stimulation at a concentration of 0.1 microM peptide and a IC50 value estimated at 30nM. For cGMP hydrolysis, the maximal effect (x25) obtained at 40nM peptide is lesser and the IC50 value is in the 10nM range. Furthermore, we verified that classical calmodulin antagonists such as calmidazolium or trifluoroperazine did not change stimulation of the calcium-calmodulin phosphodiesterase by the peptide, indicating that the myometrial peptide is different from calmodulin. To our knowledge, this is the first evidence for such a strong and selective stimulation of one isoform of the phosphodiesterase enzymatic system by a natural peptide.

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. 2. We also established that VIP did not directly stimulate the adenylate cyclase activity. The only slight stimulations were observed in non-initial rate conditions. The maximal response of this indirect effect was obtained for VIP concentrations between 1 x 10(-9) and 1 x 10(-8) mol/L and this occurred to the same extent (an approximately 1.4-fold increase) in both layers. However this response is specific, since structurally related peptides such as glucagon, gastric inhibitory polypeptide (GIP), secretin, or human growth hormone-releasing factor (hGRF) had no effect in our preparations. 3. Autoradiographic studies revealed that specific VIP binding sites were located on the vascularization of the intermediate vascular layer and on arterioles and venules distributed in the inner and outer myometrial layers. They were also present in the endometrium, but not on smooth muscle cells of either layer. 4. Such observations could provide evidence for another signal transduction pathway to mediate the biological effect of VIP. An additional intermediate step on the vascularization distributed in all of the muscle cannot be excluded.

Cisplatin-induced anemia: a potential interference with iron metabolism at erythroid progenitors level.

To elucidate the potential mechanisms of anemia induced by cisplatin (CDDP) we have evaluated hemolysis, dyserythropoiesis, ferrokinetics and cytotoxicity on erythroid progenitors in 12 patients treated by a CDDP-containing combination chemotherapy and in 6 patients treated by a similar combination but without CDDP. Eight patients, from the CDDP treated group, experienced a pronounced anemia. None had signs of hemolysis. Ferrokinetic study showed a very deep and protracted decrease of 59Fe incorporation during the chemotherapy cycle and the following 2 weeks. These results, along with a normal medullary erythroblastic cellularity, suggest that CDDP induces a deep but transient erythropoiesis alteration leading to anemia in some cases.

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.

[Multiple elemental organic components of the broncho-alveolar lavage fluid in normal subjects and in various lung pathologies (excluding silica and asbestos fibers)]. / Etude multi-élémentaire des composants inorganiques du liquide de lavage broncho-alvéolaire chez le sujet normal et dans diverses pathologies pulmonaires (silice et fibres d'amiante exclues).

Owing to a technical analysis enabling the detection of mineral elements present in trace amounts in small volumes, an analysis of the liquid obtained in 148 broncho-alveolar lavages could be studied. The elements consistently recovered were as follows: iron, copper, zinc, nickel, lead and titanium. Normal values were established. In the absence of exposure, there was no significant difference distinguishing the different pulmonary diseases studied. In occupational disease, the presence of iron in large quantities, tungsten, nickel and rare earths could be shown objectively, as well as the persistence of gold or iodine after medical absorption. However, this preliminary work does not allow one to establish a formal correlation between the alveolar concentrations obtained and the pulmonary disease observed.

A method of lead determination in human teeth by energy dispersive X-ray fluorescence (EDXRF).

A systematic sampling procedure was combined with a method of energy dispersive X-ray fluorescence (EDXRF) to study lead content and its variations in human teeth. On serial ground sections made on unembedded permanent teeth of inhabitants of Strasbourg with a special diamond rotating disk, 2 series of 500 microns large punch biopsies were made systematically in 5 directions from the tooth surface to the inner pulpal dentine with a micro-punching unit. In addition, pooled fragments of enamel and dentine were made for each tooth. On each punched fragment or pooled sample, lead content was determined after dissolution in ultrapure nitric acid, on a 4 microns thick polypropylene film, and irradiation with a Siemens EDXRF prototype with direct sample excitation by a high power X-ray tube with a molybdenum anode. Fluorescence was detected by a Si(Li) detector and calcium was used as an internal standard. This technique allowed a rapid, automatic, multielementary and non-destructive analysis of microsamples with good detection limits.

Simultaneous determination of Pt and essential trace elements in plasma by energy dispersive x-ray fluorescence (EDXRF).

Energy dispersive x-ray fluorescence allows the simultaneous determination of platinum and essential trace-elements in plasma and filtered plasma. Pharmacokinetics of platinum and the variations of trace-elements levels with respect to the normal mean values before and during the treatment are reported. Particularly during the treatment we observed that total iron concentration clearly increases in plasma, which suggests a hemolytic effect of the drug. However we can exclude this hypothesis by the simultaneous determination of potassium and rubidium levels, which do not increase as greatly as in hemolysed samples.

Characterization by DEAE-cellulose chromatography of a single molecular form of cyclic nucleotide phosphodiesterase in the myometrium of nonpregnant women.

In the longitudinal layer of nonpregnant human myometrium, cyclic nucleotide phosphodiesterase (PDE) activity from the soluble fraction is resolved by DEAE-cellulose chromatography into a single peak which presents no substrate specificity and is calcium-calmodulin-sensitive. As in the homogenate and in the soluble fraction, this peak shows two affinities for cAMP and only one for cGMP. In the soluble fraction of preterm and nearterm myometria, a similar peak is identified, but it presents a biphasic kinetic pattern towards both substrates. During pregnancy, in contrast with the nonpregnant tissue, a second peak of PDE highly selective for cAMP hydrolysis has been isolated specifically in the myometrial soluble fraction. The physiological significance of these two enzymatic forms is still unknown, particularly the role of the cAMP-specific form in the control of uterine motility during pregnancy.

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.