Effect of Pregnancy on PDE4 cAMP-Specific Phosphodiesterase Messenger Ribonucleic Acid Expression in Human Myometrium
Introduction
During human pregnancy, the uterus undergoes considerable structural and functional changes characterised by relaxation along with hypertrophy and hyperplasia of the smooth muscle fibres of the uterine wall. The purpose of these changes is to permit the normal and uninterrupted development of the foetus. Although the contribution of cyclic nucleotides cAMP and cGMP to uterine growth in pregnancy has not been completely determined, increasing evidence suggests that cAMP plays an important role in myometrial relaxation. Cyclic AMP affects intracellular calcium concentration and myosin light-chain kinase activity 1, 2, 3. These actions include the following concepts: β-adrenergic agonists can stimulate adenylate cyclase, thus increasing cAMP levels and inducing relaxation. Cyclic AMP can also directly inhibit the activity of myosin light-chain kinase and promote the storage of intracellular calcium by the sarcoplasmic reticulum. Less attention has been paid to the role of cGMP in the contraction–relaxation mechanism; however, the involvement of a nitric oxide-cGMP pathway in the control of human uterine contractility during pregnancy has been demonstrated [4].
In light of the important role of cAMP and cGMP, specific regulation of the phosphodiesterase enzymatic system responsible for cyclic nucleotide inactivation is essential. Cyclic nucleotide phosphodiesterases (PDE) constitute a complex enzymatic group that catalyses the hydrolysis of cAMP and cGMP and is therefore a critical factor in the control of intracellular concentrations of these second messengers. On the basis of gene families encoding for PDE activity, the superfamily of PDEs can be divided into at least seven subtypes: Ca2+/calmodulin–dependent PDEs (PDE1), cGMP-stimulated PDEs (PDE2), cGMP-inhibited PDEs (PDE3), cAMP-specific PDEs (PDE4), cGMP-specific PDEs (PDE5), photoreceptor PDE (PDE6) and a high affinity cAMP-specific PDE (PDE7) 5, 6, 7. Multiple related genes and different mRNA splice forms create various isoforms that are differentially expressed and regulated in individual cell types. From a pharmacological perspective, this complexity implies that individual PDEs are likely to be good targets for therapeutic intervention in diseases caused or regulated by cyclic nucleotide-modulated transduction mechanisms 8, 9.
The activities of cyclic nucleotide PDEs were previously examined in extracts of human myometria. Modifications in the kinetic behaviour of the cAMP PDE enzyme were observed in the myometrium during pregnancy and compared with non-pregnant tissue 10, 11. By means of successive ion-exchange chromatography and the use of selective PDE inhibitors, we have identified the presence of five different PDE families (PDE 1 to 5) in the soluble fraction of human myometrium [12]. Among them, the cAMP-specific phosphodiesterase family (PDE4) was shown to be the most predominant. Rolipram, which is a selective inhibitor of the PDE4 family, also was a potent in vitro inhibitor of spontaneous contractions in the near-term myometrium [13].
In this report, we focus on the cAMP-specific PDE family (PDE4). This family has received particular attention recently because its selective inhibitors have shown promising effects in a variety of disease models including heart failure, depression, asthma and other inflammatory diseases [14]. To date, four distinct PDE4 genes, PDE4A, 4B, 4C and 4D, have been identified. In addition to alternative mRNA splicing, the presence of different transcription start sites indicates a high degree of complexity in this family [15]. Differential regulation of these PDE4 variants or isoforms has been described as a means by which short- and long-term modulation of cAMP levels can be attained. Using semi-quantitative RT-PCR [16], we analysed the expression patterns of mRNAs for the four cloned human PDE4 isogenes in the myometria of pregnant and non-pregnant women. Furthermore, we identified these transcripts in human myometrial smooth muscle cells in culture. The results of the distribution analysis revealed a modification in the expression pattern in pregnant myometrial tissue.
Section snippets
Tissues
Myometria were collected from seven women undergoing hysterectomies for benign gynaecological indications (leiomyomas). Four of the surgeries were scheduled at the luteal phase of the menstrual cycle, and three were scheduled at the follicular stage. Biopsies were also obtained from seven women with normal uncomplicated pregnancy who were delivered by elective caesarean section. The caesarean section was done for previously diagnosed cephalopelvic disproportion prior to onset of labour between
Results
The expression of PDE4A, 4B, 4C and 4D genes was investigated in the myometria of pregnant and non-pregnant women. After reverse transcription of total RNAs, the resulting cDNAs were amplified by PCR with the use of external oligonucleotides specific for each PDE4 sequence. Electrophoresis on agarose gel stained with ethidium bromide revealed PCR products of the correct predicted sizes; 391 bp for PDE4A, 567 bp for PDE4B2, 816 bp for PDE4C and 642 bp for PDE4D3 (Fig. 2, lane 3). RNA from a
Conclusion
To our knowledge, this study provides the first evidence for the concurrent expression of PDE4A, 4B, 4C and 4D mRNA subtypes in human pregnant and non-pregnant myometria. We found that the PDE4D mRNA subtype is prominently expressed. PDE4A and PDE4B mRNAs also are markedly abundant, whereas lower expression is observed for PDE4C mRNA. Interestingly, by using a semi-quantitative PCR approach, we showed that transcripts of PDE4B2 are more abundant in the myometria of pregnant woman than
Acknowledgements
We are very grateful to M. Pooran and S. Allman for carefully reading the manuscript. We also thank G. Delrue (SC6 Inserm), for photographic expertise, and M. Verger, for skillful typerwriting assistance.
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