In vitro response of prostaglandin E2 receptor (EP3) in the term pregnant rat uterus and cervix to...

Prostaglandins & other Lipid Mediators 70 (2003) 317–329

In vitro response of prostaglandin E2 receptor (EP3)in the term pregnant rat uterus and cervix

to misoprostol�

Cecilia Lyonsa,c, Kay Beharrya, Yasir Akmalb,Frank Attenellob, Michael P. Nageottea,∗

a Division of Perinatal Medicine, Women’s Hospital, Long Beach Memorial Medical Center,Long Beach, CA, USA

b Department of Research Administration, Long Beach Memorial Medical Center,Long Beach, CA 90806, USA

c Department of Obstetrics and Gynecology, Irvine Medical Center, University of California,Orange, CA 92868, USA

Received 1 May 2000; received in revised form 1 June 2000; accepted 4 October 2002

Abstract

We examined and compared the in vitro effects of misoprostol (synthetic prostaglandin E1 (PGE1)analogue) on prostaglandin E2 (PGE2) secretion and EP3 receptor mRNA expression in the pregnantrat myometrium and cervix at 19 days gestation. Myometrial and cervical tissue samples wereexposed to media with or without misoprostol (50 or 100 pg/ml) and incubated for 15 and 30 min,and 1, 3, 6, 12, and 24 h. Media and tissue samples were collected for quantification of PGE2 andmRNA expression of rEP3� and rEP3� receptor, respectively. PGE2 secretion increased (P ≤ 0.05)in the myometrium exposed to 50 and 100 pg/ml misoprostol. Cervical PGE2 secretion increasedfollowing exposure to the 100 pg/ml dose only. In the myometrium, 50 and 100 pg/ml misoprostolinduced elevations in rEP3� and rEP3� receptor mRNA expression. rEP3� and rEP3� receptormRNA expression in the cervix was not different from controls. These data demonstrate that theEP3 receptor is differentially expressed in the myometrium and cervix in response to misoprostol.This may account for the ability of misoprostol to stimulate the myometrium when administeredfor cervical ripening.© 2002 Published by Elsevier Science Inc.

Keywords: EP receptor; Cervix; Misoprostol; Myometrium; Prostaglandin E2

� This paper was presented in part, at the 1999 Society for Gynecologic Investigation Annual Meeting, Atlanta,GA, USA.

∗ Corresponding author. Tel.:+1-562-933-2730; fax:+1-562-933-2737.E-mail address: [email protected] (M.P. Nageotte).

0090-6980/02/$ – see front matter © 2002 Published by Elsevier Science Inc.PII: S0090-6980(02)00146-6

318 C. Lyons et al. / Prostaglandins & other Lipid Mediators 70 (2003) 317–329

1. Introduction

It is now generally accepted that the fetus may provide a signal to the mother to commencelabor after key maturational events in fetal tissues and organs. Prostaglandin E2 (PGE2)administered intravaginally in women at 39–43 weeks gestation has been shown to inducelabor within 48 h of insertion[1]. There is considerable evidence that PGE2 plays a keyrole in the initiation of parturition[2–5]. Further studies have demonstrated that PGE2 willevoke myometrial contractions in women at early[6,7] and late[8–10]stages of gestation.The mechanism(s) by which PGs regulate myometrial contractions are still the subject ofinvestigation. Recent studies have demonstrated that ingestion of inhibitors of PG synthaseby pregnant women leads to prolongation of gestation and suppression of preterm labor[11,12]. These observations may indicate that PGs play an important role as mediators ofspontaneous labor and delivery in humans. Detailed studies of the control of PG synthesisduring labor remain the focus of investigation.

PGE2 produces a broad range of biological actions in many tissues by binding to specificreceptors. Four receptor subtypes have been classified, namely EP1, EP2, EP3 and EP4[13]. EP1 and EP3 receptors when activated mediate contraction, whereas EP2 and EP4mediate dilatation when activated[14]. EP3 receptors are known to exist abundantly in theuterus and may be responsible for contraction during parturition. In the rat, there are twosubsets of the EP3 receptor, rEP3�, which inhibit cyclic AMP (cAMP) formation[14,15]and rEP3�, which induces calcium mobilization[15].

Labor induction is frequently necessary for a number of obstetric and medical indications[16–21]. Misoprostol is a synthetic PGE1 analogue currently used in obstetric practice forcervical ripening and induction of labor[22–24]. It is a powerful drug, which if adminis-tered during the first and second trimester may result in termination of pregnancy[25,26].Misoprostol is a potent EP3 receptor agonist and may have some affinity for the EP2 re-ceptor[13,27,28]. Although misoprostol may be successful for cervical ripening, a majordisadvantage is its ability to stimulate the myometrium, an effect, which may have adverseoutcome in some patients. We, therefore, investigated the underlying mechanisms involvedwith the use of misoprostol. We examined and compared the effects of two therapeuticdoses of misoprostol on PGE2 production, and mRNA expression of rEP3� and rEP3� inthe near-term pregnant rat uterus and cervix over time. To our knowledge this is the firstreport to describe the biomolecular events associated with misoprostol in the pregnant ratmyometrium and cervix.

2. Materials and methods

2.1. Experimental design1

Twenty-four pregnant Sprague–Dawley rats at 19 days of gestation, weighing 250–300 gwere sacrificed with an overdose of carbon dioxide. Within 15 min of death, the uterus was

1 This study was approved by the Animal Care and Use Committee and the Institutional Review Board of theLong Beach Memorial Medical Center.

C. Lyons et al. / Prostaglandins & other Lipid Mediators 70 (2003) 317–329 319

removed with cervix attached and the pups were expelled by a longitudinal uterine incision.The uterus was dissected from the cervix and cleaned of fat, placenta and rinsed in ice-coldphosphate buffered saline. Each uterus, (weighing approximately 5 g), was sectioned into1 g pieces and pooled (to eliminate variability due to individual differences) in a sterile petridish (six animals per experimental day). The tissue segments were then chosen randomly,cut into 2 mm pieces and placed in sterile 6-well culture plates (1 g per well,n = 5) andallowed to stabilize in 10 ml Dulbecco’s modified Eagle’s medium (DMEM) containing25 mM HEPES buffer (pH 7.4), 1% penicillin and 1% streptomycin for 1 h pre-incubation.The cervices were sectioned into 100 mg pieces, pooled in a sterile petri dish, cut into 2 mmpieces and allowed to stabilize in 1 ml media (100 mg per well,n = 3). The rat cervix isapproximately 300 mg. Therefore, we were unable to harvest more than three samples peranimal. After stabilization, the tissues were washed and placed in fresh medium containingmisoprostol (50 pg/ml media or 100 pg/ml medium). The doses of misoprostol used inthis study was based on the pharmacokinetic data presented by Zeiman et al.[22] andthe dosing regimen used by Wing et al.[26] for cervical ripening and labor induction.The control groups were exposed to drug-free medium. Uterine and cervical samples wereobtained from 24 rats. A total of 48 (24 uterine and 24 cervical) individual culture plateswere examined. The culture plates were placed in an incubator with a 5% flow of CO2 and95% nitrogen, at 37◦C and incubated for 0, 15 and 30 min, 1, 3, 6, and 12 and 24 h. Atthe end of each experimental time, the plates were removed from the incubator, and mediasamples were collected for measurement of PGE2 levels. Tissue samples (100 mg) wereplaced immediately in ice-cold TriZol reagent (Life Technologies) and frozen immediatelyat −80◦C until extraction of total RNA. A total of 120 uterine samples (5per group pertime) and a total of 72 cervical samples (3per group per time) were assayed.

2.2. Measurement of PGE2

Media samples were collected in equal volumes of a solution containing 28 mg/ml EDTAand 40�g/ml indomethacin and immediately stored at−80◦C until assay. Fifty microlitersof samples was used to analyze PGE2 by enzyme immunoassay kits purchased from CaymanChemical Company (Ann Arbor, MI).

2.3. Isolation of total RNA

Total RNA was isolated from approximately 100 mg of myometrium and cervix by homo-genization in 1.0 ml TriZol reagent (Gibco BRL, Eggenstein, Germany) using a polytronhomogenizer (Brinkman Instruments, Inc., Westbury, NJ). The integrity of the RNA wasconfirmed by gel electrophoresis in 1% agarose gel stained with ethidium bromide (EtBr).Total RNA concentration was estimated by spectrophotometric measurements at 260 nm.Total RNA samples were then standardized to 1.0�g/�l with DEPC-treated water.

2.4. Reverse transcriptase-polymerase chain reaction (RT-PCR)

Total RNA (2�g) was reversely transcribed to cDNA. Amplification of cDNA was per-formed using specific sense and anti-sense primers for GAPDH, rEP3� and rEP3� prepared


by Life Technologies, and AmpliTaq DNA polymerase (Perkin-Elmer, Norwalk, CT). Theprimer sequences for GAPDH sense and anti-sense were 5′-TCC CTC AAG ATT GTCAGC AA-3′ and 5′-AGA TCC ACA ACG GAT ACA TT-3′, respectively. The primer se-quence for rEP3� sense and anti-sense were 5′-CTA ATT GCA GTT CGC CTG GCT-3′,and 5′-CGT CTC AAG TGC AGA GTC TTC-3′, respectively. The primer sequence forrEP3� sense and anti-sense were 5′-CTA ATT GCA GTT CGC CTG GCT-3′, and 5′-CATCGA CTG GAC CAC CAA CGT-3′, respectively. The PCR cycle profile which was carriedout using a model 480 DNA thermal cycler (Perkin-Elmer Cetus) was 94◦C for 1 min, 60◦Cfor 1 min, 72◦C for 3 min, for 35 cycles, followed by 72◦C for 7 min.

2.5. Densitometric scanning

Gel electrophoresis of the PCR products was performed on a 2% agarose gel stainedwith EtBr. The intensities of the bands were measured with the use of a GelDoc 1000Darkroom Imager and Molecular Analyst software (BioRad Laboratories, Hercules, CA).PCR fragments were identified according to their molecular mass using a DNA mass ladder(Perkin-Elmer, Norwalk, CT). The amount of DNA in each specimen was quantitated by theintegrated density of the product bands within a closed rectangle, which was then normalizedto the density of GAPDH bands. The data are expressed as mean rEP3� or rEP3�/GADPHratio± S.E.M.

2.6. Statistical analysis

Data were analyzed using GraphPad Instat (GraphPad Software, Inc., San Diego, CA).One-way ANOVA was used to compare data between control and misoprostol-treated groupsat each time interval using the Kruskal–Wallis test for significance. Statistical significancewas set at aP-value of less than 0.05. Values are reported as mean± S.E.M.

3. Results

3.1. Effect of misoprostol on PGE2 production

To examine whether misoprostol influences PGE2 production, we exposed term preg-nant rat myometrium (n = 5 per group) and cervix (n = 3 per group) to two doses ofmisoprostol over 24 h. Both doses of misoprostol resulted in rapid and sustained elevationsof PGE2 in the media from myometrium and cervix. In the myometrium, the two dosesexhibited equipotent effects (Fig. 1), however, in the cervix, the greater response occurredwith 100 pg/ml (Fig. 2). In the myometrium, 50 pg/ml misoprostol increased PGE2 at 6 h(960±146 pg/ml,P < 0.01) and 24 h (2663±112 pg/ml,P < 0.001) compared to controls(483± 78 and 408± 73 pg/ml, respectively). Myometrial exposure to 100 pg/ml misopros-tol resulted in substantial PGE2 production (pg/ml/1 g tissue) at 30 min (1311± 127 pg/ml,P < 0.01), 1 h (1123± 102 pg/ml,P < 0.01), 6 h (1029± 220 pg/ml,P < 0.01), and24 h (2030± 305 pg/ml,P < 0.001) compared to the control group (513± 104, 405± 40,483± 78, and 408± 73 pg/ml, respectively).


Fig. 1. Dose–response and time course of PGE2 secretion by rat myometrium exposed to misoprostol at 19 daysgestation. One gram of tissue was cut into 2 mm pieces and placed in sterile 6-well culture plates (n = 5 at eachtime interval) and stimulated with 50 or 100 pg/ml misoprostol. The control group was exposed to drug-free media.Data are expressed as mean± S.E.M.

Fig. 2shows that cervical exposure to 50 pg/ml misoprostol resulted in decreased PGE2synthesis (pg/ml/100 mg tissue) at 30 min (39±11 pg/ml,P < 0.01) and 3 h (53±4 pg/ml,P < 0.05) compared to control values at 30 min (157±18 pg/ml) and 3 h (118±17 pg/ml).In contrast, cervical exposure to 100 pg/ml misoprostol induced increases in PGE2 at 1 h(288± 19 pg/ml,P < 0.01) and 3 h (296± 79 pg/ml,P < 0.05) versus controls (77± 32and 118± 17 pg/ml, respectively). These transient elevations declined to control values by6 h and remained unchanged throughout 24 h of incubation.

Fig. 2. Dose–response and time course of PGE2 secretion by rat cervix exposed to misoprostol at 19 days gestation.One hundred milligrams of tissue was cut into 2 mm pieces and placed in sterile 6-well culture plates (n = 3 ateach time interval) and stimulated with 50 or 100 pg/ml misoprostol. The control group was exposed to drug-freemedia. Data are expressed as mean± S.E.M.


3.2. Regulation of EP3 receptor mRNA expression

To examine the differential effects of misoprostol on rEP3� and rEP3� receptor mRNAexpression in the pregnant rat myometrium and cervix, total RNA was extracted from100 mg myometrium and cervix following exposure to 50 and 100 pg/ml misoprostol. Inthe myometrium, 50 pg/ml misoprostol resulted in a 24% (P < 0.05), 73% (P < 0.01),90% (P < 0.01), 86% (P < 0.001) and 90% (P < 0.01) increase in rEP3� receptormRNA expression at 0.5, 1, 3, 6 and 12 h, respectively (Fig. 3). Exposure to 100 pg/mlresulted in a 66% (P < 0.05), 42% (P < 0.01) and 93% (P < 0.05) increase at 0.5, 6and 12 h, respectively (Fig. 3). The greatest expression was also noted at 6 h of incubationwith both doses. By 24 h a decrease in rEP3� receptor mRNA was noted with exposure to100 pg/ml misoprostol (Fig. 3).

In response to 50 pg/ml misoprostol, rEP3� mRNA expression in the myometrium in-creased at 1 h (77%,P < 0.05), 3 h (84%,P < 0.01), 12 h (91%,P < 0.05), and 24 h(57%,P < 0.05) (Fig. 4). A greater elevation in rEP3� mRNA expression occurred with ex-posure to 100 pg/ml dose in the myometrium at 1 h (92%,P < 0.05), 3 h (88%,P < 0.01),6 h (75%,P < 0.01), 12 h (96%,P < 0.01) and at 24 h (94%,P < 0.01) (Fig. 4). Thegreatest expression occurred at 3 h for the 50 pg/ml dose and at 6 h for the 100 pg/ml dose.In the cervix no differences were detected in the receptor mRNA expression for both EP3subtypes (Figs. 5 and 6).

Fig. 7represents mRNA expression for GAPDH, rEP3� and rEP3� in myometrium andcervix for control (A), 50 pg/ml misoprostol (B) and 100 pg/ml misoprostol (C) groups. Thisfigure represents densitometric scans of a single sample in each group at each time. Lanes1–8 represents 0, 15 and 30 min, and 1, 3, 6, 12 and 24 h of exposure, respectively. Eachlane consists of 2�g of total RNA. This figure demonstrates that at the lower concentration

Fig. 3. Ratio of relative expression of rEP3� mRNA in rat myometrium. Tissue samples were obtained frompregnant rats at 19 days gestation. One gram of tissue was cut into 2 mm pieces and placed in sterile 6-wellculture plates (n = 5 at each time interval) and stimulated with 50 or 100 pg/ml misoprostol. The control groupwas exposed to drug-free media. The relative rEP3� receptor mRNA expression in the tissue was determined asthe ratio of rEP3� mRNA/GAPDH mRNA measured by semi-quantitative densitometry. Each bar represents themean± S.E.M. of the relative rEP3� mRNA expression in arbitrary units.


Fig. 4. Ratio of relative expression of rEP3� mRNA in rat myometrium. Tissue samples were obtained frompregnant rats at 19 days gestation. One gram of tissue was cut into 2 mm pieces and placed in sterile 6-wellculture plates (n = 5 at each time interval) and stimulated with 50 or 100 pg/ml misoprostol. The control groupwas exposed to drug-free media. The relative rEP3� receptor mRNA expression in the tissue was determined asthe ratio of rEP3� mRNA/GAPDH mRNA measured by semi-quantitative densitometry. Each bar represents themean± S.E.M. of the relative rEP3� mRNA expression in arbitrary units.

of misoprostol (50 pg/ml), rEP3� receptor is more responsive in the cervix. However, atthe higher concentration of 100 pg/ml, rEP3� receptor is more abundantly expressed in themyometrium. Conversely, rEP3� receptor mRNA is highly expressed in the myometriumat both misoprostol concentrations compared to the cervix.

Fig. 5. Ratio of relative expression of rEP3� mRNA in rat cervix. Tissue samples were obtained from pregnantrats at 19 days gestation. One hundred milligrams of tissue was cut into 2 mm pieces and placed in sterile 6-wellculture plates (n = 3 at each time interval) and stimulated with 50 or 100 pg/ml misoprostol. The control groupwas exposed to drug-free media. The relative rEP3� receptor mRNA expression in the tissue was determined asthe ratio of rEP3� mRNA/GAPDH mRNA measured by semi-quantitative densitometry. Each bar represents themean± S.E.M. of the relative rEP3� mRNA expression in arbitrary units.


Fig. 6. Ratio of relative expression of rEP3� mRNA in rat cervix. Tissue samples were obtained from pregnantrats at 19 days gestation. One hundred milligrams of tissue was cut into 2 mm pieces and placed in sterile 6-wellculture plates (n = 3 at each time interval) and stimulated with 50 or 100 pg/ml misoprostol. The control groupwas exposed to drug-free media. The relative rEP3� receptor mRNA expression in the tissue was determined asthe ratio of rEP3� mRNA/GAPDH mRNA measured by semi-quantitative densitometry. Each bar represents themean± S.E.M. of the relative rEP3� mRNA expression in arbitrary unit.

Fig. 7. Messenger RNA expression of GAPDH, rEP3A and rEP3B in myometrium and cervix for control (A),50 pg/ml misoprostol (B) and 100 pg/ml misoprostol (C) groups. This figure represents densitometric scans ofa single sample taken from each group at each time. Lanes 1–8 represents 0, 0.25, 0.5, 1, 3, 6, 12 and 24 h ofexposure. Each lane consists of 2�g of total RNA.


4. Discussion

Normal spontaneous term labor and delivery is associated with increased prostaglandinproduction in amniotic fluid and maternal plasma. Up to now an increase in the productionof the uterine prostaglandins is generally thought to be a key event in the initiation andmaintenance of labor. The increased use of misoprostol for labor induction and cervicalripening necessitates determination of the optimal dosing regimens and the mechanism ofits action on the uterus and cervix remains to be defined. This study aimed to determine theunderlying biomolecular events associated with the use of misoprostol, a potent mediatorof parturition. The present data illustrate that both doses of misoprostol results in a rapidand sustained increase in PGE2 production by the myometrium (Fig. 1), whereas a higherdose of 100 pg/ml was needed to elicit a response, although transient, in the cervix (Fig. 2).The data further showed that rEP3� receptor mRNA was expressed more abundantly in theterm myometrium compared to rEP3� in response to misoprostol (Figs. 3 and 4).

The EP3 receptor subtype has been shown to play a major role in contraction of the uterus[29,30]. Studies by Goureau et al.[31] demonstrate that the EP3 receptor, when activatedby PGE2 may increase intracellular Ca2+, a precursor for uterine contraction. Other studiesshow that EP3 receptors inhibit adenylate cyclase, a second messenger for vasodilatoryprostaglandin actions[32]. EP3 receptors are, therefore, considered potent stimulators ofmyometrial contraction[33]. The rat isoforms of EP3 receptor, rEP3� and rEP3� havedifferent functions. rEP3� inhibits cyclic AMP formation[14,15]causing contraction of theuterus, whereas rEP3� receptor acts to increase uterine contraction via calcium mobilization[14]. There is some speculation that rEP3� stimulates cAMP formation by activation ofadenylate cyclase[15], however, there are no studies, which report EP3 receptor stimulationand elevation of cAMP. Negishi et al.[14] suggests that the EP3� receptor subtype inhibitsadenylate cyclase at low levels of PGE2, but this response is quickly abolished. In contrast,the EP3� receptor subtype appears to be associated with a more sustained inhibition[14].We, therefore, examined the relative expression of the two isoforms of the EP3 receptor inresponse to misoprostol.

Following oral administration, misoprostol is metabolized to misoprostol-free acid. Theparent compound appears to have a half-life of 6 min in vitro[34]. However, plasma con-centrations of the free acid as well as other metabolites decline in a biphasic manner, witha half-life of 20–40 min during initial elimination phase and a half-life of 144–177 h dur-ing the terminal elimination phase[34]. Studies by Tsai et al.[35] demonstrated that themisoprostol-free acid is the active biological form that binds to EP receptors.Fig. 1shows aninitial increase in PGE2 at 30 min of exposure to 100 pg/ml misoprostol in the myometrium,with a concomitant rise in rEP3� receptor mRNA expression (Fig. 3). However, by 24 h ofexposure, as PGE2 levels increase two-fold, there is a significant decrease in myometrialexpression of rEP3� mRNA. This effect may suggest that by 24 h the stimulatory effect ofmisoprostol on the myometrium is abolished or may be down-regulated by the high levelsof PGE2. Studies by Senior et al.[36] show that in vitro PGE2 exhibits a biphasic responsein the human myometrium. PGE2 causes stimulation of the myometrium at low concen-trations, while at high concentrations there is an initial stimulation followed by relaxation.These differential responses may be due to activation and/or down-regulation of the PGE2receptor subtypes, as well as regulation by the uterus in an effort to maintain pregnancy.


In vitro studies by Weems et al.[37] have shown that PGE2 stimulates its own secretionin late gestation. Comparing the responses between 200- and 270-day-old Brahman cowplacentas treated with PGE2, the investigators demonstrated PGE2-induced PGE2 secretionby 270-day-old placentas, but not by 200-day-old placentas, an effect was not altered bycyclooxygenase inhibitors. These findings provide further support for the important role forPGE2 as a key initiator of parturition and may explain the rise in PGE2 levels in the mediarecovered from the uterine samples (Fig. 1).

Of the four PGE2 receptor subtypes, EP3 receptors are more widely expressed in the body[27]. In early pregnancy, the levels of EP3 receptor mRNA expression in the myometriumwas reduced compared to nonpregnant myometrium[38], suggesting inhibition of EP3receptors during pregnancy may be an important regulatory mechanism for maintainingquiescence in the myometrium. Crankshaw and Gaspar[39] demonstrated that contractionsin myometrial strips from nonpregnant ewes were blocked by the EP1 antagonist AH6809indicating the presence of EP1 and EP3 receptors, and further emphasizing a role for EP3receptor inhibition in uterine quiescence and maintenance of pregnancy. The inhibitoryreceptors are thought to be EP2 and EP4, which act by stimulation of adenylate cyclase[14,40]. Therefore, it seems reasonable that the reverse may be true at term. We observedthat the lower dose of 50 pg/ml caused greater expression of rEP3� receptor mRNA, peakingat 6 h of exposure compared to the higher dose of 100 pg/ml (Fig. 3). This may indicate thatmisoprostol activates the rEP3� receptor subtype in the myometrium, causing inhibitionof adenylate cyclase and ultimately, myometrial contraction. This effect appeared to bediminished by 12 h of exposure with both doses (Fig. 3). This may be due to desensitizationof the receptor by high levels of PGE2 as suggested by Negishi et al.[14]. Conversely, thehigher dose of misoprostol caused a greater activation of the rEP3� receptor subtype, aneffect that was sustained throughout the incubation period, despite the high levels of PGE2.This finding suggests that, in contrast to the rEP3� receptor subtype, the rEP3� receptorsubtype is not desensitized by high levels of PGE2. Furthermore, these data imply that theeffect of misoprostol on the myometrium is dose dependent.

Fig. 2 demonstrates that PGE2 secretion by the cervix was induced as early as 1 h ofexposure to the 100 pg/ml dose. In contrast, no response was seen with the smaller dose.It is interesting to note that the relative expression of the two isoforms of EP3 receptorwas comparable among the groups (Figs. 5 and 6). This strongly implies that misoprostol’saction on the cervix at term may involve different PGE2 receptors, namely EP2. In fact, thelower uterine segment appears to have a greater abundance of EP2 receptors compared toEP3[41]. PGE2 is widely used to induce cervical ripening in human term pregnancy[16],however, the mechanism of PGE2 action on the cervix remains largely unclear. One majorlimitation of our study is the sample size (n = 3) of the cervical tissue. Because the ratcervix is approximately 300 mg, we were unable to harvest a sample size larger thann = 3.Although EP3 receptor mRNA is expressed in the cervix, the level of expression appearsto be generally low[42]. Our findings are in agreement with this observation.

In summary, misoprostol induces PGE2 secretion in the term pregnant rat myometrium,whereas the cervix requires a higher misoprostol dose to produce significant PGE2 secretion.High doses of misoprostol elicits a rapid expression of rEP3� and rEP3� receptor mRNA inthe myometrium, an effect that is abolished by 12–24 h of exposure. Alternatively, low andhigh doses of misoprostol do not appear to alter the two isoforms of rat EP3 receptor mRNA


expression in the cervix. Taken together, these data demonstrate that the EP3 receptor isdifferentially expressed in the myometrium and cervix in response to misoprostol. This mayexplain the stimulatory effects of misoprostol on the myometrium when used for cervicalripening and suggests an important role for EP3 in mediating those effects. Furthermore,the effects of misoprostol on the cervix may be mediated via different EP receptors, namelyEP2. These findings provide new insight on one of the mechanisms involved in the effects ofmisoprostol on the near-term pregnant rat myometrium and cervix, as well as the foundationfor further studies involving the use of EP3 receptor antagonists for uterine quiescence andmaintenance of pregnancy.

Acknowledgements

This study was made possible through a grant from the Long Beach Memorial MedicalCenter Foundation, Long Beach, CA.

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In vitro response of prostaglandin E2 receptor (EP3) in the term pregnant rat uterus and cervix to...

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