Evidence for the Presence of AH 13205-Sensitive EP2-Prostanoid Receptors in the Pregnant Baboon But Not in the Pregnant Sheep Myometrium Near Term

Objective: Our purposes were to asses the effects of prostaglandin (PG) E2 and PGF2α on myometrial contractility in pregnant sheep and baboons in an in vitro superfusion study, and to characterize further the PGE-sensitive (EP) receptor subtype involved in the myometrial response to PGE2 by using the selective prostanoid EP2 agonist AH 13205. Methods: Strip preparations of uterine muscle from 15 sheep (107-145 days' gestational age) and ten baboons (158-185 days' gestation) were studied. Cumulative concentration-response curves (CRC) were constructed to oxytocin (4.2 pmol/L to 0.42 μmol/L, PGE2 (0.1 nmol/L to 1 μmol/L), and PGF2α (1 nmol/L to 100 μmol/L), and 50% effective concentration (EC50) values (mean and 95% confidence interval) were calculated. We also tested the hypothesis that PGE2-induced myometrial relaxation in pregnant baboons could be mediated by EP2-prostanoid receptors. Myometrial strips were stimulated by oxytocin (0.42 nmol/L), and CRCs to the EP2-agonist AH 13205 (0.1 nmol/L to 10 μmol/L) were constructed. Results: Prostaglandin F2α stimulated myometrial activity in a concentration-related fashion in all preparations from both sheep and baboons. The EC50 in the sheep myometrium for PGF2α (52 nmol/L, 95% confidence interval [CI] 25-110) was significantly (P < .05) lower than that in baboon myometrium (183 nmol/L, 95% CI 93-355). Oxytocin stimulated myometrial activity in preparations of both sheep (EC50 = 0.29 nmol/L, 95% CI 0.11-0.71) and baboon (EC50 = 0.31 nmol/L, 95% CI 0.18-0.52). In contrast, responses to PGE2 were species-related: PGE2 caused concentration-related stimulation of myometrial activity in sheep tissue (EC50 = 3.2 nmol/L, 95% CI 2.0-5.0), but induced concentration-related inhibition of activity in baboon myometrium (50% inhibitory concentration [IC50] = 21 nmol/L, 95% CI 2.2-203). A concentration-related inhibitory response to AH 13205 (IC50 = 3.56 nmol/L, 95% CI 1.28-5.99) was obtained in the baboon. In contrast, AH 13205 failed to inhibit comparable myometrial strip preparations from pregnant sheep. Conclusions: The present studies suggest that both sheep and baboon myometrium contain prostanoid receptors that mediate stimulation. In addition, baboon myometrium, like that from the human, contains AH 13205-sensitive EP receptors (EP2 receptors), which mediate inhibition. The pregnant baboon may therefore represent a suitable animal model for investigations into the use of EP2 agonists for the prevention of premature labor in humans.

key4 and women. 5 Moreover, several motility-related pathophysiologic conditions such as preterm 6 or dysfunctionaf labor are associated with abnormal release ofPGs. The PGs of both the E and F series (mainly PGE2 and PGF 2 a:) appear to be among the major factors governing the contractility of the myometrium. Although PGF2a: is consistently excitatory on myometrial preparations from a11 species reported to date,8-11 the effects of PGE 2 are less consistent. Depending on thc species and experimental conditions, PGE2 has been shown to cause both excitatory and inhibitory effects on myometrial contractility. [12][13][14] The stimulatory effect of PGF 2a may be mediated by specific PGF 2a -sensitive (FP) receptors, which may act through a variety of different post-receptor mechanisms. 15 -17 Although FP receptors are present in myometrium from the rat, hamster, and probably human, they appear to be absent in myometrium from the guinea pig and cat, and in these latter preparations, PGF 2a causes its excitatory effects through interaction with the thromboxane-sensitive (TP) or even the PGE-sensitive (EP) receptor. 16 The cellular effects of PGE 2 are media ted predominantly by EP receptors, of whieh there exist several subtypes. Radioligand-binding studies have identified one or two (likely two affinity states) reeeptors for PGE 2 in uterine tissues (myometrium and/or endometrium) in several species, including the human. 18-23 However, little information is available on the subcellular pathway and/or the functional response involved in reeeptor aetivation. Funetional studies have identified at least three E-prostanoid (EP) receptor subtypes, namely EP 1 , EP 2 , and EP 3 receptors, in smooth-muscle tissues including myometrium. 24 • 25 Thus, both EPj and EP 3 receptors mediate smooth-muscle eell eontraetion by inereasing intraeellular calcium levels through phospholipase C-mediated inositol phosphate eatabolism and/or by a Gi pro teinmediated lowering of intracellular cAMP. In contrast, it appears that EP 2 receptors cause relaxant effeets in myometrial cells by a Gs pro tein-media ted inerease in intracellular cAMP. 16.26.27 The dual actions (stimulatory and/ or inhibitory) of PGE on uterine motility found in so me studies can, therefore, be explained in terms of interaction with either reeeptor subtype: the nature of the uterine contractile response to endogenous or exogenous PGE 2 depends on which EP receptors are present and functional in the plasma membrane of myometrial cells in a given physiologie situation. The hamster is the only animal species for which EP-inhibitory receptors have been identified firmly in myometrium. Experimental evidence based on diffcrences wirh thc EP2 sub type in the relative rank order of potency for selective agonists 14 has indieated that this inhibitory receptor is more likely to be ofthe recently identified EP 4 sub type. 28 So far, only human myometrium appears to contain EP 2 receptors. 12  agonists as tocolytic agents for the prevention of premature labor in pregnant women. The present study was performed on isolated, pcrfused myometrial strips obtained under halothane anesthesia from late-pregnant ewes and baboons. The objectives of the study were to evaluate the direct actions ofPGE 2 and PGF 2a on myometrial contractility and to produce evidence for inhibitory EP receptors by using the seleetive EP 2 agonist AH 13205 28 -31 on myometrial strips from both species. precontracted with oxytocin. AH 13205 is an EPz-receptor agonist with litde or no activity either at other types of EP receptor 27 or at the other types of prostanoid reeeptor (DP, FP, IP, and TP receptors, defined as receptors with the highest affinity for PGD 2 , PGF 2a , PGI 2 , and TXA 2 , respectively.).29

Processing of Myometrial Tissue
Pie ces of full-thickness uterine wall were collected at the beginning of surgical proeedures under halothane general anesthesia from pregnant animals of known gestational age. In sheep, these specimens were taken midway from the uterotubaljunction and the cervix in the antimesomctrial border (great curvature) of the fetus-bearing uterine horn. In baboons, the myometrial tissue was taken from the ventral surfaee of the uterus in the fundal region or body. The exact site depended upon the location of the placenta. We studied a total of 15 Columbia x Rambouillet cross-breed ewes (Ovis aries, 50-70 kg) between 107 and 145 days of gcstation with singleton or multiple pregnancies (term 148 days), and ten baboons (Papio cynocephalus, 24-30 kg) between 158 and 185 days of gestation and bearing a single fetus (term 184 days). Tissues were placed rapidly at 4°C in Krebs buffer of the following composition (mmol/L: NaCl, 118.0; NaHC0 3 , 25.0; KCI, 5.0; KH 2 P04, 1.0; glucose, 11.0; and CaClz, 1.3. The cyclo-oxygenase blocker indomethacin (3 ~mol/L) was added to the buffer to prevent post-collection synthesis of endogenous PGs, which could interfere with the contractility of the collected tissue. Studies were approved by the Cornell University Institutional Animal Care and Use Committee.

Tissue Preparation
The endometrium was removed gently with the aid of a glass slide and dissection forceps. Myometrial tissue was then cut with a scalpel blade. Individual strips (approximately 0.4 x 1 cm) were prepared in the direction of the longitudinal muscle fibers. A long cotton thread was sutured to thc upper end ofeach musdc strip for attachment to a force transducer (UFl, Pioden Controls Ltd., Canterbury, UK), and the lower end was anchored to the bottom of thc superfusion chamber. The distance between the two sutures, measured under 1 g of tension , was found to be in the range of9-12 mm for a11 thc strips J Soc Gynecol Invest Vol. 2, No. I, Jan.-Feb. 1995 used in the study. Care was taken not to allow the tissues to dry during preparation.

Muscle Superfusion System
The superfusion system used was simi!ar to those described previous!y. 13.32 In each study, eight strips of myometrium were placed in individual chambers. Oxygenated (95%/5% 02/C02) Krebs buffer (35-37°C), of the composition described above and containing indomethacin (3 fLmol/L), was supcrfuscd onto thc myometrial strips using eight channels of a 16-channel individual-cartridge peristaltic pump (eR 07618-60, Ismatec, Cole-Parmer, Chicago, IL) equipped with calibrated vinyl tubing (2.5 mm interna! diameter). The system achieved a flow rate of 2.0 mLlminute. Ca re was taken that the nutrient buffer dripped down the cotton thread and spread over the whole strip in each chamber. A lower hole in the chamber was connected to a waste collector. Drugs were instilIed into the flow of nutrient buffer using the remaining eight channels of the peristaltic pump, which were equipped with narrow-bore calibrated vinyl tubings (0.25 mm internal diameter) to pro du ce a rate of flow for drug solutions 11100 of that for the buffer. This dilution was taken into account when determining the final concentration of drug that actually came in contact with the superfused tissues.

Contractility Data Acquisition
After placement in the superfusion chamber, a resting tension of 1 g was applied to each myometrial strip, which was then allowed to equilibrate for 1-2 hours until a regular contractility pattern developed. Tension changes produced du ring the studyranged from 1-20 g, and were sampled at 32 Hz using a Data Acquisition System 33 connected to an IBM dass PC computer. The computer was programmed for real-time analysis, and printers were used to output strip-chart representations of muscle activity and integrated drug-induced effects. Baseline contractility activity for each channel was defined as the average tt:nsion (g) computed for the 10minute period immediately before administration of a drug. Drugs were administered for 12 minutes. The differences between baseline myometrial activity and the levels achieved during the final 10 minutes of the 12minute period of drug treatment were computed on-line. Drug concentrations used covered the range from that producing no effect to that achieving a maximal activity response, as determined in previous pilot studies .

Experimental Schedule
In the first study, myometrial strips were superfused with PGE2 at concentrations ranging from 100 pmol/L to 1 fLmol/L (six sheep and four baboons), and with PGF 20 from 1 nmol/L to 100 fLmol/L (nine sheep and five baboons).

Garcia-Villar et al
In the second study, to evaluate the effects of AH 13205, the musc1e strips were precontracted. This was considered necessary because in tracheal smooth muscle, PGE 2 has been shown to be stimulatory on low-tone preparations but consistently inhibitory on precontracted strips.34 Thus, coneentration-response eurves (CRCs) to oxytoein were constructed cumulatively in myometrial strips from five sheep and five baboons using coneentrations of 4.2 pmol/L to 0.42 fLmollL. Myometrial activity was driven by adding oxytocin up to the 50% effective concentration (EC50) into the nu trient Krebs buffer. For the study of AH 13205, the CRCs were eonstrueted over the range of 0.1 nmol/L to 10 fLmol/L of AH 13205 in muscle stimu!ated wirh 0.42 nmollL oxytocin.

Curve Fitting
Contractility da ta from the different replieates (two to eight for each eoncentration in each animal were averaged and normalized to pereent of maximal efTect. A nonlinear regression eurve-fitting program (GraphPad V2.0, ISI software, Philadelphia, PA) was used for fitting the replieates for each drug-concentration point for all the animals of each experimental group to a sigmoid curve, using the logistic equation: in which E = the effeet of a given concentration of drug; Emax = the maximal aehievable effect (top ofthe eurve); [D] = the eoneentration of drug; EC50 = the concentration of drug that aehieves 50% of Emax (for investigations of inhibitory efTects, the term EC50 was replaced by lCSO, with a similar definition); and exponent s = the slope faetor. From this overall CRC fit, drug poteneies were caleulated for each species as the EC50 or its negative logarithm (-log EC50 or pD 2 ).

Spontaneous Contractility Pattern
After equilibration, all myometrial strips studied displaycd a characteristic pattern of contractility_ The pattern consisted of the cydic occurrence of contractilc episodes of slightly less than 1 minute in duration, followed by relatively Ion ger periods of quiescence_ In sheep myometrium, episodes of 51.6 ± 5 _0 seconds and a mean amplitude of 5_37 ± 0_34 g occurred at intervals of 143.4 ± 10_6 seconds (mean ± SEM, n = 10)_ The mean values calculated for each sheep were obtained from at least 20 contractile episodes per animaL In baboon myometrium, episodes lasted 33_1 ± 8_3 seconds, had a mean amplitude of1.9 ± 0.3 g, and occurred at intervals of144.4 ± 53_7 seconds (mean ± SEM, n = 5). The mean values calculated for each baboon were obtained from at least ten contractile episodcs per animaL Effects of PGE 2 and PGF 2llt on Myometrial Contractility Table 1

Effects of Oxytocin
As expected, oxytocin (4 _2 pmal/L to 0.42 ~mol/L) caused concentration-related increases in myometrial activity in both species (Figure 2)_ In sheep myometrium, the EC50 for oxytocin was 0_29 nmol/L (95% CI 0_11-0_71) , and in baboon myometrium, the EC50 value was 0.31 nmol/L (95% CI 0_18-0_52) ( cin (0.42 nmol/L) consistently increased the activity of myometrial strips from both baboons and sheep by two to three times the spontaneous activity level. This effect was maintained for at least 2 hours (data not shown).
Consequently, this concentration of oxytocin was chosen to drive myometrial motility, and the activity level obtained was considered as 100% baseline activity for the following study.

DISCUSSION
The present study, using an in vitro smooth musc!e superfusion system, confirms the already well-documented stimulant action of PGF 2a on both pregnant sheep and baboon myometrium in late gestation. Myometrial contractile responses to PGF 2a analogues have been reported both in vi va and in vitro , e. g. , in humans,lO non-human primates,8 shecp, 9 and rats_ 11 Howcver, our data also show a species-dependent effect of PGE 2 on myometrial contractile activity_ Prostagiandin E 2 caused a dear concentration-related stimulation of motility in sheep myometrial strips, but caused a concentration-related inhibition of motility in comparable strips from the baboon myometrium_ A species-specific, concentration-related effect of the selective prostanoid EP 2 agonist, AH 13205, on oxytocin-induced contractile activity was demonstrated in myometrial muscle from the pregnant baboon. In contrast, there was no effect of AH 13205 on the motility of  comparable strips from pregnant sheep myometrium.
Because AH 13205 has been shown to be essentially inactive at other potentially inhibitory prostanoid receptors, such as IP or DP receptors,16 our findings suggest that functional EP receptors are present on the plasma membrane of myometrial cells and mediate the inhibitory effect of AH 13205 in the baboon. In contrast, EP 2 receptors are either absent or not functionally coupled in the myometrium of late-pregnant ewes. The species-specific myometrial response to PGE 2 is interesting. Prostagiandin E 2 is thought to exert its cellular effects primarily by an action on prostanoid EP receptors, of which there are at least three subtypes: EP!, EP z , and EP). The interaction of endogenous and/or exogenous PGE2 with these receptor subtypes initiates different intracellular regulatory cascades in myometrial smooih-muscle cells . Both EP! and EP 3 receptor subtypes mediate excitatory responses by increasing intracel- lular calcium levels and/or by lowering intracel1ular levels of cAMP. Conversely, EP 2 receptor activation leads to inhibitory responses, which appear to be secondary to increases in intracel1ular levels of cAMP. 16 Recently, however, another EP-receptor subtype, termed EP 4' has been identified in the hamster myometrium. 14 Like the EP z receptor, this receptor is inhibitory and appears to produce its effects through incrcases in intracellular cAMP, but it difTers from EP z by different rank order of potency of relatively selective agonists. 14 These difTerences between species, in overall activity as wen as in the nature of the inhibitory receptor subtypes (EP2 or EP 4)' clearly demonstrate the need for an experimental model that resembles as closely as possible the pregnant human myometrium. It is likely that not aB species possess all of the EPreceptor subtypes, or if they do , they are not all func-tionaBy coupled to their intracellular signaling pathways. Functional EP1 and EP) receptors havc been characterized pharmacologicaBy in the myometrium of a variety of animal species, including guinea pig, rat, hamster, cat, and human. 16 Conversely, to date, experimental evidence  for the presence of functional EP2 receptors in the myometrium has been restricted to the human. 13 Drawing a comprehensive picture of uterine prostanoid receptors would have important implications in clinical obstetrics. Indeed, PGE 2 has been used successfully to improve the rate of cervical ripening during term labor in humans, 35 and it has been shown to soften the ovine cervix in vitro. 36 However, the lack of consistency ofPGE2 etIects on uterine contractility (inhibition or activation) remains a concern for the c1inician. Synthetic analogues of PGE 2 would be most likely to be effective if their stimulatory effects (cervical ripening and myometrial stimulation) and their inhibitory etIects (myometrial tocolysis) could be dissociated. This could be achieved if these effects were mediated by different receptor sub types and if selective agonists and antagonists for each receptor subtype become available. Thus, EP 1 and/or EP 3 agonists could be used to improve management of problems due to inadequate myometrial activity during parturition, whercas EP2 agonists could be intended for clinical use as toCQlytics in the treatment of premature labor. In addition, EP2 agonists such as AH 13205 might be useful when PGE2 is used to induce cervical dilatation, if the effect~ of PGE2 on the cervix were unaffected by AH 13205, while permitting AH 13205 to inhibit any unwanted uterotonic action of PGE 2 . So far, the lack of an appropriate animal model for human myometrial tissue has impeded progress in the development of EPrreceptor selective drugs.
In conclusion, the PGF 2n -induced stimulation of the myometrium of both pregnant baboons and sheep is in agreement with results obtained with a wide range of species, including the human. In view ofits high potency, PGEz-induced stimulation of sheep myometrium can be explained most likely by an interaction with stimulatory EP 1 and/or EP 3 receptors. Because PGF2n alone caused contraction of baboon myometrium, it is tempting to speculate that FP receptors are involved. However, PGF 2n demonstr<lted a rather low absolute potency in this effect. Thus, further experiments with other more selective agonists and antagonists will be required to characterize definitively the receptors involved in the baboon myometrial contractile response to PGF2n . We hypothe~ size, however, that the stimulatory response ofthe myometrium to PGF 2n is mediated by TP or even EP 1 and/or EP 3 receptors, as in other tissues and species. 16 The data obtained in the second part of our study, featuring oxytocin-induced stimulation of myometrial strips from both pregnant baboons and sheep and sub se quent challenge with the selective EP 2 -receptor agonist AH 13205, provide pharmacologic evidence that the prostanoid receptor(s) involved in the inhibitory etIect ofPGE 2 in baboon myometrium is of the EP2 subtype.
Further in vitro experiments testing other selective EP agents in parallel functional and radio ligand binding studies in baboon myometrium would provide more definitive evidence as to the nature of the different EP receptors actuallY present in the myometrium (stimulatory and/or inhibitory), and would determine any regional or gestational-age-related changes in both non-human and human myometrium. However, the present work clearly indicates that the baboon may represent a suitable animal model for investigating the potential of EP 2 agonists in the control of myometrial hyperactivity in humans. A major advantage of this model is the ability to study activity in vivo using chronic recording of myometrial electrical activity and changes in intrauterine pressure 37 in the pregnant baboon. Thus, in vitro contractility of myometrial preparations can be equated precisely with the in vivo function of the same specimens.