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INSERM, U-361, Maternité Port Royal Hôpital Cochin, Université René Descartes (C.M., G.T., E.D., D.C., F.F., M.-J.L.), 75014 Paris, France; and Division of Reproductive Biology, Stanford University School of Medicine (C.M.), Stanford, California 94305-5317
Address all correspondence and requests for reprints to: Dr. Marie-Josèphe Leroy, INSERM, U-361, Pavillon Baudelocque, 123 boulevard Port Royal, 75014 Paris, France. E-mail: leroy-zamia{at}u361.cochin.inserm.fr
Abstract
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 ß-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 ß-mimetic activation. Accordingly, coapplication of a selective PDE4 inhibitor might greatly improve the usefulness of ß-mimetic in tocolysis.
PRETERM BIRTH CONSTITUTES the leading cause of perinatal morbidity and mortality in humans. Although its etiologies are multifactorial, preterm labor is an univocal syndrome clinically defined by alterations of the uterine cervix and painful and intense contractions (1). Tocolysis, pharmacological intervention in preterm labor, consists in reducing the intense contractions with the aim to postpone preterm birth, knowing that each additional day in utero upgrades the perinatal management, allowing glucocorticosteroid treatment to enhance fetal lung maturation and in utero transfer to a specialized care facility (2).
Worldwide, ß2-adrenergic agonists are the most commonly used tocolytic agents (3). The rationale for using these compounds is based on their ability to increase the cAMP level in the smooth muscle of the uterus or myometrium through binding to specific receptors linked to the stimulatory G protein-coupled, Gs, which activates adenylyl cyclase (4, 5). The rise in the cAMP level activates specific kinases, which, in turn, phosphorylate a large range of substrates and finally diminish the interaction between myosin and actin that is responsible for shortening of the cells (6). However, the loss of responsiveness to ß-adrenergics, which accounts for the main failures of tocolysis, occurs physiologically in late pregnancy myometrium and has been described after long-term treatment with ß-mimetics or in advanced preterm labor in response to certain proinflammatory mediators such as cytokines or PGs (7, 8, 9). The processes governing desensitization are multiple, e.g. down-regulation of the ß2-adrenoceptor and uncoupling of the receptor from Gs (5, 9). Another mechanism that may account for the reduced responsiveness of cells is the enhanced capacity to degrade cAMP (10). This mechanism, which is readily manipulated pharmacologically, surprisingly remains largely ignored in obstetrics, even though one can predict that intervention with this phenomenon could improve tocolytic therapies.
Indeed, the intracellular concentration of cAMP is critically dependent on its rate of degradation, which is catalyzed by the superfamily of cyclic nucleotide phosphodiesterases (PDEs). PDEs comprise 11 related gene families that have been classified according to their amino acid sequences, substrate specificities, and sensitivities toward endogenous regulators and pharmacological compounds. These enzymes are differentially expressed in different human tissues and are targeted to discrete locations within organs and cells (11). Among the PDE families, the PDE4 family, which includes various isoforms encoded by four genes (PDE4A–D), specifically hydrolyses cAMP (12, 13). Compelling evidence in numerous systems, in particular isolated immune and proinflammatory cells, indicates that up-regulation of PDE4 activity would reduce cell sensitivity to factors that act through activation of adenylyl cyclase (10, 14, 15, 16, 17).
We previously reported that the PDE4 family is the major PDE family expressed in human myometrium and that selective PDE4 inhibitors, rolipram or RP 73401, completely abolish the in vitro contractile activity of human myometrium (18, 19, 20). Moreover, we noticed that PDE4 participation in cAMP degradation rises in near-term pregnancy myometrium compared with nonpregnant tissue. This can be explained by an up-regulation of at least one PDE4 isoform, PDE4B2 (20). These data strongly suggest that isoforms of the PDE4 family may participate in dampening the actions of uterorelaxant agents at the end of pregnancy.
Considering this hypothesis, we conducted the present study firstly to assess the enhanced participation of PDE4 in diminishing the efficiency of ß-mimetics to inhibit the in vitro contractility of near-term myometrium compared with nonpregnant tissue. Secondly, using both a model of human myometrial cells in culture and functional contractile studies, we examined whether PGE2, a potent myometrial stimulant, could up-regulate PDE4 activity that, in turn, would reduce the activation of the cAMP pathway.
Materials and Methods
Biological samples
Myometrial samples were obtained from nonpregnant cycling women who were undergoing hysterectomy. Myometrial strips from normal muscle (myometrial longitudinal layer) were dissected free of serosa. Biopsies of the myometrium were obtained from pregnant women who presented normal uncomplicated pregnancies but were delivered by elective caesarian section before the onset of labor (38–40 wk of pregnancy), because of previously diagnosed cephalopelvic disproportion. Myometrial strips were excised from the longitudinal layer at the antiplacental site. Written informed consent was obtained from all donors. This study was approved by the comité consultatif de protection des personnes pour la recherche biomédicale (Paris-Cochin, France).
In vitro contractile studies
Segments of myometrium (8–12 x 2–3 mm) were suspended for isometric tension recordings using Bioscience UF1 tension transducers (Phymep, Paris, France), in 6-ml organ baths containing aerated (95% O2/5% CO2) Krebs buffer (11.1 mM glucose, 6.2 mM KCl, 144 mM NaCl, 2.5 mM CaCl2, 0.5 mM MgCl2, 1 mM NaH2PO4, and 30 mM NaHCO3) maintained at 35 C. An optimum resting tension of 900 mg was applied to each segment, and a spontaneous tone was allowed to develop. The myometrial strips, after equilibration for 2 h in Krebs solution with washing every 15 min, presented spontaneous contractions of regular frequency and intensity. Dose-relaxation curves were constructed with the cumulative addition of salbutamol (10-9–10-4 M, final concentration), freshly dissolved in water, twice with a 10-min interval. Addition of RP 73401 (3 x 10-9 M), a selective PDE4 inhibitor, was performed 30 min before the first injection of salbutamol. In some experiments myometrial strips were incubated for 18 h at room temperature in aerated Krebs buffer containing 10 µM PGE2 and then suspended. Only one dose-response curve was recorded for each strip. Strips showing unstable responses or not responding to KCl (80 mM) at the end of experiments were discarded. Areas under the tension curve were measured for a given time and processed using the Maclab/8e software package (ADInstruments Ltd., Hastings, UK). Results are expressed as a percentage of total relaxation obtained with 10-4 M RP 73401 as previously described (20). Dimethylsulfoxide, used for the dilution of RP 73401, was shown to have no effect on contractility over the ranges used in this study. Spontaneous contractile activity was controlled to remain constant throughout the experiment, and RP 73401 (3 x 10-9 M) alone did not abolish the contractions during the total incubation period (data not shown).
Cell culture and drug treatments
After collection, the biopsies of myometrium were placed in DMEM
supplemented with 100 IU/ml penicillin and 100 µg/ml streptomycin.
Myometrial cells were prepared by the explant method as previously
described by Cavaillé et al. (21).
Experiments were performed during the fourth to sixth subcultures of
the cells, cultured in DMEM/10% FCS. At confluence, cells were placed
in a serum-free medium for 72 h, allowing the expression of smooth
muscle markers: 
-smooth muscle actin, myosin heavy chain isoforms
(SM1 and SM2), and desmin (22).
Cells were incubated for the indicated times with vehicle (ethanol/NaCO3, 1:7 vol/vol, 1%) or PGE2 (10-8–10-5 M). The presence of vehicle was controlled so as to have no effect on PDE activity. When using actinomycin (5 µg/ml) or cycloheximide (50 µM), it was dissolved in water and added at the start of the incubation period.
cAMP PDE assays
Cells were harvested by scraping in ice-cold homogenization buffer [100 mM Tris-HCl (pH 7.4), 2 mM MgSO4, 2 mM EDTA, 10% glycerol, 1 mM ß-mercaptoethanol, and a protease inhibitor cocktail: 1 µM leupeptin, 10 µg/ml aprotinin, 25 µg/ml Pefabloc, 130 µg/ml benzamidine, and 50 µg/ml soybean trypsin inhibitor). After sonication, samples were immediately stored at -20 C until use.
cAMP PDE activity was determined using the Kincaid and Manganiello method (21) as previously described (23). Activities were measured with 1 µM [3H]cAMP as substrate. PDE4 activity was gauged as the fraction of cAMP PDE activity inhibited by 10 µM rolipram. Protein concentrations were determined by protein assay (Bio-Rad Laboratories, Inc., Richmond, CA), with BSA as a standard.
cAMP assay
Cells previously incubated with PGE2 or vehicle were exposed to ice-cold 10% trichloroacetic acid to stop the reaction (24), scraped, and stored at -20 C. At the time of the cAMP assays, the precipitated proteins were separated from the soluble extracts by centrifugation at 300 x g for 10 min at 4 C. Trichloroacetic acid was removed by four successive extractions with water-saturated ethyl ether. cAMP content was measured using a commercially available RIA kit, Biotrak (Amersham Pharmacia Biotech, Little Chalfont, UK).
RT-PCR analysis
As described previously (23), extraction of total RNA from myometrial cells using the TRIzol reagent method, RT using Moloney murine leukemia virus reverse transcriptase, and PCR reactions using Taq DNA polymerase were performed under the conditions recommended by the manufacturer (Life Technologies, Inc., Cergy Pontoise, France). The primers and the PCR conditions for each studied PDE4 were previously described in detail (23). An aliquot from the PCR mixture was resolved by electrophoresis on a 3% Nusieve agarose gel (FMC Bioproducts, Rockland, ME) and visualized by ethidium bromide staining under UV light. To verify the sizes of the PCR products, a DNA molecular mass standard ladder (123-bp DNA ladder, Life Technologies, Inc.) was concomitantly subjected to electrophoresis. Additional control of validity was achieved by Southern blot analysis and restriction mapping of the PCR product as previously described (24) (data not shown). Amplification of an endogenous marker, human ß2-microglobulin cDNA (25), was performed as an internal control.
Immunodetection of PDE4 isozymes
Samples (40 µg proteins) were prepared in 1 x Laemmli buffer (Sigma, St. Louis, MO), subjected to electrophoresis on an 8% SDS polyacrylamide gel, and blotted onto nitrocellulose membrane (Amersham Pharmacia Biotech). Western blot analysis was performed using either the polyclonal antibody K118 (1:500) that recognizes the carboxyl-terminal region of PDE4B or the monoclonal antibody 61D10E (1:10,000) that recognize the carboxyl-terminal region of PDE4D as described in detail previously (20, 23). Blots were further treated with appropriate horseradish peroxidase-linked antibodies (Amersham Pharmacia Biotech) at a dilution of 1:5,000 and with ECL detection reagents (Amersham Pharmacia Biotech).
Other sets of antibodies used for additional controls were 96G7A murine monoclonal antibodies directed against the PDE4B subtype and M3S1 murine monoclonal antibodies directed against the PDE4D subtype (23).
For densitometric analysis, blots were scanned with a StudioScan Iisi instrument (Agfa PhotoScan software, Agfa-Gevaert, Morstel, Belgium), and analysis was performed using the public domain NIH Image program (developed at NIH, http://rsb.info.nih.gov/nih-image/).
Materials
RP 73401 was a gift from Aventis (Dagenham, UK), and rolipram (racemate) was a gift from Schering AG (Burgess Hill, UK). K118 and M3S1 were donated by Dr. M. Conti (Stanford University, Stanford, CA); 61D10E and 96G7A were donated by Dr. S. Wolda (ICOS Corp., Seattle, WA). All compounds, except for Pefabloc, were purchased from Sigma. Pefabloc was obtained from Interchim (Montluçon, France). [3H]cAMP was purchased from Amersham Pharmacia Biotech.
Data analysis
Dose-response curves were analyzed with commercially available software (InPlot, GraphPad Software, Inc., San Diego, CA) and used to generate pD2 (-log [IC50]) and the maximally effective concentration (Emax). Significance of difference was assessed by one-way ANOVA, followed by t test (two-tailed for paired samples). For comparison of cAMP PDE activities and cAMP concentrations in cultured myometrial cells, the nonparametric Wilcoxon-Mann-Whitney test for paired samples was applied. The difference was considered significant when P < 0.05.
Results
Pretreatment with RP 73401 potentiated the relaxant effect of salbutamol on myometrial strips from near-term pregnant women
Application of salbutamol (ranging from
10-9–10-4 M)
to muscle strips caused partial relaxation of the spontaneous
contractile activity of myometrium obtained from late pregnant women
(Figs. 1
and 2). The inhibition was dose
dependent, reaching 79%, with a mean
potency of -6.8 ± 0.2 (Table 1). Conversely, this drug produced
a complete relaxation of strips from nonpregnant myometrium with
no apparent difference in potency (Fig. 2). After 30 min of
pretreatment with RP 73401 (3 x 10-9
M), a potent-selective PDE4 inhibitor, the salbutamol
dose-response curve was significantly shifted to the left, and the
ß-mimetic achieved total relaxation (Fig. 2 and Table 1). This result
was also seen when using a low dose of rolipram, another PDE4 inhibitor
(data not shown). In contrast, RP 73401 pretreatment did not potentiate
the effect of salbutamol on myometrial strips from nonpregnant woman
(Fig. 2 and Table 1), indicating a nondesensitized state to salbutamol
for the nonpregnant tissue.
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PGE2, a potent uterotonic agonist efficient
either at term or in preterm labor, has been shown to induce
heterologous desensitization of the ß-adrenergic receptor system
(8). To reproduce an in vitro functional model
of heterologous desensitization, we incubated nonpregnant strips in the
presence of 10 µM
PGE2 for 18 h. Figure 3 shows the effect of this
long-term PGE2 treatment on the responses to
salbutamol of nonpregnant myometrial strips. Incubation of tissues with
PGE2 for 18 h caused a significant reduction
of myometrial relaxation induced by salbutamol in terms of both potency
and efficiency (Table 2), indicating
impairment of the ß-adrenergic signaling pathway. Preincubation
of the tissues with RP 73401 (3 x 10-9
M) restored the salbutamol dose-response curve,
although it had no effect on the relaxation of vehicle-treated strips
induced by the ß-mimetic (Fig. 3 and Table 2), suggesting an
up-regulation of PDE4 activity in the
PGE2-treated group.
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To determine whether elevated levels of PGE2
might up-regulate PDE4 activity, cultured myometrial cells were treated
from 2–18 h with 10 µM PGE2. As
shown in Table 3, the treatment led to a
significant increase in PDE4 activity at 2 h, which remained
constant at 5 h, and a significantly different value from control
cells was still seen at 18 h. Examination of the PDE4 activity in
cells incubated for 5 h with a range of 0.001–10 µM
PGE2 revealed the dose-dependency of the rise in
PDE4 activity, reaching a maximum with 10 µM
PGE2 (Fig. 4). The
increase in PDE4 activity induced by PGE2 was
sensitive to both actinomycin D, an inhibitor of mRNA synthesis, and
cycloheximide, an inhibitor of protein synthesis (Table 3), although
only cycloheximide completely blocked the up-regulation of PDE4
activity.
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To determine which discrete PDE4 gene products are manipulated by
PGE2 within a 5-h incubation, we examined by
RT-PCR the steady state level of the mRNAs of the four PDE4 subtypes,
PDE4A, PDE4B, PDE4C, and PDE4D, in treated and untreated cells (Fig. 5). Initially, we employed sets of
generic primers for amplifying fragments present in a unique region of
each PDE4 subtype but common to all known products of a particular
PDE4 gene. As illustrated in Fig. 5A, in RT-PCR preparations from
PGE2-treated cells compared with matched control
cells, no difference in signal intensity was seen for the PDE4A and
PDE4C products. A slight elevation of the band intensity for PDE4D
products and a noticeable increase in the PDE4B PCR signal were
observed. We verified successful normalization of RNA inputs by
obtaining equivalent intensity for the
ß2-microglobulin signal in control
vs. treated cells.
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).
Western blot analyses were performed to confirm at the protein level
the up-regulation of PDE4B and PDE4D gene products observed in
PGE2-treated cells. As illustrated in Fig. 6, four bands were labeled with the
PDE4B-specific antibodies in extracts from untreated myometrial cells.
The two upper bands migrated with apparent molecular masses of 105 and
98 kDa, respectively, and might correspond to the PDE4B long forms,
PDE4B3 and PDE4B1, as previously described (23). The two
lower bands migrated as a 76- and a 71-kDa protein, respectively. The
76-kDa signal was also detected in untreated cells when using the
monoclonal antibody, 96G7A, raised against a different PDE4B-specific
epitope (23), and has the same migration in SDS-PAGE as
the short-form PDE4B2 detected in human monocytes (15).
The 71-kDa signal remained unidentified and might correspond to an
unspecific signal, in that it was not detected when using the other
antibody set (23). Upon treatment with 10 µM
PGE2 for 5 h, the intensity of the 76-kDa
signal was stronger, whereas no difference in intensity was seen for
the other bands.
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). The apparent 105- and 93-kDa proteins have the same
migration behavior on SDS-PAGE that the long forms, PDE4D5 and PDE4D3,
respectively, detected in HeLa cells (26). In
PGE2-treated cells, two additional signals were
detected at 72 and 67 kDa, identical in size to those of PDE4D1 and
PDE4D2, respectively (23, 26), whereas the signal
intensities of the long-form PDE4D5 remained unchanged, and that of
PDE4D3 decreased. PGE2 increased the cAMP concentration in myometrial cells
Knowing that long-term regulation of PDE4 activity by its own
substrate can occur in human myometrial cells, we measured the cellular
cAMP content in untreated and PGE2-treated cells
to determine whether PGE2 is able to increase
cAMP content within our experimental conditions. As shown in Fig. 7, PGE2 produced a
5-fold increase in cAMP content within 1 h of incubation.
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The present study provides evidence that the PDE4 family participates in the heterologous desensitization to activators of the relaxant cAMP-signaling pathway, which takes place in human late pregnancy myometrium. We demonstrated that a PDE4 inhibitor can act synergistically with a ß-adrenergic receptor agonist to relax spontaneous contractions of pregnant myometrial strips, whereas it did not affect the capacity of salbutamol to relax nonpregnant strips. Moreover, incubation of nonpregnant strips with PGE2, a potent myometrial effector, whose level dramatically increases during preterm and term labor, led to a significant reduction of the ability of a ß-mimetic to provoke in vitro relaxation, which was reversed by the addition of a PDE4 inhibitor. Concomitantly, using the model of human myometrial cells in culture, we assessed that PGE2 is able to augment PDE4 activity in human myometrial cells. This up-regulation appeared to account for the induction of discrete PDE4 isoforms, PDE4B2, PDE4D1, and PDE4D2. Collectively, these data suggest that the induction of PDE4 activity is one of the mechanisms by which labor effectors lessen the cAMP pathway activation involved in the maintenance of myometrial quiescence.
In human myometrium, we previously reported an increase in PDE4 activity in late pregnancy, compared with that in nonpregnancy tissue (20), through at least an accumulation of PDE4B2 mRNA and protein (20, 24). Furthermore, we demonstrated that PDE4 isozymes are involved in the myometrial contractile process (19, 20). Recently, pretreatment with rolipram has been shown to potentiate the in vitro relaxant response to salbutamol of near-term myometrial strips (27). However, this potentiation might be due to an additive effect of both compounds: on the one hand a pharmacological increase in cAMP synthesis, and, on the other hand, a decrease in the cyclic nucleotide degradation. In the present study we demonstrated that an up-regulated PDE4 activity is functionally implicated in limiting the salbutamol relaxant effect in near-term myometrium. Indeed, pretreatment of nonpregnant strips with a low dose of RP 73401 did not improve the relaxant index of salbutamol, although it caused a significant shift to the left of the salbutamol dose-response curve of pregnant myometrial strips. In addition, pretreatment of nonpregnant strips with PGE2 shifted the ß-mimetic dose-response curve to the right, which can be reversed by a low dose of RP 73401. This shift did not appear to reflect a different effect of RP 73401 alone, as we have already shown that the relaxant effect of RP 73401 was similar in strips from pregnant and nonpregnant myometrium (20). Actually, the low dose of RP 73401 used for the pretreatment of the strips appears to differently affect the cAMP accumulation induced by the ß-mimetic in both tissues. These data suggested that in late pregnancy, up-regulated PDE4 activity is implicated in regulating the pool of cAMP driven by ß-adrenoceptor agonists and that a potent labor effector such as PGE2 can induce this up-regulation.
To further examine the biological relevance of such phenomenon, we determined whether elevated concentrations of PGE2 could up-regulate PDE4 activity in cultured myometrial cells. We previously demonstrated that, as in the whole myometrium, PDE4 activity contributed to the predominant cAMP-hydrolyzing activities in these cells (23). When challenged long term with PGE2, the cells exhibited increased PDE4 activity. This augmentation was sensitive to treatments with actinomycin and cycloheximide, suggesting that PGE2 affected PDE4 activity at least through induction of PDE4 protein synthesis. Furthermore, an additional posttranscriptional mechanism may exist, as a coincubation with actinomycin D partially prevented the rise of PDE4 activity. The up-regulation of PDE4 activity was confirmed by examination of the PDE4 mRNA steady state level and protein content. In PGE2-treated cells, we observed an accumulation of the mRNAs and proteins of discrete PDE4 short forms, namely, PDE4B2, PDE4D1, and PDE4D2. Two general types of PDE4 activity regulation are now well described (12). The first mechanism, designated short-term regulation, involves phosphorylation of a preexisting protein and modulation of enzymatic kinetic constants. The second general mechanism, called long-term regulation, requires protein synthesis. In the present study we focused on the long-term regulation, which may alter cell responsiveness throughout pregnancy. Numerous investigations, pioneered by the Conti laboratory, have established that elevation of the cAMP content resulted in increased expression of selected PDE4 subtypes, and within a single PDE4 subtype of only certain splice variants. Actually, the four PDE4 genes present similar organizations of the transcription units and can differentially generate long- and short-form variants (28, 29, 30). Within the PDE4D subtype, for example, a prolonged elevation of the cAMP concentration increases the expression of the short forms, but not the long forms (12). This possibly occurs with activation of an intronic promoter that contains cAMP- and hormone-responsive elements (31). In human myometrial cells we already demonstrated that cAMP-elevating agents manipulate PDE4 activity through selective induction of PDE4 and PDE4D short forms (23). Four subtypes of PGE2 receptors have been identified, namely EP1, EP2, EP3, and EP4 (33). Among them, EP2 and EP4 have the capability to trigger elevation of intracellular cAMP. Knowing that the presence of the four EP subtypes has been assessed in human myometrial cells (34), that PGE2 is able to augment cAMP levels in myometrial cells, and that either PGE2 or cAMP-elevating agents induced the same PDE4 short-form variants in these cells, we reasonably hypothesized that PGE2 elevated PDE4 activity through activation of the cAMP pathway. However, examination of the transducing mechanisms by which PGE2 induced PDE4 activity was beyond the scope of this study.
Alterations in the amount of PDE4 protein can affect their
responsiveness to endogenous and exogenous stimuli. During late
pregnancy, a heterologous desensitization to relaxant elevators of cAMP
content takes place. This phenomenon, which includes, for example,
modification of adenylyl cyclase activity or conversion from a
stimulatory (protein Gs-mediated) coupling to
an inhibitory (Gi-mediated) system
(35), can be the result of the chronic activation of a
specific pathway and is particularly damaging in the case of
pharmacological treatment. In this study we described an additional
mechanism of desensitization in the human myometrium through the
selective induction of PDE4 activity. In this particular case, we
evaluated the potential of PGE2 to induce
heterologous desensitization of the adenylate cyclase-stimulating
pathway. In addition, in human myometrium, Berg and co-workers
(36) previously reported that PDE activity was induced
in vivo after a prolonged tocolytic treatment with
terbutaline, a ß-mimetic compound. Therefore, chronic administration
of ß-mimetic may be directly responsible for the elevation of PDE4
activity, and subsequently, PDE4 inhibition will prevent or delay the
development of tolerance to such drugs (10). The
identification of this additional desensitization mechanism during
human late pregnancy is promising for the improvement of present
tocolytic therapies.
To conclude, our results demonstrated that up-regulated PDE4 activity is involved in the refractoriness to ß-adrenergic agonists of human near-term myometrium and may lessen their tocolytic effects. Additionally, we showed that PGE2 elevation, which occurs during late pregnancy and labor (8), induces selective PDE4 subtypes that might influence rates of cAMP hydrolysis in myometrial tissue. In light of these results we propose that a coapplication of ß-mimetics and the new generation of PDE4 inhibitors might greatly improve the tocolytic efficiency of these drugs to prevent preterm delivery.
Acknowledgments
We thank Dr. J. Hough (Aventis) for kindly providing RP 73401. We acknowledge Dr. S. Wolda (ICOS Corp.) and Dr. M. Conti (Stanford University) for kindly providing the antibodies used in these studies. We are indebted to the medical staffs of Maternity Port Royal and Department of Gynecology, Cochin-Port Royal, for assistance in obtaining uterine tissues. We are grateful to Dr. T. Schmitz for clinical expertise, and to C. Spencer for editorial work on the manuscript.
Footnotes
Abbreviations: Emax, Maximally effective concentration; PDE, phosphodiesterase.
Received October 2, 2001.
Accepted July 25, 2001.
References
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)
or after a 30-min pretreatment with RP 73401 (3 x
10-9 M; •) were added to muscle strips from
either near-term pregnant women (A) or nonpregnant women (B). Results
are expressed as a percentage of complete relaxation obtained with RP
73401 (10-4 M). Strips from the same patients
were subjected in parallel to treatments. Data are the mean ±
SEM for strips isolated from myometrium of five different
pregnant women and three different nonpregnant women. Significance of
differences from salbutamol alone values: *, P <
0.05; **, P < 0.01.
) or 10 µM PGE2 (
) for 1 h. The
cAMP content was determined as described in Materials and
Methods. Data are given as the mean ± SEM of
five separate experiments using cell preparations from different
explants.