Two types of endothelin B receptors mediating relaxation in the guinea pig ileum

Life Sciences, Vol. 54, No. 10, pp. 645-652, 1994 Copyright © 1994 Elsevier Science Ltd Printed in the USA. All rights reserved OO24-32O5/94 $6.O0 + .00

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TWO TYPES OF ENDOTHELIN B RECEPTORS MEDIATING R E L A X A T I O N IN T H E G U I N E A P I G I L E U M Masatoshi Hori, Sri Agus Sudjarwo, Kyoko Oda*, Yoshihiro Urade* and Hideaki Karaki Department of Veterinary Pharmacology, Faculty of Agriculture, The University of Tokyo, Bunkyo-ku, Tokyo 113 and *International Research Laboratories, Ciba-Geigy Japan, Takarazuka 665, Japan (Received in final form December 13, 1993) Summary In guinea pig ileum, binding assays showed the existence of endothelin (ET) receptors of ETA (isopeptide-selective) and ETB (nonselective) subtypes. ETs induced relaxation followed by contraction. ET-1 induced greater contraction at lower concentrations than ET-3. An ETA antagonist, BQ-123, shifted the concentration-response curves for ETs to the right. An ETB antagonist, IRL 1038, shifted the concentration-response curve for ET-3 to the right and downwards with little effect on the curve for ET-I. In contrast, ET-1 and ET-3 induced relaxation at similar concentrations. The relaxation induced by ETs was composed of an initial transient relaxation followed by sustained relaxation. Only the transient phase was inhibited by IRL 1038 in a concentration-dependent manner. These results suggest that the ET-induced relaxation is mediated by two types of ETB receptor; transient and sustained relaxations are mediated respectively by IRL 1038-sensitive and IRL 1038-insensitive subtypes of ETB receptor. In contrast, the contractile effect seems to be mediated mainly by the ETA receptor and partially by an IRL 1038-sensitive subtype of ET8 receptor. It has been shown that the family of potent vasoconstrictor peptides, endothelins (ETs) (1), activates two types of receptor. The ETA receptor is relatively selectively activated by ET-1 (2) and selectively inhibited by BQ-123 (3). In contrast, the ETB receptor is activated nonselectively by ET-1, ET-3 (4) and an ETB-selective agonist, IRL 1620 (5,6), and selectively inhibited by IRL 1038 (7,8). In arteries, the ETA receptor mediates the contractile effects (1,2) whereas the ETB receptor mediates the relaxant effects of ETs on the smooth muscle. The relaxant effects of ETs are mediated via release of a relaxant factor, nitric oxide, from the vascular endothelium (9-12). In the intestinal smooth muscle of guinea pig ileum, ETs cause transient relaxation (13-16). The ETB receptor seems to mediate this relaxation because ET-1 and ET-3 show similar potency (15,16). Following the transient relaxation, ETs induce contraction; ET-1 induces greater contraction at lower concentrations than ET-3 (1416), suggesting the involvement of the ETA receptor. However, part of the contractile effect is inhibited by the ETB antagonist, IRL 1038, suggesting that the ETa receptor

Correspondence to: H. Karaki, Department of Veterinary Pharmacology, Faculty of Agriculture, The University of Tokyo, Yayoi, Bunkyo-ku, Tokyo 113, Japan.

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may also mediate contraction (7). To further characterize the ET receptors involved in the regulation of intestinal movement, we examined the effects of agonists and antagonists of ETA and ETB receptors in the isolated guinea pig ileum. Methods

Male, white guinea pigs were killed by a sharp blow to the neck and exsanguination. The ileum was removed and the longitudinal muscle layer isolated. Physiological salt solution contained (mM): NaCI 136.9, KCI 5.4, CaCI2 1.5, MgCI2 1.0, NaHCO3 23.8, EDTA 0.01 and glucose 5.5. High K+ solution was made by adding 40 mM KCI. These solutions were saturated with 95% 02 and 5% CO2 mixture at 37oC to maintain the pH at 7.4. Force of contraction was recorded isometrically. Muscle strips, 2-3 cm long, were attached to a holder under a resting force of 5 mN and equilibrated for 60-90 min. During this period, high K+ was applied repeatedly until the peak force was reproducible. To examine the contractile effects, agonist was added cumulatively. Relaxant effects were examined by a single application of agonist during the sustained contraction induced by 100 nM carbachol, because the relaxant effect was observed only transiently and the cumulative addition of ET did not induce graded relaxation. In both experiments for contraction and relaxation, antagonist was added 20 min before the addition of agonist. The plasma membrane of ileal longitudinal smooth muscle (2 ~g of protein) was incubated at 37oC for 1 h with 50 pM [1251]ET-1 or [1251]ET-3 in the presence or absence of various concentrations of unlabeled ligands in a total volume of 1 ml of 20 mM HEPES (pH 7.4), 145 mM NaCI, 5 mM KCI, 3 mM MgCI2, 1 mM EGTA, 1 mg/ml bovine serum albumin and 0.2 mg/ml bacitracin. After the incubation, unbound [1251]ETs were separated by centrifugation and the radioactivity in the membrane pellet was measured. Nonspecific binding was defined as the membrane-associated radioactivity determined in the presence of a saturating concentration (200 nM) of ETs. Nonspecific binding (approximately 20% of the total binding) was subtracted from the total binding measured in the absence of unlabeled ligands. The difference was defined as specific binding. Total binding was always less than 5 % of the total radioactivity added. For Scatchard analysis, the binding to the ETB receptor was measured with 5 to 90 pM [1251]ET-1 or [1251]ET-3. The binding to the ETA receptor was determined with 5 to 180 pM [1251]ET-1 in the presence of 100 nM unlabeled IRL 1620, which masked the ET8 receptor binding sites. Agonists and antagonists for ET receptors used in the present experiments were as follows: ET-1, a nonselective agonist of the ETA and ETB receptors; ET-3 and IRL 1620 (5,6), selective agonists of the ETB receptor; BQ-123 (3), a selective antagonist of the ETA receptor; and IRL 1038 (7,8), a selective antagonist of the ETB receptor. ET-1 and ET-3 were purchased from the Peptide Institute (Osaka, Japan). IRL 1620 {Suc[Glug, Ala11,15] ET-1 (8-21)}, IRL 1038 {[Cysll-Cys is] ET-1 (11-21)} and BQ-123 were synthesized in our laboratory. [1251]ET-1 and [1251]ET-3 (74 TBq/mmol) were purchased from Du-Pont New England Nuclear (Boston, MA, U.S.A.). Carbachol was purchased from Wako Pure Chemicals, Tokyo, Japan. Each experiment was repeated in at least four preparations isolated from different animals. Results of the experiments are plotted as a logarithmic concentrationresponse relationship. The concentration needed to induce the half-maximum response and S.E.M. were expressed as mean + S.E.M. in -log M. In some cases, these values were converted to linear values and expressed as the geometric mean with its corresponding S.E. range in pM or nM. Student's t - test was used for the statistical analysis of the results. P values equal to or less than 0.05 were considered to be significant.

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Results Saturable and specific binding sites for [1251]ET-1 and [1251]ET-3 were detected in membranes of the longitudinal smooth muscle of guinea pig ileum. In competitive binding assay with 50 pM [1251]ET-3 (Fig. 1A), unlabeled ET-1, ET-3, IRL 1620, IRL 1038 and BQ-123 showed monophasic inhibition curves. The half-maximum inhibitory concentration (IC5o) was 40 pM for ET-1, 40 pM for ET-3, 200 pM for IRL 1620, 26 nM for IRL 1038 and 5.7 #M for BQ-123. When 50 pM [1251]ET-1 was used, unlabeled ET-1 also showed a monophasic inhibition curve with an IC5o of 60 pM whereas unlabeled ET-3, IRL 1620, IRL 1038 and BQ-123 showed biphasic curves (Fig. 1B). About 80 % of the [1251]ET-1 binding was displaced by lower concentrations of ET-3 (<1 nM), IRL 1620 (<10 nM) and IRL 1038 (<1 I~M), while the remaining 20 % was by higher concentrations of ET-3 (>1 nM), IRL 1620 (>10 nM) and IRL 1038 (>1 I~M). The former binding sites, which were considered to be ETa receptors, were displaced by higher concentrations of BQ-123 (>1 #M) and the latter binding sites were ETA receptors displaced by lower concentrations of BQ-123 (<1 I~M). By Scatchard analysis, the

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012 11 10 9 8 7 6 5 Unlabeled ligands added (-log M)

Competitive binding of 50 pM [125I]ET-3 (A) and [1251]ET-1 (B) to the guinea pig ileal membrane by unlabeled ET-I (O), ET-3 (e), IRL 1620 (A), IRL 1038 (A) and BQ-123 (El). Each value is expressed as a percentage of the specific binding. A

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Scatchard analysis of the specific binding of [1251]ET-1 in the absence (O) or presence (e) of 100 nM IRL 1620. The Scatchard plot of [125I]ET-1 binding in the presence of 100 nM IRL 1620 is shown on an expanded scale.

binding of [1251]ET-1 (Fig. 2) and [1251]ET-3 (not shown) to ETB receptors showed linear regression lines with dissociation constants (Kd) of 10 and 15 pM, and maximum binding capacities (Bmax) of 790 and 950 fmol/mg of protein, respectively. On the other hand, the binding of [1251]ET-1 to the ETA receptor, which was measured in the presence of 100 nM IRL 1620 to mask the binding sites of ETa receptors, also showed

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ETB Receptor Subtypes in Ileum

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a linear regression line with Kd of 90 pM and Bmax of 360 fmol/mg of protein. Based on the Kd value for [1251]ET-3 (15 pM), the apparent binding inhibitory constant (Ki) of ETa receptors for each analog was calculated from the IC5o to be 9 pM for ET-1, 9 pM for ET-3, 50 pM for IRL 1620, 6 nM for IRL 1038 and 1.3 #M for BQ-123. The Ki values of ETA receptors for these analogs were about 40 pM for ET-1, 40 nM for ET-3, 300 nM for IRL 1620, 2 I~M for IRL 1038 and 50 nM for BQ-123. In the resting ileum, spontaneous rhythmic contraction was observed. Addition of 30 nM ET-1 transiently inhibited the spontaneous contraction followed by an increase in muscle tone (Fig. 3A). ET-3 and IRL 1620 (100 nM each) showed similar effects as ET-1 although the contractile effect was weaker than that of ET-I. Fig. 4 shows the concentration-response relationship for the contractile effects of ET-1, ET-3 and IRL 1620. Concentrations needed to induce a half-maximum contraction (EC50) are listed in Table 1. The maximum level of contraction induced by ET-1 was greater than that induced by ET-3 or IRL 1620. In addition, the EC5o value for ET-1 (1.2 nM) was approximately 30 times lower than that for ET-3 (32 riM) or IRL 1620 (24 nM). In the presence of 3 I.tM BQ-123, the concentration-response curve for ET-1 was shifted to higher concentrations by more than 100-fold (EC5o > 100 nM). In contrast, contractions induced by 300 nM ET-3 and 100 nM IRL 1620 were not inhibited by 3 #M BQ-123 (n=4 each, data not shown). In the presence of 3 #M IRL 1038, the contractile effects of lower concentrations of ET-1 (<1 nM) were inhibited. In contrast, 3 #M IRL 1038 shifted the concentration-response curve for ET-3 to approximately 10-fold higher concentrations (EC5o = 281 nM) and inhibited the maximum contraction (Fig. 4 and Table 1). The contractile effect of IRL 1620 was inhibited by IRL 1038 in a similar manner to ET-3 (Table 1).

A

ET-1.

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CCh

BQ-123

BQ-123 + IRL1038

FIG. 3 Effects of ET-1 in guinea pig ileum. ET-I (30 nM) was added in resting (A) or 100 nM carbachol (CCh)-stimulatexi ileum in the absence (B) or in the presence of 3 I~M BQ-123 (C, D) and 3 ~M IRL 1038 (D). Traced from typical result out of 4 experiments.

As shown in Fig. 3B-D, addition of 100 nM carbachol induced sustained contraction in the ileum. Subsequent addition of 30 nM ET-1 induced transient relaxation which rapidly returned to a level higher than that before the addition of ET-1 (Fig. 3B). ET-3 and IRL 1620 also induced transient relaxation in carbachol-stimulated ileum. The maximum relaxation induced by these peptides was approximately 60 % of the carbachol-induced contraction. Fig. 5A shows the concentration-response relationship for the relaxant effects of ET-1, ET-3 and IRL 1620. Table 2 shows the concentrations of these peptides needed to induce a half-maximum relaxation. These peptides were approximately equipotent in inducing relaxation. In the presence of 3 #M BQ-123, 30 nM ET-1 induced rapid relaxation with similar magnitude as that observed in the absence of BQ-123 (Figs. 3C and 5B). The ECso value for ET-1 was not affected by BQ-123 either (Table 2). In the presence of BQ-123, however, the duration of the ET-l-induced relaxation became longer (Fig. 3C). The ET-l-induced relaxation seemed to be composed of an initial rapid phase followed by sustained relaxation (Fig. 3C). In the presence of 3 #M IRL 1038, ET-1 induced only

Vol. 54, No. 10, 1994

80

A

ETB Receptor Subtypes in Ileum

tO ,m O

o

Concentration-response curves for the contractile effect of ET-1, ET-3 and IRL 1620 in the absence or presence of 3 ~M BQ-123 or 3 ~M IRL 1038. The contractions were expressed as percentages of the high K+-induced contraction. Each point represents the mean of at least 4 experiments. S.E.M. is shown by vertical bar. * : Significantly different from the value in the absence of antagonist with P<0.05.

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649

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TABLE 1 Concentrations of ET-1, ET-3 and IRL 1620 needed to induce the half-maximum contraction (ECs0) in the ileum.

Condition

ECs0 (nM) ET-3

ET-1

Control BQ-123 (3 IxM) IRL 1038 (3 ~tM)

1.19 (0.95-1.50) >100" 2.43 (1.88-3.13)

IRL 1620

31.6 (24.0-33.1) 1) 281 (234-354)*

24.3 (19.3-30.5) 2) >300*

Each value represents the geometric mean of at least 4 experiments. The corresponding S.E. range is shown in parentheses. *: Significantly different from respective control with P<0.05. 1) Contraction induced by 300 nM ET-3 was not affected. 2) Contraction induced by 100 nM IRL 1620 was not affected.

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FIG. 5 Relaxant effects of ETs. (A) Concentration-response curves for the maximum relaxant effect of ET-1, ET-3 and IRL 1620 in the ileum precontracted by 100 nM carbachol. (B) Effects of 3 ~tM BQ-123 and 3 taM IRL 1038 on the maximum relaxation induced by ET-1. 100 % represents relaxation to the resting muscle tone prior to addition of carbachol. Each point represents the mean of at least 4 experiments. S.E.M. is shown by vertical bar. * • Significantly different from the value in the absence of antagonist with P<0.05.

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ETB Receptor Subtypes in Ileum

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a sustained relaxation without the initial rapid phase (Fig. 3D), suggesting that IRL 1038 selectively inhibits the initial rapid relaxation. To confirm this, the effects of IRL 1038 on the relaxation induced by ET-3 were examined. Fig. 6 shows the effects of various concentrations of IRL 1038 on the relaxation induced by 30 nM ET-3 in the carbachol-stimulated ileum. The initial rapid relaxation induced by ET-3 was inhibited by IRL 1038 in a concentration-dependent manner. The IC5o for IRL 1038 was approximately 490 nM. In contrast, the sustained relaxation induced by ET-3 was not affected by IRL 1038. IRL 1038 shifted the concentrationresponse curves to the right and increased the EC50 values for ET-1, ET-3 and IRL 1620 (Fig. 5B and Table 2). TABLE 2 Concentrations of ET-1, ET-3 and IRL 1620 needed to induce the half-maximum relaxation (ECs0) in the 100 nM carbachol-stimulated ileum. ECs0 (nM) Condition

ET- 1

Control BQ-123 (3 rtM) IRL 1038 (3 I~M)

ET-3

0.78 (0.59-1.02) 0.54 (0.48-0.60) 3.72 (2.51-5.50)*

IRL 1620

1.07 (0.66-1.74) 0.51 (0.38-0.69) 16.2 (11.2-23.4)*

1.82 (1.41-2.34) 1.20 (0.98-1.48) 5.62 (4.27-7.41)*

Each value represents the geometric mean of at least 4 experiments. The corresponding S.E. range is shown in parentheses. *: Significantly different from respective control with P<0.05. FIG. 6 ET-3

100

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B

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¢,-

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40 0

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7.5

I

7

I

6.5

IRL 1038

I

6

5.5

(-log M)

Effect of IRL 1038 on the relaxation induced by 30 nM ET3 in the ileum precontracted by 100 nM carbachol. As shown in the inset, ET-3 induced transient (A) followed by sustained relaxation (B). Magnitudes of transient (O) and sustained relaxation (O), are plotted. 100 % represents the magnitude of transient relaxation in the absence of IRL 1038. IRL 1038 was added 20 rain before the addition of ET-3. Each point represents the mean of at least 4 experiments. S.E.M. is shown by vertical bar. * : Significantly different from the value in the absence of antagonist with P<0.05.

Discussion Binding assays showed that there are two types of ET receptors in guinea pig ileum; isopeptide-selective ETA receptors and isopeptide-nonselective ETB receptors. To understand the functional role of these receptors, we examined the effects of ETs on contractile force. ET-1 and ET-3 induce transient relaxation as reported previously (15-

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18). EC50 values for the relaxant effect of ET-1 and ET-3 were 0.8 nM and 1.1 nM, respectively. The selective ETB agonist, IRL 1620, also induced relaxation at the similar concentrations to ET-1 or ET-3 (EC50 = 1.8 nM). The relaxant effects of ET-1, ET-3 and IRL 1620 were not inhibited by the ETA antagonist, BQ-123. These results, taken together, suggest that the relaxation is due to activation of the isopeptide-nonselective ETB receptor. The relaxation induced by these peptides is composed of two phases; initial rapid relaxation and following sustained relaxation. The ET8 antagonist, IRL 1038, selectively inhibited the initial rapid relaxation with little effect on the following sustained relaxation. The IC50 for IRL 1038 on the initial rapid relaxation was approximately 490 nM, which is similar to those for the inhibition of the ETg-mediated release of nitric oxide from vascular endothelium (650 - 1000 nM) (6,8,12). In swine pulmonary and rabbit saphenous vein, ET-1, ET-3 and IRL 1620 induce contraction within a similar concentration range, suggesting the involvement of ETB receptors (17-19). However, since IRL 1038 did not inhibit contraction in these veins, ETa receptors might be divided into two subtypes on the basis of their sensitivities to IRL 1038 (18,19). Present results also support the possibility that there are two subtypes of the ET8 receptor; one is being sensitive to IRL 1038 (tentatively named ETB1) whereas the other (ET82) is not. In the ileum, the initial rapid relaxation induced by ETs is mediated by the ETB1 receptor whereas the following sustained relaxation is mediated by the ETB2 receptor. The existence of two subtypes of the ETB receptor is also suggested by competitive binding assays between [1251]ET-3 and unlabeled ligands to the ETa receptors (Fig. 1A). When the competition curves were analyzed using pseudo-Hill plots, the slope factors were less than unity for IRL 1620 (0.58) and IRL 1038 (0.80) whereas the values were almost equal to unity for ET-1 (1.10), ET-3 (1.03) and BQ-123 (1.03). These results indicate that the binding of IRL 1620 and IRL 1038 to these ETa receptor subtypes may be heterogeneous while the binding of other ligands seems to be homogeneous. In contrast to the relaxant effect, the EC50 for the contractile effect of ET-1 was approximately 30 times less than that of ET-3. The maximum contraction induced by ET-1 was greater than that induced by ET-3. Furthermore, the effects of ET-1 and ET-3 were antagonized by BQ-123. These results suggest that the contractile effects of ETs in ileum are due mainly to the activation of the ETA receptor. However, since a selective ETB agonist, IRL 1620, induced contraction and since the contractions induced by ET-3 and IRL 1620 were antagonized by IRL 1038, the ETB1 receptor may also mediate part of the contractile effect. In several types of smooth muscle, it has been shown that stimulation of the ETA receptor results in an increase in cytosolic Ca 2+ levels followed by contraction (10). In vascular endothelium, stimulation of the IRL 1038-sensitive type of ET receptor (ETB1) also results in an increase in cytosolic Ca 2+ level followed by release of endotheliumderived relaxing factor (6,8,12). In the present experiments, we did not examine the site of action of ET (either directly act on smooth muscle or indirectly through release of transmitters) nor mechanisms of ET-induced relaxation and contraction. In the ileum, it has been suggested that the relaxant effect is at least partly due to activation of Ca 2+activated K+ channels resulting from the increase in cytosolic Ca 2+ levels (14,20) whereas the contractile effect is also due to the increase in cytosolic Ca 2+ levels (10). In conclusion, we suggest that the ET-induced transient and sustained relaxation in the guinea pig ileum is mediated respectively by IRL 1038-sensitive (ETa1) and IRL 1038-insensitive subtypes (ETB2) of ETB receptors. In contrast, the contractile effect of ETs seems to be mediated mainly by the ETA receptor and partially by the ETB1 receptor.

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AcknQwl~O~ments We are grateful to Drs. T. Okada and A. F. James, Ciba-Geigy Japan, for helpful discussion and critical reading of the manuscript. This work was supported by Grant-inAid for Scientific Research from the Ministry of Education, Science and Culture, Japan.

R~f~r~n¢~ 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

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