Human myometrial biopsies were taken at near term labour (approximately 39 weeks) from the upper margin of the uterine lower segment incision at the time of elective caesarean section prior to the onset of labour

Human myometrial biopsies were taken at near term labour (approximately 39 weeks) from the upper margin of the uterine lower segment incision at the time of elective caesarean section prior to the onset of labour. response to cAMP. We found that MAPK activation mediated the cAMP effects on COX-2 expression and that PGE2 acts through EP-2 to activate MAPK and increase COX-2. These data provide further evidence in support of a dual role for cAMP in the regulation of myometrial function. cAMP to induce myometrial relaxation. However, therapeutically in the management of preterm labour (PTL), 2-agonists are limited both by severe, potentially life threatening side effects and tachyphylaxis, the latter mediated by 2-agonist-induced down-regulation of myometrial -adrenergic receptors [5, 6]. Consequently, other mechanisms to increase myometrial cAMP levels have been explored and a recent publication showed that using the phosphodiesterase Eprodisate Sodium type 4 inhibitor, rolipram, successfully reduced rates of PTL in a mouse model [7]. Prostaglandins (PGs) play a critical role in the onset of preterm and term labour, ripening the cervix [8] and promoting myometrial contractility [9]. These properties have been successfully exploited therapeutically on the one hand to induce labour and on the other by using Adam30 inhibitors of PG synthesis to prevent PTL [9-11]. The key enzyme in PG synthesis is COX, and at least three isoforms are present in human myometrium, of which COX-2 is the most important. COX-2 is Eprodisate Sodium highly regulated by both transcriptional and post-transcriptional mechanisms [12, 13] and can be stimulated by growth factors, cytokines and endotoxins; its expression is increased in myometrium and amnion with the onset of labour [14, 15]. We have previously reported that the inflammatory cytokine, IL-1, promotes COX-2 expression NF-B activation and that both IL-1 and mechanical stretch also act MAPK to up-regulate COX-2 expression [16]. Previous studies have shown that cAMP is able to down-regulate both NF-B and MAPK activity in a variety of tissues [17, 18] and could therefore be therapeutically useful in the prevention of PTL. However, interestingly, a recent study found that activation of the cAMP/PKA pathway by PGI2 leads to increased expression of the contraction associated proteins, connexin 43, -SMA, h-caldesmon, calponin and SM2-MHC, suggesting that in response to certain signals, cAMP might promote the process of myometrial activation prior to the onset of labour [19]. Given that cAMP has the potential to be an effective tocolytic, but may also in some circumstances promote myometrial contractility, we have carried out this study to define whether cAMP increases myometrial COX-2 expression and studied the mechanisms responsible. Eprodisate Sodium Materials and methods Tissue collection The local ethics committee approved the study and women donating tissue gave informed written consent. Human myometrial biopsies were taken at near term labour (approximately 39 weeks) from the upper margin of the uterine lower segment incision at the time of elective caesarean section prior to the onset of labour. The samples were processed for myocyte isolation and culture. Isolation and culture of human myometrial cells The myometrial tissue was carefully dissected and washed in ice-cold PBS several times. The tissue samples were digested for about 45C60 min. at 37C in a collagenase solution 0.5 mg/ml collagenase 1A (Sigma-Aldrich Co. Ltd., Poole, Dorset, UK), 0.5 mg/ml collagenase XI (Sigma-Aldrich Co. Ltd.), 1 mg/ml bovine serum albumin in DMEM (Sigma-Aldrich Co. Ltd.). Digestion was stopped by addition of DMEM supplemented with 7.5% foetal calf serum (FCS; Sigma-Aldrich Co. Ltd.). The myometrial tissues suspension was agitated to further disperse the cells. The resulting suspension was then passed through a cell strainer (70 m nylon cell strainer) and individual cells were collected by centrifugation at 3000 r.p.m. for 5 min. After washing, cells were grown in DMEM with supplementation of 7.5% FCS, 1% l-glutamine and 1% penicillinCstreptomycin at 37C and 5% CO2. The myometrial cells were Eprodisate Sodium used at either second or third passages. The culture medium was changed after 24 hrs and then every other day. Cells were exposed to different treatments as described for individual experiments. Cells were serum starved overnight prior to initiation of experiments. Materials In the following experiments, we use the different treatments and the final concentration of them was: forskolin 100 M.