Mol. Hum. Reprod. Advance Access originally published online on February 18, 2005
Molecular Human Reproduction 2005 11(4):279-287; doi:10.1093/molehr/gah158
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Identification and localization of prostaglandin E2 receptors in upper and lower segment human myometrium during pregnancy
1Biomedical Research Institute, Department of Biological Sciences, University of Warwick and Coventry CV4 7AL, 2Walsgrave Hospital, Clifford Bridge Road, Coventry CV2 2DX, UK
3 To whom Correspondence should be addressed at: Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK. E-mail: s.astle{at}warwick.ac.uk, E-mail: d.m.slater{at}warwick.ac.uk
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Abstract |
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Prostaglandin E2 (PGE2) plays a key role in the maintenance of human pregnancy and labour onset. PGE2 can elicit diverse actions within the uterus depending on the PGE2 receptors (EP1, EP2, EP3 and EP4) expressed. By signalling through different intracellular pathways the EP receptors may inhibit or promote smooth muscle contractility. Nine different EP3 receptor splice variants have been identified with divergent signalling pathways. RT–PCR and immunohistochemistry were utilized to identify and localize EP receptor isoforms within the upper segment (US) and lower segment (LS) myometrium. EP1 was significantly increased in the LS myometrium with term labour. EP3 (and EP3 splice variants EP3I(1b), EP3II, EP3III and EP3IV) was down-regulated in pregnancy (US and/or LS) with a further decrease at term labour in the LS. Overall, expression of EP2 was significantly higher in the LS while EP3 was significantly higher in the US. No significant EP4 changes were observed. Consistent with the RT–PCR results, immunohistochemistry confirmed the presence and, interestingly, showed nuclear localization of EP receptors in the myometrium with higher EP1 expression and lower expression of EP3. The differential regulation of EP receptors within the myometrium indicates that they may play a role in controlling the onset and maintenance of human labour.
Key words: labour/myometrium/nuclear/prostaglandin E2 receptors
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Introduction |
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During pregnancy and labour the uterus undergoes profound physiological and biochemical changes, which require functional differentiation of the different regions of the pregnant uterus. The upper segment (US) region of the uterus must expand to accommodate the growing fetus and then at labour contract to cause expulsion of the fetus, while the lower segment (LS) must relax to allow passage of the fetus.
The molecular changes underlying the complex transition from uterine quiescence to labour are not fully understood, however, the prostaglandins are one of the key factors that have long been implicated in the parturition process. Inhibitors of prostaglandin synthesis have been utilized to lengthen gestation and delay human labour onset (Lewis and Schulman, 1973
). The onset of labour is associated with increased prostaglandin synthesis within the uterus. Prostaglandin E2 (PGE2), produced in large quantities by the fetal membranes and decidua, is believed to play a key role in the onset and maintenance of labour in humans, mediating cervical ripening and myometrial contractions (Skinner and Challis, 1985; Gibb, 1998). Clinically, a PGE2 analogue is widely used for the induction of labour although the individual response is variable. This may be due to the diversity in PGE2 receptor signalling pathways.
Prostaglandin E2 exhibits a particularly wide spectrum of physiological actions depending on which of the PGE2 receptors (EP) are present. Four EP receptor subtypes EP1, EP2, EP3 and EP4 have been identified pharmacologically that are encoded by separate genes (Coleman et al., 1994; Negishi et al., 1995). The EP receptors act through different intracellular pathways, for example EP1 and EP3 are coupled to calcium influx and inhibition of adenylate cyclase, respectively (Funk et al., 1993; Kotani et al., 1995), whereas, EP2 and EP4 both stimulate adenylate cyclase (Bastien et al., 1994; Regan et al., 1994). Consequently in the uterus, activation of EP1 and EP3 are reported to cause smooth muscle contraction, while stimulation of EP2 and EP4 are more likely to lead to relaxation. To add to the complexity at least eight different EP3 receptor isoforms have been identified, encoded by nine transcripts (EP3-I(1a), EP3-I(1b), EP3-II, EP3-III, EP3-IV, EP3-V, EP3-VI, EP3-e and EP3-f), generated by alternative splicing at the carboxyl-terminal tail (Schmid et al., 1995; Kotani et al., 1997; 2000). As the carboxy-terminal end of the receptor is important for mediating G protein coupling, changes in this region appear to alter the coupling affinity. EP3 is mainly coupled to Gi and hence tends towards a contractile phenotype, however, splice variants (EP3-II and EP3-IV) may couple Gs and/or Gq leading to different cellular responses (Namba et al., 1993; Kotani et al., 1995).
Our hypothesis is that differential expression of the EP receptors may be important for regulating uterine activity in the US and LS both throughout pregnancy and during labour. Various studies have previously investigated the expression of the EP receptors in a number of animal/primate models (Brodt-Eppley and Myatt, 1998; Ma et al., 1999). These include two studies in the baboon, that have compared expression in the upper and lower regions of the uterus (Smith et al., 1998, 2001), that found expression of EP1 and EP3 significantly higher in US, whereas EP2 was significantly lower in this region. However in humans, studies have so far been limited to LS biopsies (Matsumoto et al., 1997; Brodt-Eppley and Myatt, 1999; Leonhardt et al., 2003; Wing et al., 2003). Thus, to date there is a lack of information regarding expression of all the EP receptor subtypes (including EP3 isoforms) within the US and LS of the myometrium in human pregnancy and labour (term and preterm).
The purpose of this study was to determine whether there were any changes in the expression of EP receptors in pregnant human myometrium, in association with labour at term or preterm. We therefore examined the expression of all four EP receptor subtypes, EP1, EP2, EP3 and EP4 including EP3 splice variants, by looking at the receptor mRNA levels in the US and LS. In addition, immunohistological localization was performed to determine any difference in the cellular localization of EP receptors between US and LS myometrial samples.
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Materials and methods |
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Patient samples
Paired upper (fundal) and lower uterine segment myometrial tissue was collected from non-pregnant and pregnant women with informed written consent and institutional ethics committee approval (LREC 02(C)07/98 Walsgrave Hospital Trust). Non-pregnant myometrium (n=3) was obtained from pre-menopausal women undergoing hysterectomy for dysmenorrhea.
Pregnant myometrium was collected from women undergoing Caesarean section for fetal distress, breech presentation, previous section, placental praevia, maternal request or failure to progress. LS uterine samples were collected from the upper margin of the LS uterine incision while US uterine samples were taken using ovarian biopsy forceps introduced into the uterine cavity through the incision. Biopsies were collected from women preterm prior to the onset of labour (PTNL, 28–36 weeks, n=9), preterm following the onset of labour (PTL, 27–35 weeks n=4), term not in labour (TNL, 37–41 weeks n=11) and term in labour (TL, 39–42 weeks, n=8). Labour was defined as regular contractions (<3 min apart) plus membrane rupture and cervical dilation (>2 cm) with no augmentation (oxytocin or prostaglandin administration). For RNA extraction, tissues were rinsed in phosphate buffered saline, decidual tissue was removed from the myometrium, snap-frozen in liquid nitrogen and stored at –80 °C until use. For immunohistochemistry, biopsies were fixed in 4% paraformaldehyde overnight, washed in saline (adherent decidual tissue was removed unless otherwise stated) and then in 70% ethanol until processing, and paraffin embedding.
RT–PCR
Total RNA was isolated using the SV Total RNA Isolation system (Promega, Southampton, UK) as recommended by the manufacturer. RT–PCR was used for semi-quantitative analysis of RNA expression. RT of the RNA (total 100 ng) was carried out using random hexanucleotide primers (0.25 µg), which were denatured at 65 °C for 5 min followed by RT with Superscript II (Invitrogen Life technologies, Paisley, UK) for 60 min at 37 °C. The resultant complementary DNA used as template for PCR, using gene specific primers for the EP receptors or control ‘housekeeping’ genes. The PCR primers (5'–3') and product sizes are described in Table I. In addition to the EP3-generic primers which amplified a region of the receptor common to all splice variants, the generic 5'(sense) primer was used in combination with splice variant specific 3'(antisense) primer which was designed to a unique region of the c-terminal tail of each splice variant allowing individual EP3 splice variants to be identified. It was possible to amplify EP3-1b and EP3-IV using the same 5' primer due to a size difference in the transcripts caused by the deletion of an exon in EP3-1V. The cycling parameters were; denaturing, 94 °C, 30 s; annealing 30 s at primer specific annealing temperature; extension, 72 °C, 30 s for an appropriate number of cycles followed by a 72 °C, 5 min extension.
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For each gene analysed (EP receptors and ß-actin) a ‘cycle profile’ was performed to determine the linear range of amplification where product formation is related to starting template. To do this an aliquot from each myometrial (upper or lower) cDNA sample was taken and pooled and these ‘pooled’ samples were used as template for PCR cycle profiles. The cycles were carried out from 24 to 42, at two cycle intervals. Following amplification 10µl aliquots of the reactions were separated on a 1% agarose gel stained with ethidium bromide and visualized under ultraviolet light. Abundance of the amplified product was performed by densitometric analysis of the gel using Total Lab software (Newcastle upon Tyne, UK). Densitometric units were plotted against cycle number to establish the exponential phase of amplification. The appropriate cycle number within the linear range of amplification was chosen for subsequent analysis of each gene. Once the optimal cycle number for each gene had been determined all the individual upper and lower cDNA samples were amplified using the same PCR master mix, separated on the same agarose gel and analysed densitometrically using Total Lab software. No RT–PCR products were observed in the RT negative (indicating no genomic contamination of the RNA) or blank PCR controls. All RT–PCR products were of the expected size and all were sequence verified (and correlated 100% with the published sequences). Expression levels of EP receptor genes in the individual samples were normalized relative to the expression of the housekeeping gene, ß-actin. The expression level of ß-actin was quantified and showed no significant differences among patient groups, US or LS, when statistically analysed. Each RT–PCR reaction for each gene investigated was performed at least two times.
Statistics
Data are presented as mean±SEM, throughout. Comparison of two means was made using unpaired, two tailed t-test. Comparison of more than two means was made using analysis of variance (ANOVA) followed by Tukey's multiple comparison test using PRISM statistics software (GraphPad software Inc., San Diego, CA). A value of P
0.05 was considered significant.
Immunohistochemistry
Slices (5 µm) of wax embedded myometrial tissue were deparaffinized and hydrated in xylene and graded alcohol series, respectively. Antigen retrieval was performed using 1% antigen unmasking solution (Vector laboratories, Burlingame, USA) incubated at 96 °C for 60 min. To localize the EP receptors the Vectastain Elite ABC detection kit was used, following the manufacturer's protocol. Briefly, endogenous peroxidase activity was quenched by incubation in 3% hydrogen peroxide for 15 min, the slides were then blocked in 1% goat serum in phosphate-buffered saline at room temperature for 60 min and incubated overnight with the desired rabbit polyclonal primary antibody at 4 °C. Primary antibodies (EP1, EP2, EP3 and EP4, Cayman chemicals, Michigan, USA) were diluted 1:75 in 1% goat serum/phosphate-buffered saline or as a control incubated with the respective blocking peptides as recommended by the manufacturer. Incubation of the sections with pre-absorbed antibody or omitting of the primary antibody gave no staining signals. The colour reaction was developed by the use of a biotinylated secondary antibody (30 min, room temperature), addition of avidin/biotinylated complex (30 min, room temperature) followed by incubation with 3,3'-diaminobenzidetetrahydrochloride solution with metal enhancer (Sigma, Poole, UK). Sections were then counterstained using Harris haematoxylin (Sigma), dehydrated in an increasing ethanol series, cleared in xylene and mounted in DPX mounting media (Sigma).
Immunostaining was scored by three independent observers who where blinded to the status (i.e. US or LS myometrium, non-labouring or labouring, gestation) of the tissue and the receptor staining performed on the tissue. The staining intensity, taking into account both the strength and proportion of positively stained cells, for each region of tissue was assessed by each observer and the average calculated. The staining intensity was graded according to the scale;++++=very strong staining;+++=strong staining;++=moderate staining;+=weak staining, (+) diffuse staining.
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Results |
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Expression of EP receptors in the upper and lower myometrial segments
Expression of EP receptors was analysed in human LS and US myometrium obtained from non-pregnant and pregnant women. All of the generic EP receptor mRNA, (EP1, EP2, EP3 and EP4) and four of the EP3 splice variant mRNA (EP3-II, EP3-III, EP3-1b and EP3-IV), were detected by RT–PCR, in US and LS myometrium of both non-pregnant and pregnant women (Figure 1).
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To give an overall expression profile in the US and LS the expression values from all the pregnant patients were combined. When the overall expression level of each receptor was compared in the pregnant upper and lower myometrium samples, EP2 was found to be significantly higher in the LS of the pregnant samples (Figure 2b) and this trend was observed in the PTNL, PTL and TNL groups (Figure 3c,d). In contrast, EP3 was higher in the US in the combined pregnant samples (Figure 2c) but this only reached significance in the TNL group during pregnancy (Figure 3e,f). A similar expression profile for EP2 and EP3 was also observed in the NP samples albeit a smaller sample group (Figure 3). EP1 and EP4 expression was not significantly different between the two regions.
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Significantly (P<0.05) different in the US versus LS as compared using unpaired t-test.
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Pregnancy and labour associated changes in the expression of EP1 and EP3
Within US myometrium, expression of EP3 (generic) was significantly down-regulated in pregnant samples compared to that of the non-pregnant samples (NP versus all other groups, P<0.05, Figure 3e).
The expression of individual EP3 splice variants followed a similar pattern to generic EP3 expression, with three (EP3-III, EP3-1b and EP3-IV) of the four identified splices variants being significantly down-regulated in pregnant samples compared to non-pregnant samples (NP versus all other groups, respectively, Figure 4) in the US. No significant EP1 expression changes were observed in the US.
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Within the LS, however, expression of EP1 was significantly increased at term in association with labour (Figure 3b). The expression of EP3 (generic) followed a similar profile to that observed in the US with expression significantly down-regulated in pregnancy (NP versus PTL, NP versus TL, P<0.05), with a further significant decrease in labouring term samples (TNL versus TL, P<0.05, see Figure 3f).
For the EP3 splice variants, EP3-II was significantly higher in the non-pregnant samples while EP3-III, EP3-1b and EP3-IV were significantly down-regulated in TL compared to TNL in the LS (Figure 4).
Pregnancy and labour associated changes in the expression of EP2 and EP4
Expression of EP2 within US was significantly increased in the term labour group (TL versus PTNL, TL versus PTL) with the lowest expression observed in the preterm labour group (Figure 3c).
Within the LS there were no significant changes in the expression of EP2. No marked changes in EP4 were observed in pregnancy or labour onset, in the US or LS.
Receptor localization
Immunohistological analysis demonstrated a distinct localization pattern in the staining for the generic EP receptors (Table II summary). All four EP receptor proteins (EP1, EP2, EP3 and EP4) were identified in both US and LS myometrium. Localization of EP1 showed that the receptor was highly expressed in myometrial smooth muscle cells of both US and LS samples (Figure 5b,d, respectively) and abundant in the vascular endothelial cells, vascular smooth muscle (Figure 5h) and glandular epithelial cells (data not shown).
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EP2 (Figure 5e,i and l) and EP4 (Figure 5f,nFigure 5f,n) receptor protein expression was present in myometrial smooth muscle and glandular cells, with less intense staining being observed in vascular smooth muscle and endothelial cells.
Localization of EP3 receptor protein was most abundant within vascular smooth muscle and endothelial cells (Figure 5j), with little expression observed in myometrial smooth muscle cells (Figure 5m). In addition to the distinct pattern of EP receptor localization within the myometrium, it was also observed that EP1, EP2, EP3 and EP4 were abundantly expressed within the decidual component. This pattern of receptor (EP2, EP3 and EP4) expression is clearly shown in Figure 5l,m and n where LS samples were taken with adherent decidua to highlight the different intensity of staining in the decidua and myometrial smooth muscle cells.
For all the receptors the level of overall staining and receptor localization appeared uniform in the different sample groups with no obvious changes observed with gestational age or labour. However, expression of EP receptors in myometrial smooth muscle cells (EP1, EP2 and EP4) tended to be predominantly localized to the nucleus especially of LS samples, while in the US staining appeared to be in the nuclear and peri-nuclear region.
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Discussion |
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There is an ever-expanding amount of data implicating the role of prostaglandins in the initiation and maintenance of labour (term and preterm). However, there is a paucity of information regarding the expression of the prostaglandin receptors themselves within the upper and lower regions of the human uterus, and the role they play in functional regionalization of the uterus during pregnancy and labour. We have identified a distinct pattern of EP receptor localization and a number of interesting pregnancy and labour associated changes within the human myometrium.
In the present study the expression of the functionally diverse family of prostaglandin E2 receptors was determined and compared in both US and LS myometrium, with the aim of determining which receptor(s) are important for controlling/regulating uterine activity during pregnancy and how this may alter at labour. We have identified that EP receptors (EP1, EP2, EP3 and EP4) are present within both the US and LS regions of the uterus. This is in keeping with data reported in the human LS, (Matsumoto et al., 1997; Brodt-Eppley and Myatt, 1999; Leonhardt et al., 2003; Wing et al., 2003) rat (Brodt-Eppley and Myatt, 1998), sheep (Ma et al., 1999) and US and LS data reported in the baboon uterus (Smith et al., 1998; 2001). However, this is the first time EP receptor (generic EP1, EP2, EP3 and EP4 and EP3 splice variants) expression and localization has been extensively investigated in paired upper and lower uterine samples in human pregnancy and labour. These data suggest that the effects elicited by prostaglandin E2 are not likely to be mediated by a single specific receptor subtype but rather by a combination (or balance) of all four of the major subtypes (EP1–4).
There was no significant pregnancy, labour associated or spatial (upper versus lower) changes observed in the expression of EP4. This is similar to the findings in baboon (Smith et al., 2001), and suggests that EP4 has little role in the regulation of PGE2 mediated signalling changes in association with labour.
However, significant changes were observed in the expression of the other reported relaxatory EP receptor, EP2. Overall, EP2 mRNA expression, was higher in the LS compared to the US in the pregnant samples. These results are again consistent with the observations in the baboon that showed highest EP2 expression in the lower uterine segment (Smith et al., 1998). Previous studies have indicated that EP2 expression declines with increasing gestational age in the LS samples collected from women not in labour (Brodt-Eppley and Myatt, 1999; Leonhardt et al., 2003). However no gestational age related changes were observed in the baboon (Smith et al., 2001) and no such correlation was observed with our samples. Functionally, there is also more evidence to suggest that the EP2 is the predominant relaxatory EP subtype present in the lower myometrium as shown by in vitro characterization of the prostanoid receptors in the pregnant term myometrium using a combination of EP agonists and antagonists (Senior et al., 1993). These findings suggest that EP2 may play a role in relaxation of the lower uterine segment necessary to allow delivery of the fetus.
Within the US, EP2 was higher in term labour samples and lower in preterm labour samples compared to term non-labour. This expression profile is difficult to explain, as it does not correspond with the current dogma of a contractile phenotype of the US during labour. However, these observations may indicate that the EP2 perhaps plays a role in controlling the timing of labour-onset possibly by maintaining quiescence until the excitatory influences become more dominant allowing a shift to a contractile phenotype. This hypothesis is supported by the observation that EP2 was significantly reduced in preterm labour in the US as this may favour premature contractility.
For the receptor subtype EP1, which is assumed to activate contractile signalling pathways in the uterus, the general expression profile between the US and LS showed no significant difference. However, LS expression was significantly higher in term labour samples, indicating that EP1 may play a functional role in the labour process at term. No significant increase in EP1 was observed in the preterm labour group, however this could be due to the relatively small number in this sample group or an indication of the difference in the aetiology of preterm labour. It is difficult to reconcile why expression of a contractile receptor would increase in the LS during term labour, but maybe expression of this receptor plays a role in post-partum contraction.
The expression of EP3 (generic) was generally higher in the US during pregnancy and may contribute to the contractile phenotype of the US. Within both the US and LS, EP3 was down-regulated in pregnancy with a further significant decrease in LS labour samples collected at term. When comparing these differences it is important to remember that our non-pregnant samples were taken from patients suffering from dysmenorrhoea and this condition may have an adverse effect on EP expression. However, these results are consistent with previous results that indicated down-regulation of EP3 expression during pregnancy (Matsumoto et al., 1997) and the observation that EP3 receptor stimulants are less active on myometrium from pregnant subjects than from non-pregnant donors (Senior et al., 1993). We also investigated which of the EP3 splice variants were expressed in the US and LS myometrium. We found expression of the EP3-1b, EP3-II, EP3-III and EP3-IV isoforms in both non-pregnant and pregnant upper and lower myometrium. Consistent with generic EP3, the individual EP3 splice variants followed a similar pattern. The majority of splice variants were significantly down-regulated during pregnancy and showed lower expression in LS term labour samples. Our results were consistent with results of Wing et al., who found that EP3-II was significantly lower in the pregnant compared to the non-pregnant LS myometrium while EP3-III was relatively unchanged. Interestingly, functional analysis has previously shown that these four variants (EP3-II, EP3-III, EP3-1b and EP3-IV) are coupled to multiple second messenger systems. In expression studies it was found that splice variants EP3-I and EP3-III are likely to signal through pathways that stimulate a characteristic EP3 excitatory (or contractile) response while EP3-II and EP3-IV exhibit mixed properties by activating a number of cellular responses, which may cause muscle contraction or relaxation (Kotani et al., 1995). If these signalling pathways are also activated in the myometrium, this may suggest that any relaxatory effects PGE2 may have on the myometrium, that were previously believed to be mediated by EP2 and EP4, may also be mediated in part by EP3-II and EP3-IV. Although, as our expression data shows, these splice variants are generally down-regulated in myometrium during pregnancy and/or term labour the functional significance of these receptors during labour is likely to be limited.
Perhaps the answer to the role of the EP receptors in the uterus during pregnancy and labour lies not only with the expression levels but also with the precise cellular and intracellular localization of the receptors (summarized in Table II). Immuno-localization confirmed the presence of all the four receptor subtypes in the myometrium. Staining for EP1 receptor protein was consistently strong in myometrial smooth muscle cells and in numerous other cell types (see Table II) found to be present in the upper and lower uterine samples. Conversely, whereas EP3 showed positive staining in vessels, decidua and glandular epithelial cells, staining was generally weak and diffused in the pregnant myometrial smooth muscle cells. This is consistent with the mRNA data, which demonstrates that EP3 expression is significantly lower in the pregnant myometrium. However, the presence of EP3 in the decidua may suggest a paracrine role for the receptor in these tissues. Generally, our immuno-localization results are in accordance with those published by Leonhardt et al 2003 in the LS. However, our work also compared the localization of the receptors in the LS with the US myometrium. We found that the EP receptors, when expressed in the myometrial smooth muscle cells, tended to be nuclear or perinuclear localized especially in the LS. Others have previously demonstrated that functional EP receptors (EP1, EP2, EP3
and EP4) are co-localized in the nuclear membranes of a variety of cell types and tissues and have been reported to regulate gene expression (Bhattacharya et al., 1998; 1999; Gobeil et al., 2002). The enzymes involved in the biosynthesis of prostaglandins, namely COX-1, COX-2 and PLA2, have also been localized to the nuclear envelope (Schievella et al., 1995; Spencer et al., 1998) and a specific prostaglandin transporter that facilitates the influx of prostaglandins has been identified (Kanai et al., 1995; Schuster et al., 1998). Thus evidence exists for an alternative mechanism of prostaglandin signalling whereby prostaglandins synthesized and retained within the cell, or imported by a specific transporter protein, are able to exert their effects on target receptors located on the nuclear membrane. In the rat myometrium the FP receptor was found to be localized in the plasma membrane, cytoplasm and around the nucleus, with the ratio of the receptor in the cytoplasmic to membrane fractions changing significantly throughout gestation suggesting a dynamic movement of the receptors (Al-Matubsi, et al. 2001). Although such dynamic movement of the EP receptors was not observed in our sample groups using immunohistochemistry, further investigation using more sensitive quantitative detection and cell fractionation techniques, may show such changes. However, taken together these observations indicate that this novel intracrine signalling mechanism may also be present in the pregnant myometrium and may also provide novel targets for pharmacological intervention.
It is also worthwhile to consider the complexity of the signalling systems present in the myometrium and remember that while PGE2 plays an important role in regulating myometrial contractility other uterotonic agents such as prostaglandin F2 and oxytocin are also very important in regulating myometrial activity and it is likely to be a balance of these factors and signalling pathways that ultimately regulate uterine activity.
In conclusion, we believe that changes in the relative expression and localization of PGE2 receptors play a role in human parturition. EP2 and to a lesser extent EP4 are present in the myometrium although at present their functional role is unclear. EP1 appears to be highly expressed and shows an up-regulation with labour, in contrast to EP3 that is down-regulated and thus EP1 is more likely to mediate the contractile effects of PGE2 in the myometrium.
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Acknowledgements |
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This work was supported by grants from Wellbeing of Women (192) and The Wellcome Trust.
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References |
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Submitted on December 9, 2004; accepted on January 24, 2005.
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