Stimulatory (EP1 and EP3) and inhibitory (EP2) prostaglandin E2 receptors in isolated ileal smooth muscle cells

European Journal of Pharmacology, 237 (1993) 131-137 © 1993 Elsevier Science Publishers B.V. All rights reserved 0014-2999/93/$06.00

13 I

EJP 53092

Stimulatory (EP 1 and EP 3) and inhibitory (EP 2) prostaglandin E 2 receptors in isolated ileal smooth muscle cells A l a i n B o t e l l a a, M i c h e l D e l v a u x a,b, J e a n F i o r a m o n t i a, J a c q u e s F r e x i n o s ~,b a n d L i o n e l B u e n o ~' '~ Department of Pharmacology, INRA, B.P. 3, F-31931 Toulouse, France and h Laboratory of Digestice MotiliO,, CHU Rangueil, F-31054 Toulouse, France Received 25 June 1992, revised MS received 15 February 1993, accepted 2 March 1993

Isolated smooth muscle cells from the circular layer of pig and guinea-pig ileum were used to study the effect of prostaglandin E 2 (PGE 2) and three PGE 2 receptor (EP) agonists; iloprost (EP~), butaprost (EP~) and enprostil (EP3). In pig cells, PGE 2 and enprostil induced cell contraction (22.1 and 21.5% shortening of cell length, obtained at 10 nM for PGE 2 and 1 nM for cnprostil, respectively). Iloprost and butaprost had no contractile effect. However, the cholecystokinin octapeptide (CCK-8; 10 nM)-induced contraction was inhibited when cells were preincubated with iloprost or butaprost. In guinea-pig cells, PGE 2, butaprost and iloprost induced cell contraction, whereas enprostil had no effect (23.1% for 10 nM PGE 2, 22.8% for 1 nM butaprost and 22.6% for 10 nM iloprost). Preincubation with SC19220 (EPI antagonist) inhibited the PGE2-, butaprost- and iloprost-induced contractions. When the contractile effect of PGE 2, butaprost and iloprost was inhibited by addition of SC19220, these agents inhibited the cell contraction induced by CCK-8 (1 riM). Smooth muscle cells from guinea-pig and pig ileum express two PGE 2 receptor subtypes that induce opposite effects. EP 1 and EP 3 receptors mediate cell contraction in guinea-pig and pig, respectively, whereas EP 2 receptors mediate cell relaxation in both species. PGE2; Smooth muscle cells; EP receptors

1. Introduction

Prostaglandins (PG) are synthesized in large amounts and are widely distributed throughout the gastrointestinal tract (Ahlquist et al., 1982; Wallace et al., 1984; Whittle and Vane, 1987). There is increasing evidence that PG act as local regulatory agents controlling smooth muscle contractile activity at different levels of the digestive tract, in particular the small intestine (Bueno et al., 1985; Sanders, 1984; Staumont et al., 1990). This action is extremely variable, depending on the type of PG, the concentration, the organ, the species, and even the muscle layer studied (Eglen and Whiting, 1988; Gardiner, 1986; Staumont et al., 1990). PG of the Ez type have been shown to contract intestinal longitudinal smooth muscle and relax circular smooth muscle in various animal species (Gardiner, 1986; Sanders, 1981). P G E 2 acts through specific receptors which have been subdivided into EP 1, EP 2 and EP 3 subtypes on the basis of the relative potency of selective agonists and antagonists (Coleman et al., 1985; Coleman et al.,

Correspondence to: M. Delvaux, Department of Pharmacology, INRA, B.P. 3, F-31931 Toulouse C~dex, France. Fax 33.51 28 53 10.

1987a; Coleman et al., 1987b) in both functional and binding studies (Senior et al., 1991). To date, butaprost (TR 4979) appears to be the most selective agonist for the EP 2 receptor subtype (Gardiner, 1990). Enprostil, a synthetic dehydroprostaglandin E 2, is more active at the EP 3 receptor (Coleman et al., 1988), while iloprost displays potent EP 1 activity (Sheldrick et al., 1988). SC19220 is an antagonist known to block the EP~ receptor (Sanner, 1972), but as yet antagonists for EP 2 and EP 3 receptors have not been described. In vitro experiments have shown that EP l receptors mediate contraction of the longitudinal smooth muscle from guinea-pig ileum, fundus and trachea (Coleman et al., 1985). EP 2 receptors mediate relaxation of cat and guinea-pig trachea (Coleman et al., 1986). EP3 receptors trigger contraction of the chicken ileum (Coleman et al., 1987c). Previous studies have shown that P G E 2 influences intestinal motility by acting at a neuronal level (Bueno et al., 1985; Fargeas et al., 1984). In the present study, we investigated the possibility of a direct myogenic effect of P G E 2 on intestinal smooth muscle ceils. We studied the effects of P G E 2 and their various agonists on cells isolated from the circular muscle layer of pig and guinea-pig ileum. The effects observed were compared to those of the extensively studied contracting

132 agent (CCK-8) of these cells. EP receptor subtypes involved in these effects were then characterized.

2. Materials and methods

2.1. Material Stock solutions of prostanoids were prepared in ethanol and stored at -20°C. The prostanoids were obtained from Schering (iloprost), Bayer (butaprost) and Syntex (enprostil). SC19220 (1-acetyl-2-(8-chloro10,11-dihydrodibenz(b,f)(1,4)oxazepine-10-carbonyl)hydrazine) was a gift from Searle, USA. Pronase was purchased from Boehringer Mannheim Ltd (Meylan, France). Penicillin G and Streptomycin G were from Specia (Paris, France). Collagenases (Type I, IV, V), sulfated C-terminal octapeptide of CCK (CCK-8), PGE2, vasoactive intestinal peptide (VIP) and all other reagents were obtained from Sigma (St Louis, MO, USA).

2.2. Cell dispersion 2.2.1. Cell dispersion from pig ileum Cells were dispersed as described previously (Delvaux et al., 1991). Smooth muscle cells were isolated from the circular muscle layer of the ileum of a 6month-old large white male pig. Muscle strips were removed from an area 10 cm proximal to the ileo-caecal junction. After removal of serosa, longitudinal muscle and mucosa-submucosa layers, small muscle strips from the circular muscle layer were incubated for two successive periods of 30 min at 31°C in a medium (132 mM NaC1, 5.4 mM KC1, 5 mM N a 2 H P O 4, 1 mM NaHzPO4, 1.2 mM MgSO4, 1 mM CaC12, 25 mM Hepes, 0.2% glucose (w/v), 0.2% bovine serum albumin (w/v); pH 7.4, bubbled with 95% 02-5% CO 2 and supplemented with antibiotics, penicillin G 100 I U / m l and streptomycin 50 # g / m l ) containing 0.38 I U / m l collagenase (type I), 0.2 m g / m l pronase and 0.2 m g / m l soybean trypsin inhibitor. At the end of the second incubation, the medium was filtered and the party digested muscle strips were washed four times with enzyme-free medium. These strips were then transferred to fresh enzyme-free medium and left to stand for 20 rain to allow the muscle cells to disperse spontaneously under very slow mechanical agitation. Cells were harvested through a 500-~m nylon filter. It should be emphasized that only those cells that had dissociated spontaneously in enzyme-free medium were used for functional measurements. 2.2.2. Cell dispersion from guinea-pig ileum Cell dispersion was as described previously (Botella et al., 1992a). Intestinal samples were obtained from an

albino male guinea-pig (250-300 g body weight). After removal of serosa, longitudinal muscle layer and mucosa-submucosa layers from ileum, small muscle strips from the circular muscle layer were incubated for two periods of 15 min at 31°C in the same medium as used for pig strips, containing collagenase 0.1 m g / m l (type IV) and 0.1 m g / m l (type V). At the end of the second incubation, the medium was filtered and the partly digested muscle strips were washed four times with enzyme-free medium. These strips were then transferred to fresh enzyme-free medium and left to stand for 10 min to allow the muscle cells to disperse spontaneously under very slow mechanical agitation. Cells were harvested through a 500-/~m nylon filter. It should be emphasized that only those cells that had dissociated spontaneously in enzyme-free medium were used for functional measurements.

2.3. Measurement of contractile response Cell suspensions were usually studied within 30 min of dispersion. The density of the suspension was about 250,000 cells/ml, as assessed by cell counting on a Malassez slide. Aliquots of 250 /.tl of cell suspension were added to 250/M of solution containing the agent to be tested, thereby ensuring rapid mixing, and incubated for 30 s at 3 I°C. The reaction was interrupted by addition of glutaraldehyde to a final concentration of 2.5%. In control experiments, 250/,1 of the same medium was substituted for the test agent. To measure cell length, an aliquot of cells fixed with glutaraldehyde was placed on a Malassez slide and the length of the first 50 cells encountered at random in successive microscopic fields was measured. Only whole undisrupted cells were measured.

2.4. Experiments of inhibition or relaxation For relaxation experiments, cells were first incubated in the presence of various concentrations of relaxing agent or antagonist for 1 min and the contracting agent was then added for 30 s, as described previously (McHenry et al., 1991). At the end of the incubation, cells were fixed by addition of glutaraldehyde as described above.

2.5. Expression of results The contractile response is defined as the decrease in the average cell length of a population of muscle cells exposed to a test agent in comparison to controls. Cell contraction is expressed as the percentage decrease in cell length from control. The decrease in cell length was calculated using the following formula: ((LO - L x ) / L O ) * 100, where LO is the mean length of

133 In order to demonstrate the direct action of P G E 2 and CCK-8 on smooth muscle cells, the effect of tetrodotoxin (TTX) (10 ~zM) was evaluated on the contraction elicited by P G E 2 and CCK-8 of smooth muscle cells isolated from pig and guinea-pig ileum. T T X failed to inhibit cell contraction induced by P G E 2 (10 nM) as well as that induced by CCK-8 (10 nM) in both species (data not shown).

cells during the resting state and Lx, the mean length of treated ceils. In relaxation experiments, the extent to inhibition is expressed as the percentage decrease in the contractile response from the maximal response observed in the absence of inhibitors, taken as 100%. Throughout this paper, n refers to the number of experiments performed with cells from different animals. Statistical evaluation was done with Student's t-test, and the normality of the cell samples was assessed by the normal law test.

3.2. Contractile effect of t'arious synthetic EP-receptor agonists on isolated smooth muscle cells Enprostil contracted cells isolated from pig ileum in a concentration-dependent manner, while butaprost and iloprost had no effect. The maximal contraction induced by enprostil was observed at 1 nM and corresponded to a 21.5 + 2.1% decrease in cell length from control. The ECs0 value was 80 pM (fig. 2A; mean _+ S.E.M. of five separate experiments with cells from different animals). In contrast, iloprost and butaprost contracted cells isolated from guinea-pig ileum in a concentration-dependent manner, while enprostil had no effect. The maximal contraction was observed at 10 nM for iloprost and 1 nM for butaprost, corresponding to a 22.6 +_ 2.2 and 22.8 +_ 3.6% decrease in cell length from control, respectively. The ECs0 value was 20 pM for iloprost and 4 pM for butaprost (fig. 2B; mean _+ S.E.M. of five separate experiments with cells from different animals).

3. Results

3.1. Effect of PGE 2 and CCK-8 on isolated smooth muscle cells from pig and guinea-pig ileum P G E 2 and CCK-8 contracted cells isolated from pig ileum in a concentration-dependent manner. The maximal contraction was observed at 10 nM P G E 2 and 10 nM CCK-8 and corresponded to a 22.1 + 1.8 and 24.6 _+ 1.4% decrease in cell length from control, respectively. The concentration inducing a half-maximal contraction (ECs0) was 100 pM for P G E 2 and 80 pM for CCK-8 (fig. 1A; mean _+ S.E.M. of five separate experiments with cells from different animals). P G E 2 and CCK-8 contracted cells isolated from guinea-pig ileum in a concentration-dependent manner. The maximal contraction was observed at 10 nM P G E 2 and 1 nM CCK-8 and corresponded to a 23.1 + 2.1 and 24.8 _+ 2.6% decrease in cell length from control, respectively. The ECs0 value was 50 pM for P G E 2 and 8 pM for CCK-8 (fig. 1B; mean _+ S.E.M. of five separate experiments with cells from different animals).

3.3. Effect of SC19220 on contraction induced by PGE 2 and their analogs In cells isolated from pig ileum, SC19220 failed to inhibit the contraction induced by enprostil (1 nM) at

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Fig. 2. Cell contraction induced by three agonists of EP-receptors in isolated circular smooth muscle cell from pig (A) and guinea-pig (B) ileum. Cells were stimulated for 30 s in the presence of various concentrations of iloprost ( • ) , enprostil (*) or butaprost ([]) at 3 I°C. Cell contraction is expressed as the percentage decrease in cell length from control. Points are means+S.E.M, for five experiments with cells from different animals.

c o n c e n t r a t i o n s r a n g i n g f r o m 1 p M to 100/.tM ( d a t a not shown). In cells i s o l a t e d f r o m g u i n e a - p i g ileum, SC19220 i n h i b i t e d t h e c o n t r a c t i o n i n d u c e d by P G E 2 (10 nM), iloprost (10 n M ) a n d b u t a p r o s t (1 n M ) in a c o n c e n t r a t i o n - d e p e n d e n t m a n n e r (fig. 3; m e a n +_ S.E.M. o f five s e p a r a t e e x p e r i m e n t s with cells f r o m d i f f e r e n t animals). C o n c e n t r a t i o n s i n d u c i n g a half-maximal inhibition w e r e 100, 8 a n d 40 p M in the p r e s e n c e of P G E 2, i l o p r o s t a n d b u t a p r o s t , respectively. T h e PGE2-, iloprost- a n d b u t a p r o s t - i n d u c e d c o n t r a c t i o n s w e r e a b o l i s h e d at 1 /xM, 10 n M a n d 1 /,tM of SC 19220, respectively. M o r e o v e r , t h e specificity of SC19200 was a s s a y e d a g a i n s t the c o n t r a c t i o n i n d u c e d by 10 n M C C K - 8 in b o t h species. SC 19220 failed to inhibit the c o n t r a c t i o n i n d u c e d by 10 n M C C K - 8 at c o n c e n t r a t i o n s r a n g i n g from 10 fM to 1 ~ M . T h e results o b t a i n e d with cells i s o l a t e d from g u i n e a - p i g ileum are shown in fig. 3.

was 100 p M for P G E 2 , 3 n M for iloprost and 6 n M for b u t a p r o s t and the maximal inhibition was o b s e r v e d at 100/,tM P G E 2 , iloprost a n d b u t a p r o s t (fig. 4B; m e a n _+ S.E.M. of five s e p a r a t e e x p e r i m e n t s with cells from d i f f e r e n t animals).

4. Discussion In this study, we d e m o n s t r a t e that P G E 2 can contract o r relax s m o o t h muscle of the ileum by acting directly at the level of muscle cells. T h e s e effects of

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I n c u b a t i o n of cells i s o l a t e d from pig i l e u m for 1 min with i n c r e a s i n g c o n c e n t r a t i o n s of i l o p r o s t or b u t a p r o s t i n h i b i t e d the c o n t r a c t i o n i n d u c e d by C C K - 8 (10 n M ) in a c o n c e n t r a t i o n - d e p e n d e n t m a n n e r . T h e ICs0 value was 60 p M for iloprost a n d 20 p M for b u t a p r o s t . T h e C C K - 8 i n d u c e d - c o n t r a c t i o n was a b o l i s h e d at a c o n c e n t r a t i o n of 1 # M for b o t h a g e n t s (fig. 4A; m e a n _+ S.E.M. o f five s e p a r a t e e x p e r i m e n t s f r o m d i f f e r e n t animals). In cells i s o l a t e d from g u i n e a - p i g ileum, w h e n t h e c o n t r a c t i o n s i n d u c e d by P G E 2, i l o p r o s t a n d b u t a p r o s t w e r e i n h i b i t e d by a d d i t i o n of 1 /zM SC19220, P G E 2, i l o p r o s t a n d b u t a p r o s t i n h i b i t e d cell c o n t r a c t i o n ind u c e d by C C K - 8 (1 n M ) in a c o n c e n t r a t i o n - d e p e n d e n t m a n n e r , while e n p r o s t i l h a d no effect. T h e ICs0 value

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Fig. 3. Inhibition by the specific antagonist of the EP~ receptor SC19220 of cell contraction induced by PGE 2 (*), iloprost ([]), butaprost ( I ) and CCK-8 (©) in cells isolated from guinea-pig ileum• Cells were preincubated for 1 min at 31°C in the presence of various concentrations of SC19220 and then the contracting agent (10 riM) was added for 30 s. Results are expressed as the percentage cell contraction observed in the absence of antagonist, taken as 100%. Points are means+_ S.E.M. for five separate experiments.

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Fig. 4. Effects of P G E 2 a n d / o r agonists of the EP-receptor on CCK-8-induced contraction of circular smooth muscle cells isolated from pig and guinea-pig ileum. Cells from pig ileum were preincubated for 1 min at 31°C in the presence of various concentrations of butaprost ( , ) or iloprost ([:3) and then the contracting agent (CCK-8 10 nM) was added for 30 s (fig. 4A). Cells from guinea-pig ileum were preincubated for 1 rain at 3l°C in the presence of butaprost (U), enprostil ( , ) , iloprost (11) or P G E 2 (~>), and then the contracting agent (CCK-8 l n M ) was added for 30 s (fig. 4B). The results are expressed as the percentage cell contraction observed in the absence of P G E 2 or the EP agonists, taken as 100%. Points are m e a n s + S.E.M. of five separate experiments.

P G E 2 are not sensitive to TTX, a selective blocker of nerve depolarization, ruling out a possible indirect effect of P G E 2 via a neurogenic pathway. Membrane receptors for various hormones and neurotransmitters have been described on isolated smooth muscle cells from small intestine, with some receptors eliciting contraction and others eliciting relaxation (Berry et al., 1991; Makhlouf, 1987). Isolated cells have been used for many years to study receptors. The stoichiometry of the contraction in this model is characterized by the following main features (Bitar and Makhlouf, 1982a, b; Collins and Gardner, 1982): (i) broad dose-response curves, (ii) high sensitivity to agonists with significant responses in the range of 0.01 to 1 pM, (iii) maximal responses that are similar for peptides and non-peptide agonists, (iv) a tendency for the response to decrease at supramaximal doses. The high sensitivity to agonists is one feature of the response of isolated smooth muscle cells that is not found in smooth muscle strips, and it implies the presence and operation of high-affinity receptors. A possible explanation for the exclusive presence of these receptors in isolated cells is the lack of neurohormonal circuits in these preparations (Makhlouf, 1987). In the present study, we observed that P G E 2 and its agonists and antagonists were active at concentrations typical for the model used. Consequently, comparison of the present results with those previously obtained for muscle strips in terms of potency of the drugs is too difficult and will not be discussed further. Nevertheless, previous studies led to similar conclusions about the receptor types involved by PGE2, as discussed below.

The present results strongly suggest that receptors for P G E 2 are also present on muscle cells of the guinea-pig and pig ileum. Indeed, the concentrations at which P G E 2 and the synthetic analogs contracted the cells are in the same range as those observed for other stimulating agents on the same cell type (Delvaux et al., 1991; Berry et al., 1991) and in other cell types (Makhlouf, 1987; Bitar and Makhlouf, 1982a). These concentrations are compatible with a membrane receptor-mediated action (Coleman et al., 1985; MacIntyre, 1985). Furthermore, the concentration-response curve showed a slight decrease in contractile response at supramaximal concentrations, a pattern frequently observed in receptor-mediated actions (Berry et al., 1991; Grider and Makhlouf, 1987; Botella et al., 1992a, b). Moreover, SC19220, a specific antagonist of the P G E 2 receptor (EP 1 subtype), inhibited the effect of P G E 2 and its synthetic analogs in the guinea-pig. PGE2 receptors have been divided into three subtypes, according to their selective affinity for various synthetic analogs (Coleman et al., 1985; Coleman et al., 1987a; Coleman et al., 1987b; Senior et al., 1991). The results of previous studies suggest that different receptor subtypes could be involved in the effect of P G E 2 on digestive motility, depending on the organ and the species studied (Staumont et al., 1990; Coleman et al., 1985; Gardiner, 1986; Horton and Main, 1963; Clegg, 1966; Bennet et al., 1975; Sanders, 1978; Sanders, 1981). The EP receptor subtypes involved in the myogenic contractile effect of P G E 2 are different in pig and guinea-pig ileum. In guinea-pig ileum, we observed the presence of an EP 1 receptor: P G E 2, butaprost and iloprost, the most EP1 selective agonist, induced a

136

contraction, while the EP 3 agonist enprostil did not. The EPI antagonist SC19220 selectively inhibited the PGE2-, butaprost- and iloprost- induced contractions, in a concentration-dependent manner. These results are in agreement with previous results suggesting the presence of EP 1 receptors in smooth muscle of the guinea-pig ileum, since the response to prostanoids was selectively antagonized by the EP t antagonists A H 6809 and SC19220 (Coleman et al., 1985). Moreover, the complete inhibition of the iloprost-induced contraction by SC19220 ruled out a side-effect of iloprost on IP receptors in this model, despite the fact that, in other cell types, iloprost has been shown to be as potent an agonist at IP receptors as at EP1 receptors (Sheldrick et al., 1988). The contractile effect of P G E 2 on cells isolated from pig ileum has not been previously described. In the present study, only P G E 2 and enprostil induced cell contraction, while iloprost and butaprost did not. This effect was not antagonized by SC19220, indicating that, in pig ileum, the PGE2 receptor mediating contraction resembles that mediating contraction in chicken ileum, namely, the EP3-receptor (Coleman et al., 1988). In further experiments, we studied the relaxing effects of P G E 2 and its synthetic analogs in both species. EP 2 receptors are known to mediate relaxation in cat and guinea-pig trachea (Coleman et al., 1986) and in chicken ileum smooth muscle (Coleman et al., 1987c). In cells isolated from pig ileum, both iloprost and butaprost inhibited the CCK-8-induced contraction. Similarly, in cells isolated from guinea-pig ileum, after inhibition of the PGE2-, butaprost- and iloprost-induced contractions by blockade of EP~ receptor with SC19220, P G E 2, iloprost and butaprost inhibited the contraction induced by CCK-8 (10 nM) in a concentration-dependent manner. However, since an antagonist of EP 3 receptors is not available, similar experiments could not be performed with cells isolated from pig ileum. In the absence of inhibition of the direct contractile effect of P G E 2 or enprostil in these cells, we could not investigate whether P G E 2 and enprostil were able to inhibit the CCK-8-induced contraction. The receptor subtype which mediates cell relaxation displays the pharmacological properties of the EP 2 receptor. These results suggest that smooth muscle cells from pig and guinea-pig ileum express two types of receptor for P G E 2, one mediating contraction and showing species specificity, and the other mediating relaxation; the latter receptor was of the EP 2 type in both species studied. The specificity of action of the various agonists at the different EP receptors is not absolute. This observation was especially obvious for butaprost, which had an EC50 value of 4 pM at the EP1 receptor but an EC50 value of 6 nM at the EP2 receptor. This differ-

ence of 3 log units in potency is quite different from the results obtained with P G E 2, which displayed quite similar potency in inducing both contraction (50 pM) and relaxation (100 pM). These results suggest that the synthetic agonists may vary in potency when interacting with the various EP receptor subtypes because of many factors, such as access to the receptors and differential unmasking of the receptors in isolated cells. The rather similar potency of the natural ligand P G E 2 for all types of receptor shows that these receptors have strict EP properties. In the literature, the presence of two receptors for one hormone or neurotransmitter on the same cell type has been described only for histamine (Morini et al., 1985; Waldman et al., 1977) and serotonin (Kuemmerle et al., 1990). Indeed studies with smooth muscle cells demonstrated that histamine induces cell contraction via the H 1 receptor and relaxation via the H2 receptor after blockade of the H~ receptor (Morini et al., 1985; Waldman et al., 1977). This situation is quite similar to what we observed with P G E 2. The receptor responsible for contraction, being species specific, could predominant, while the relaxing effect could be mediated by a less predominant receptor whose effect is observed only after blockade of the predominant one. Results obtained from in vivo or in vitro studies with whole organs have shown that the regulation of digestive motility by P G E 2 is the sum of several effects. Most of the time, P G E 2 induces contraction of the longitudinal muscle layer and relaxation of the circular muscle layer (Bennett et al., 1975; Sanders, 1981; Coleman et al., 1987c). However, in some experiments, P G E 2 has been observed to contract the circular muscle layer (Mishima and Kuriyama, 1976; Sakamoto and Nasu, 1987). In the present study, we showed that the myogenic effects of PGE2 results in a complex regulation involving several receptors types, each of which mediates a specific effect.

Acknowledgements This work was supported by grants from the Fondation pour la Recherche Mddicale and from the Conseil R6gional Midi-Pyr6n6es. The authors thank the staff of the St. Gaudens slaughter-house for providing gut samples from pig.

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