Br. J. Pharmacol. (1991), 102, 747-753

C Macmillan Press Ltd, 1991

In vitro characterization of prostanoid EP-receptors in the non-pregnant human myometrium 'J. Senior, K. Marshall, R. Sangha, G.S. Baxter & *J.K. Clayton Postgraduate Studies in Pharmacology, University of Bradford, Bradford, BD7 lDP and *The Royal Infirmary, Bradford, BD9 6RJ 1 Prostaglandin receptors of the PGE type have been characterized in the non-pregnant human myometrium in vitro according to the scheme of Coleman et al. (1984) by use of the agonists PGE2, sulprostone, rioprostil, AY23626, butaprost, misoprostol, 16,16-dimethylprostaglandin E2, enprostil and iloprost, and, the antagonist AH6809. 2 All prostanoids tested were active in non-pregnant human myometrium either as stimulators and/or inhibitors of spontaneous activity or both. Biphasic responses to PGE2 indicate that at least two receptor types of the EP-receptor exist, one mediating relaxation and the other mediating contraction. 3 Further evidence for the EP-receptor mediating excitation and relaxation was provided by the action of the EP2-/EP3-receptor selective prostanoids rioprostil, AY23626 and misoprostol, and the EP1-/EP2-receptor selective agonist 16,16-dimethylprostaglandin E2. 4 Butaprost, an EP2-receptor selective agonist, produced potent inhibition of spontaneous activity in the tissue which was generally longer-lasting than that evoked by the natural prostanoid PGE2. 5 The EP1-/EP3-receptor selective agonist sulprostone and the EP3-receptor agonist enprostil produced potent contractile responses supporting the presence of contractile EP3-receptors in the non-pregnant human myometrium in vitro. 6 The EP,-/IP-receptor selective agonist, iloprost, produced mixed responses in non-pregnant human myometrium. The contractile response was inhibited by the EP,-receptor antagonist AH6809. However, responses to the EP1-/EP3-receptor selective agonist sulprostone were unaffected by AH6809 which may indicate that only a small population of EP1-receptors is present. 7 Therefore it would seem that a heterogeneous population of EP-receptors is present in the nonpregnant human myometrium.

Introduction

Prostanoids appear to be involved in most aspects of uterine physiology and have been implicated in a multitude of pathophysiological conditions such as dysmenorrhoea, menorrhagia, preeclampsia and preterm labour. Indeed they have found their major therapeutic application in obstetrics and gynaecology. However, wide use of prostanoid medicines is limited by a high incidence of side-effects. Side-effect liability can be reduced by the development of drugs which have a selective action at a given receptor mediating the beneficial but not the deleterious effects. It is for this reason that the classification of prostanoid receptors is important. Evidence for the existence of prostanoid receptors is abundant. They are highly potent in many biological systems and both their potency and profile of action are sensitive to small changes in chemical structure. In addition, the profile of activity is often species-, tissue-, or prostanoid-specific and in many cases, may be altered selectively by use of antagonists. There are distinct receptors for the naturally-occurring prostanoids prostaglandin D (PGD), PGE (Jones, 1976), PGF, PGI and thromboxane which are designated 'P-receptors' (Kennedy et al., 1982; 1983; Coleman, 1983; Coleman et al., 1984). Furthermore the EP-receptor can be arbitrarily subdivided into EP1-, EP2- and EP3-receptors on the basis of studies with selective agonists and antagonists (Coleman, 1987; Coleman et al., 1985; 1987a,b). The classification is based on agonist and antagonist activity in both functional and binding studies. There are now many selective agonists and antagonists for the EP-receptor but there are few which show absolute selectivity for the individual EP1-, EP2- and EP3-subtypes. In general, conclusions must be drawn on the basis of results obtained from various combinations of receptor selective compounds, and a process of elimination. To date, butaprost (TR4979) (Gardiner et al., 1986) appears to be

the most selective agonist for the study of EP-receptors in that it shows high potency and selectivity at the EP2-receptor subtype, with little or no activity for the EP1- or EP3-receptors. Other PGE analogues such as sulprostone, rioprostil and misoprostol are useful but show activity at two EP-receptor types. Thus sulprostone (Hess et al., 1979), a stable acyl sulphonamide analogue of PGE2 shows selectivity for both EP1and EP3-receptors (Coleman et al., 1987a,b; 1988), and rioprostil (Kluender & Woesser, 1979) shows selectivity for the EP2- and EP3-receptors (Reeves et al., 1988; Coleman et al., 1988). Misoprostol is a potent anti-secretory prostaglandin, structurally related to PGE1 (Collins et al., 1985). It is believed that it exerts its gastric anti-secretory effects via EP3-receptors (Reeves et al., 1988). Some degree of EP2-receptor activity has also been reported with misoprostol (Coleman et al., 1988). 16,16-dimethyl prostaglandin E2 has a selectivity of action within EP-receptors, being at least 10 times more potent on EP1- than on EP2-receptors (Coleman, 1987). Some action via the EP3-receptor has also been recorded (Reeves et al., 1988). Enprostil, a synthetic dehydroprostaglandin E2 has activity at the EP3-receptor and also at the FP- and TP-receptors (Eglen et al., 1989) and EP,-receptor (Coleman et al., 1988). The combined use of these compounds in conjunction with EP,-receptor blocking drugs can yield useful information about receptor populations. Iloprost, which displays potent EP1-, but negligible EP2- or EP3-receptor activity (Sheldrick et al., 1988) may then corroborate evidence obtained with other EP-receptor selective agonists. Iloprost is also an IP-receptor agonist. SC19920 (Sanner, 1972), though a weak antagonist at the EP,-receptor, does appear to be selective in its prostanoid receptor blocking activity. AH6809, has a similar profile of EP,-receptor blocking activity though it is approximately

748

J. SENIOR et al.

10-40 times more potent with demonstrated pA2 values ranging between 6.5-6.8 (Coleman et al., 1985). AH6809 does have weak action at other receptors including the DP- and TP-receptors (Keery & Lumley, 1988) yielding pA2 values at each site of 5.4 and 4.4 respectively. In this study AH6809 was used at a concentration of 10pM to eliminate effectively any EP1-receptor involvement. The selectivity profiles of the agonists can be summarised as follows: rioprostil/AY23626/misoprostol-EP3> EP2 > EPI -EP3 > EP, > EP2 16,16-dimethyl PGE2 sulprostone -EP3 z EP, > EP2 z 0 enprostil -EP3 > EP, > EP2 butaprost -EP2 > EP1 & EP3 > 0 iloprost -EP1(=IP) > EP2 & EP3 In addition, 16,16-dimethyl PGE2 and enprostil are known to have a similar level of FP-receptor agonist activity. In vitro PGE2 predominantly inhibits spontaneous myometrial activity in corporal specimens (Bygdeman & Eliasson, 1963; Bygdeman, 1964) though other workers have observed mild excitation (Pickles et al., 1965) and more recent evidence has reported a biphasic response (Wiqvist et al., 1983; Maigaard et al., 1986) consisting of stimulation of spontaneous myometrial activity followed by inhibition at higher doses. In non-pregnant human myometrium in vivo early work has shown a potent stimulatory effect after intravenous, intramuscular or subcutaneous administration of PGE2 (Karim et al., 1971) which originally suggested that a discrepancy existed between the effect of PGE2 on the non-pregnant myometrium in vitro as compared to in vivo. However, later work demonstrated an inhibitory effect after intrauterine administration of PGE2 (Toppozada et al., 1974) which coincides with in vitro results. We have previously reported a biphasic response with PGE2 in the spontaneously active human myometrium in vitro (Clayton et al., 1986a). Generally, these effects are consistent with a physiological role for prostanoids in the regulation of uterine motility throughout the menstrual cycle and the identification of the EP-receptors will confer clinical advantages in treatment based upon PGE2 and its analogues.

Methods

Non-pregnant human myometrium was obtained from premenopausal patients undergoing hysterectomy for the treatment of various benign disorders such as menorrhagia and fibroids. Patients with underlying malignancy were excluded from the studies. Samples of non-pregnant human myometrium were taken from the anterior wall of the corpus uteri. The phase of the menstrual cycle was assessed by histological examination of the endometrium by the pathology department of St. Luke's Hospital, Bradford. After surgery, human tissue samples were placed in Krebs solution at room temperature and transported to the laboratory where experimental preparations were completed within a 60 min post-operative period. Samples of human myometrium were cut in the direction of muscle fibres to produce strips (20 x 3 x 3mm) of predominantly longitudinal muscle. Care was taken to ensure that each strip was free from either endometrium or serosa. The tissues were placed under an initial passive tension of 2 g and superfused at a rate of 2 ml min- with Krebs solution aerated with 95% 02/5% CO2 at 37°C as previously described (Massele & Senior, 1981). Preparations were allowed to equilibrate for a period of not less than 2 h and at least until spontaneous activity had become regular. The Krebs solution passed through a section of silicon tubing which served as a

drug injection site, just prior to passing over the tissue contained within the organ chamber.

Measurement of responses Responses were measured via isometric transducers (Dynamometer UFI) linked first to a Grass Polygraph (Model 7D) chartpen recorder and then to a BBC Master microcomputer running integration software which allows measurement of area of tension and peak height of a response (Eltec Computer Ltd., Bradford).

Analysis of results All agonists were injected directly into the flow of the superfusion fluid as a bolus dose and dose-effect curves constructed sequentially. Agonists were injected immediately after a spontaneous contraction to avoid superimposing responses on this activity. The profile of spontaneous activity and sensitivity to agonists often changed markedly throughout the course of the experiments. For this reason, agonist dose-effect curves were not repeated, and comparisons were made between preparations in a non-paired manner. Where antagonists were used, they were dissolved in the Krebs superfusion solution and were in contact with tissues for a minimum contact period of not less than 30 min before agonist dose-effect curves were constructed. The quantification of agonist responses in non-pregnant human myometrium presented several problems in this study, partly because of the variability of intrinsic spontaneous activity which occurs throughout the menstrual cycle. From examination of traces it appeared that the greater the magnitude of background contractions, the greater the magnitude of the excitatory response. Thus the results were expressed as T/B ratios of 'test' (T) agonist responses as a ratio of background (B) contractions. The inhibitory actions of prostanoids in nonpregnant human myometrium were expressed in terms of how they modified the periodicity of spontaneous activity, i.e., for how long (min) they extended the period between contractions. The time period between spontaneous contractions was measured in addition to the periods from the point of agonist addition to the re-occurrence of a spontaneous contraction of at least 80% of the peak height of the background contraction. The period of inhibition was then corrected for background frequency and expressed in absolute terms. Responses were often mixed, consisting of initial excitatory responses followed by inhibition. When this occurred, both aspects of the response were treated independently. The use of T/B ratios to express agonist responses, provides a convenient method of deriving their potencies in individual preparations. As these values are effectively multiples of background activity it is possible to express potency in terms of the dose of agonist required to attain a response which reaches a predetermined value. For the purposes of this study, agonist potency was determined as the dose required to cause a response of equal magnitude to background activity, i.e. at the level where T/B ratio equalled 1. It must be made clear that a T/B value of 1 does actually represent an agonist response, and not simply the occurrence of the next spontaneous contraction. The T/B ratio of 1 was chosen as most agonists reach this level of response. The dose of agonist required to attain this level of response was defined as its ED1 (Excitatory Dose) value. ED1 values were obtained from individual dose-effect curves of T/B ratio against agonist concentration, and expressed as geometric means with 95% confidence limits. For agonist-induced inhibition of spontaneous activity, potencies were expressed as ID4 values i.e. the dose of agonist required to extend the normal periodicity of spontaneous contractions by 4min. This 4min period was chosen as this represents an extension of approximately two times the normal periodicity of spontaneous activity in non-pregnant human myometrium,

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EP-RECEPTORS IN NON-PREGNANT HUMAN MYOMETRIUM

as derived from pooled data (i.e. grouping data from all stages of the menstrual cycle). ID4 values were obtained from individual dose-effect curves of inhibition (min) against agonist concentration and expressed as geometric means with 95% confidence limits. Statistical comparisons were made by an unpaired Student's t test (Snedecor & Cochran, 1979).

a

0 Co

Drugs Compounds used, their vehicles and sources of supply are outlined in Table 1 (all final dilutions were in 0.9% sodium chloride solution (normal saline)). 0.01

Results The spontaneous activity of non-pregnant human myometrium was not influenced by the presence of indomethacin (2.79 pM) in the Krebs solution, hence it was included to inhibit endogenous prostanoid synthesis in all experiments. We have previously shown that human myometrium contains thromboxane receptors (Clayton et al., 1983; 1986b). In order to antagonize any TP-receptor activity of the synthetic analogues, GR32191 was used at a concentration of 1pM (which is approximately 1000 times its pA2 in human uterine artery (Baxter et al., 1989)) in all experiments.

E

201-5

0

C

1 O2

0.001

-10l

0.01

II

0.1

iiil 1

Stock vehicle

Source Upjohn

3% ethanol 0.01% tween

Schering

Ethanol 1% NaHCO3

Ortho Ayerst

Butaprost TR4979

Ethanol

Bayer

Iloprost

0.9% normal s,;aline

Schering

Misoprostol

Ethanol

16,16-dimethyl PGE2

1% NaHCO3

Searle Glaxo

Enprostil

Ethanol Distilled water

Glaxo

Rioprostil AY23626 1

1-desoxy-PGEO ZK36 374

GR32191

Syntex

([lR-[[ta(z),2fl,3f,5oa]] -( + )-7-[5[[1,1-biphenyl)-4-yl) methoxy]-3-hydroxy-2(1-piperidinyl)cyclopentyl]-4heptanoic acid hydrochloride 1% NaHCO3 6-isopropoxy-9-oxaxanthene-2-carboxylic acid 1% NaHCO3 Indomethacin

AH6809

10

III

Indi

itim

100

1000

taneous activity (ID4= 1.4nmol) (Figure 3). The profile of response was qualitatively similar to that produced by PGE2 except that at lower doses there was a time lag between agonist injection and onset of the excitatory response. This time lag disappeared as the dose increased. As an excitatory agonist, rioprostil showed considerable variability in potency;

1% NaHCO3

PGE2 Dinoprostone Sulprostone

IIIII1

Dose (nmol) Figure 1 Mean dose-effect curves to stimulant (a) and inhibitory (b) responses to prostaglandin E2 (PGE2) in the absence (El) n = 8, and in the presence (*) n = 8 of GR32191 (1 pM). Data are expressed as arithmetic means and vertical bars represent s.e.mean.

Table 1 Compounds used

Compound

1000

C

Agonists Agonist potencies (ED1 and ID4) and maximal responses together with group sizes are given in Table 2. PGE2 had a dose-related activity in non-pregnant human myometrium which was not affected by the presence of GR32191 (Figure 1). The response comprised both excitation and inhibition. The excitatory component of the biphasic response was generally phasic in nature. The inhibitory response ranged from a reduction in peak height of spontaneous contractions at lower concentrations, to a complete inhibition of activity for periods in excess of 20 min. Sulprostone was a potent agonist, and caused purely excitatory responses (ED1 = 0.03 nmol) (Figure 2a). Responses developed rapidly and consisted of an increase in baseline tonus upon which frequent phasic contractions were superimposed. Like sulprostone, rioprostil was a potent excitatory agonist (EDI = 0.02 nmol) (Figure 2a). However, over the range 1.4-140 nmol, it caused dose-related inhibition of spon-

100

10

0.1

Glaxo

Sigma

J. SENIOR et al.

750

Table 2 Mean ED1, ID4 and maximum responses for excitation and inhibition by prostanoids Inhibition

Excitation ED1

(95% CL) (s.e.mean)

Prostanoid PGE2

0.02

Sulprostone

(0.001-0.58) 0.03

Rioprostil

(0.006-0.16) 0.02

AY23626

(0.001-0.46) 0.16 (0.07-0.83)

n

(95% CL)

max

n

3.34 (1.69) 4.2 (0.83) 2.17 (0.83) 1.7 (0.5) NR

8

0.8 (0.2-2.9) >110

21.3 (4.2) NR

8

1.4

33.9 (3.4) 13.1 (2.5) 88.0 (21.4) 24.0

>120

Butaprost

Misoprostol

Enprostil Iloprost

6 6

(0.54-3.6) 6.2

6

(1.3-30.0) 6

2.2 (0.63) 1.8 (0.5) 7.0 (1.0) 2.3 (0.3)

0.07 (0.005-0.134) 0.02 (0.004-0.34) 0.02 (0.0125-0.025) 2.74 (1.03-7.3)

16,16-dmPGE2

ID4

max

(s.e.mean)

0.43 (0.06-2.9) 2.11 (1.04-3.64) 3.53 (2.97-4.26)

6 6

-

I12

7

0.94

(0.2-4.9)

6 6

8 6

7

(3.0)

7

5

(1-0) 7.6 (2.3)

7

NR = no response. ED1 and ID4 values are expressed as geometric means (nmol) with 95% CL in parentheses. Maximum responses for excitation are expressed as multiples of background activity (i.e. maximum T/B ratios) and maximum responses for inhibition are expressed in min.

similar variability was observed with PGE2 as we have previously reported (Clayton et al., 1989). Like rioprostil, AY23626, also demonstrated biphasic activity (Figures 2b and 3). Inhibitory responses produced an ID4 value of 6.2 nmol, with a mean maximum inhibitory response of 13.1 min. Butaprost did not cause excitation at any dose as reported in earlier work (Clayton et al., 1989), but was a potent inhibia

tor of spontaneous activity (ID4= 0.43 nmol) producing

a

maximum inhibition of approximately 90 min (Figure 3). Misoprostol, at low doses, is a potent excitatory agonist (Figure 2b). However, at higher doses misoprostol produced a dose-related inhibition of spontaneous activity with maximum 2.11 nmol). 16,16-dimethylmean inhibition of 24min (ID4 prostaglandin E2 provoked a response qualitatively similar to misoprostol i.e. at the high doses employed the inhibitory response was seen with little excitation, however the maximum inhibition was lower at 5min (ID4= 3.53 nmol) (Figures 2b and 3). Enprostil produced a dose-related contractile response reaching a maximum at 2.5 nmol (Figure 2a). The EP1-/IP-receptor selective prostanoid, iloprost, evoked a biphasic response comprising an initial excitation (ED1 = mean

=

0

Co

I._ 4@

120 100

_

80

E60

-

C

0

n0

-

0

co

._

0.001

0.01

0.1

1

10

100

1000

1 -201 0.001

1 1

miill

0.01

Dose (nmol)

I

I "I'ill 0.1

li

I

1111

1

I I

"'Ii

I I

10

"'I'lll

100

"I'mli

1000

Dose (nmol)

Figure 2 Mean stimulatory dose-effect curves to: (a) prostaglandin E2 (PGE2), (0) n = 8, rioprostil (*) n = 6, sulprostone (U) n = 6 and enprostil (A) n = 12; (b) PGE2 (O) n = 8, AY23626 (*) n = 6, misoprostol (U) n = 6 and 16,16-dimethylPGE2 (0) n 6. Data are expressed as arithmetic means and vertical bars represent s.e.mean. =

Figure 3 Mean inhibitory dose-effect curves to prostaglandin E2 (PGE2) (E) n 8, rioprostil (*) n 6, butaprost (A) n = 6, AY23626 (A) n 8, misoprostol (U) n = 7 and 16,16-dimethyl PGE2 (0) 7. Data are expressed as arithmetic means and vertical bars repn =

=

resent s.e.mean.

=

EP-RECEPTORS IN NON-PREGNANT HUMAN MYOMETRIUM

2.74 nmol, max. 2.3 x background) followed by an inhibition of spontaneous activity (ID4 = 0.94 nmol), to maximum of 7.6 min. These biphasic responses were qualitatively similar to biphasic responses produced by PGE2, although iloprost was considerably less potent as an excitatory agonist (Figure 4).

Antagonists

3

20

01

0.01

0.1

1

10

1I11II11100

Dose (nmol) Figure 4 Mean stimulatory dose-effect curves to iloprost in the absence (C]) n = 7 and in the presence (*) n = 5 of AH6809 (1O0UM). Data are expressed as arithmetic means and vertical bars represent s.e.mean. * P < 0.05; **P < 0.005. 5

4-

032-

O~~~~~~~~~~~~ 'I1 " ''"'''10 Al' I

I

il 11100 '"" 1000

Dose (nmol) Figure 5 Mean stimulatory dose-effect curves to sulprostone in the absence (El) n = 5 and in the presence (*) n = 5 of AH6809 (lOMm). Data are expressed as arithmetic means and vertical bars represent s.e.mean.

response appears to be associated with a slight potentiation of the inhibitory component, although this was not statistically significant (Figure 4). Similar effects were found with the less potent EP1-receptor antagonist SC19220 at a concentration of 10 5M.

Discussion

The effect of the EP1- receptor blocking drug AH6809 on responses to sulprostone and iloprost was also investigated. At a concentration of 10paM, approximately 30-80 times its reported pA2 on EP,-receptor containing preparations (Coleman et al., 1985), AH6809 failed to affect dose-effect curves to sulprostone (Figure 5), but it did cause a rightward shift of excitatory dose-effect curves to iloprost (control ED1 (95% CL) = 2.74 (1.03-7.3)nmol and in the presence of AH6809 = 22.8 (19-27)nmol). This shift in the excitatory

V.

751

All prostanoids tested were active on non-pregnant human myometrium, either as excitatory agonists, inhibitors of spontaneous activity or both. The existence of EP-receptors mediating excitation was supported by data obtained with the selective EP,-/EP3-receptor agonist sulprostone, which caused excitation (ED1 = 0.03 nmol) over a similar range to PGE2 . The observation that the mean maximum response obtained with sulprostone was greater than that obtained with PGE2 may be explained by the functional antagonism between dual inhibitory/excitatory response to PGE2. It is possible that the excitatory component is opposed by the inhibitory component resulting in a lower maximum response. Sulprostone, on the other hand, did not cause inhibition at any concentration used and therefore reached its full excitatory potential. Further evidence for EP-receptors mediating excitation, and additionally for EP-receptor-mediated inhibition in nonpregnant human myometrium is provided by the action of the EP2-/EP3-receptor selective prostanoid, rioprostil. Rioprostil, with an ED1 value of 0.02 nmol, was of a similar potency to both PGE2 and sulprostone as an excitatory agonist, and of a similar potency to PGE2 as an inhibitor of spontaneous activity (ID4 = 1.4 and 0.8 nmol respectively). Like PGE2, rioprostil produced a lower mean excitatory response than sulprostone which may result from a functional antagonism of excitation by the inhibitory component of its response. The mean maximum inhibition however, was greater with rioprostil than PGE2 (max inhibition = 33.9 and 21.3 min respectively) and why this should be so is not clear. It is possible that rioprostil is simply more lipophilic than PGE2 and remains in the vicinity of the receptor for a longer time. AY23626 which is also an EP2-/EP3-receptor selective compound had a qualitatively similar profile of activity to both PGE2 and rioprostil. As an inhibitor of spontaneous activity AY23626 was approximately 3 times weaker than both PGE2 and rioprostil. The observed inhibitory response of AY23626 in non-pregnant human myometrium is in line with that found in other tissues where it has been shown to be only 14 or 1.2 times less potent than PGE2 at EP2-receptors in guinea-pig ileum (circular muscle) and cat trachea respectively (Coleman et al., 1987c). The EP2-/EP3-receptor agonist misoprostol showed a biphasic response in this study; at high doses the inhibitory component of this response appeared dose-related (ID4 = 2.11nmol). At lower doses misoprostol is a potent EP3-receptor agonist (Reeves et al., 1988). The EP2-receptor mediated response has been found in cat trachea where misoprostol is approximately 3 to 4 times weaker than PGE2 (Coleman et al., 1988). The compound 16,16-dimethyl PGE2 had a response qualitatively similar to misoprostol but as an inhibitor of spontaneous activity it was found to be weaker (ID4 = 3.53 nmol). 16,16-dimethyl PGE2 is a potent agonist at the EP,-receptor where it has been shown on guinea-pig fundus to be approximately twelve times more potent than PGE2 (Coleman et al., 1988). In addition to its EP1-/EP2-receptoractivity 16,16dimethyl PGE2 also has activity at the EP3-receptor (Reeves et al., 1988). Enprostil, which is a potent EP3-receptor agonist produced purely excitatory responses in non-pregnant human myometrium as previously reported (Clayton et al., 1987). This compound has been reported to mediate action via EP1-receptors also in the guinea-pig fundus preparation where it is approximately two times weaker than PGE2. In addition to its EP-receptor activity, enprostil has moderate

752

J. SENIOR et al.

action on the FP- and TP-receptors which has been observed in dog iris and rat aorta respectively (Coleman et al., 1988). With respect to TP-receptors, we have observed some action with enprostil using selective TP-receptor antagonists (unpublished observation). With the data obtained with sulprostone, rioprostil, misoprostol, enprostil and to a lesser extent with AY23626 and 16,16-dimethyl PGE2, it appears that EP3-receptors may mediate excitation in non-pregnant human myometrium, as activity at this site is common to all three compounds. Sulprostone and rioprostil have been shown to be 0.16 and 1.1 times as potent as PGE2 in guinea-pig vas deferens, and AY23626 approximately 5.5 times as potent in chick ileum, preparations thought to contain a homogeneous population of EP3-receptors (Coleman et al., 1987c; Reeves et al., 1988). However, sulprostone and 16,16-dimethyl PGE2 also have activity at the EP,-receptor and an additional contribution of this site to the excitatory response is possible. An involvement of EP1-receptors is indicated by the excitatory action of the EP1-/IP-receptor selective agonist, iloprost. This compound caused mixed responses in non-pregnant human myometrium of excitatory (ED1 = 2.74 nmol, max. 2.3 x background) and inhibitory (ID4 = 0.94 nmol, max. = 7.6 min) effects most probably mediated via EP,- and IP-receptors respectively. The involvement of EP1-receptors is further supported by the action of the EP1-receptor blocking drug, AH6809 (10pM), which caused an apparent 10 fold shift of dose-effect curves to iloprost. The data obtained with AH6809 and the EP-selective agonists suggest that EP1- and EP3-receptors may co-exist in non-pregnant human myometrium. Iloprost has been shown to be a partial agonist at EP1-receptors on the bullock iris, guinea-pig trachea and rat fundus, but it is approximately equipotent with PGE2 (Dong et al., 1986). Therefore, the rather low potency of iloprost as an excitatory agonist in non-pregnant human myometrium may indi-

cate that only a small population of EP1-receptors are present. Further support for this finding lies in the observation that dose-effect curves to sulprostone were unaffected by AH6809 (10pM). Although this prostanoid possesses potent EP1-receptor activity (Coleman et al., 1988), this does not appear essential for its action in non-pregnant human myometrium. The inhibitory actions of rioprostil, AY23626, misoprostol and 16,16-dimethyl PGE2 which show selectivity for the EP2-receptors and the lack of inhibitory effect with sulprostone and enprostil, which do not possess EP2-receptor activity (Reeves et al., 1988), suggest that EP2-receptors may mediate the observed inhibition of spontaneous activity. The effect of the selective EP2-receptor agonist, butaprost (Gardiner, 1986) also supports the proposal that the inhibitory effect of PGE2 on non-pregnant human myometrium was through the EP2-receptor. The data presented in this paper are consistent with the presence in non-pregnant human myometrium of a heterogeneous population of prostanoid receptors of the EP-type mediating both excitation and relaxation of spontaneous activity. Based on data obtained by use of the receptor selective agonists and the antagonist AH6809, it is suggested that the prostanoid receptor subtypes present on non-pregnant human myometrium, which mediate excitation, are EP1 and EP3-receptors with a greater proportion of the EP3-subtype being present in this tissue, and those which mediate inhibition are of the EP2-receptor subtype. We thank all suppliers of compounds listed in Table 1. G.S.B. was supported by the Glaxo Group Research Ltd., Ware. We are grateful to Dr R.A. Coleman of Glaxo Group Research Ltd., Ware and Dr R.L. Jones of the Pharmacology Department, University of Edinburgh for discussion. We also thank Dr P.J. Gardiner of Bayer (U.K.) Ltd for the first EP-selective agonist.

References BAXTER, G.S., CLAYTON, J.K., COLEMAN, R.A., MARSHALL, K.M. &

COLEMAN, R.A., HUMPHRAY, J.M., SHELDRICK, R.L.G. & WHITE, B.P.

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(Received June 27, 1990 Revised November 7, 1990 Accepted November 14, 1990)

In vitro characterization of prostanoid EP-receptors in the non-pregnant human myometrium.

1. Prostaglandin receptors of the PGE type have been characterized in the non-pregnant human myometrium in vitro according to the scheme of Coleman et...
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