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Incomplete inhibition of endothelin-1 pressor effects by an endothelin ETA receptor antagonist
European Journal of Pharmacology, 240 (1993) 295-298
295
© 1993 Elsevier Science Publishers B.V. All rights reserved 0014-2999/93/$06.00
EJP 21305
Short communication
Incomplete inhibition of endothelin-1 pressor effects by an endothelin ETa receptor antagonist J. E i l e e n Bird a n d T h o m a s L. W a l d r o n Department of Pharmacology, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ 08543-4000, USA Received 25 March 1993, revised MS received 21 June 1993, accepted 29 June 1993
Incomplete inhibition of endothelin-l-induced pressor effects by FR-139317, a novel, potent, ETA receptor antagonist, was observed in conscious, normotensive rats. Maximum inhibition by FR-139317 of the endothelin-1 pressor response (0.1, 0.3, 1.0 nmol/kg) was 49 + 7, 41 :i: 3, 62 + 5%, respectively. Two ETa-selective receptor ligands induced pressor responses in conscious rats. A portion of the endothelin-1 pressor response may be mediated by ET a receptors, and ETa-mediated vasoconstriction may contribute to incomplete inhibition of the pressor response to endothelin-1 by an ETA-selective receptor antagonist. Endothelin-1; Mean arterial pressure; Endothelin receptor antagonist; Sarafotoxin $6c
1. Introduction
Endothelin, a 21-amino acid peptide, was first described by Yanagisawa et al. (1988) as a potent vasoconstrictor. Three distinct endothelin isopeptides, endothelin-1, endothelin-2, and endothelin-3 have been characterized (Inoue et al., 1989). There are at least two receptor subtypes: E T A receptors originally found in smooth muscle (Arai et al., 1990), and E T a receptors originally described in endothelial cells (Sakurai et al., 1990). Endothelin-1 has similar binding affinity for E T A and E T a receptors (Aramori and Takanishi, 1992). Ihara et al. (1992) suggested that E T A receptors mediate vasoconstriction and E T a receptors mediate vasodilation. Pressor effects of the ETa-selective agonist, sarafotoxin $6c were observed in rats (Williams et al., 1991), suggesting that E T a receptors mediate pressor responses. Whether the response to endothelin-1 is vasoconstrictive or vasodilatory appears to be dependent on the dose and vascular bed studied (Warner et al., 1989). FR-139317, ((R)2-[(R)-2-[(S)-2-1-(hexahydro- 1Hazepinyl)]carbonyl] amino-4-methylpentanoyl]amino-3[-3-(1-methyl- 1H-indoyl)] propionyl]amino-3-(2-pyridyl)propionic acid), is a novel, modified tripeptide, ETA-selective receptor antagonist (Eur. Pat. App., 1991) with E T A / E T a K i values of 0.5 _+ 0.1 n M / 8 8 + 10 ~zM
Correspondence to: E. Bird, Department of Pharmacology, BristolMyers Squibb Pharmaceutical Research Institute, P.O. Box 4000, Princeton, NJ 08543, USA_. Tel. 1-609-252-4803, fax 1-609-252-6609.
(Dr. Maria Webb, Bristol-Myers Squibb). We characterized the effects of FR-139317 on endothelin-l-induced vascular responses in conscious, normotensive rats, and measured the vascular responses to two E T a receptor ligands, sarafotoxin $6c and [Alal~'15]endo thelin-l-(8-21), the truncated linear peptide analog of endothelin-1 (Saeki et al., 1991).
2. Materials and methods
Male, normotensive Sprague-Dawley rats (9 weeks of age) were prepared surgically according to the method of Weeks and Jones (1960). Two weeks later, the rats were prepared for direct measurement of mean arterial pressure (MAP). Vehicle (10% ethanol + 90% 5% N a H C O 3) was administered prior to an injection of endothelin-1 (0.1, 0.3, or 1.0 n m o l / k g ) as a control challenge (n = 4 - 1 0 / g r o u p ) . At 90 min intervals, consecutive i.v. injections of vehicle or FR-139317 ( 0 . 0 3 - 3 0 . 0 / z m o l / k g ) were made prior to endothelin-1 (Peptides International, Louisville, KY) challenges. MAP was allowed to return to baseline prior to the next challenge. In a separate study, the depressor and pressor effects of i.v. administration of 0.03, 0,1, 0.3, 1.0, and 3.0 n m o l / k g sarafotoxin $6c (Peninsula Laboratories, Belmont, CA) and 0.03, 0.1, 0.3, 1.0, 3.0, 10.0, and 30.0 n m o l / k g [Ala~l'lS]endothelin-l-(8-21) (Bristol-Myers Squibb, Princeton, NJ) were measured. MAP was tabulated during the first 6 min and at 10 min intervals after administration of endothelin-1. Re-
296 suits are expressed as means + S.E.M. The mean % inhibition of the pressor response to endothelin-1 after administration of increasing doses of FR-139317 (Bristol-Myers Squibb, Princeton, NJ) were compared, and mean values of pressor and depressor responses to sarafotoxin $6c and [Ala~l'~5]endothelin-l-(8-21) were compared to responses after vehicle administration by one-way analysis of variance.
20
3. Results 0.01
Relatively low doses of FR-139317 inhibited the pressor response to endothelin-1 (0.1 n m o l / k g i.v.) in conscious rats (fig. 1). The calculated dose of FR139317 to inhibit the endothelin-l-induced pressor response by 25% was 0.065 / x m o l / k g i.v. The dose-response curve for inhibition of the endothelin-1 pressor response was linear at doses of FR-139317 from 0.033.0 ~ m o l / k g . However, the extent of inhibition (28 + 11%) noted after the highest dose of FR-139317 administered (30.0 /~mol/kg) was significantly less (P < 0.05) than the inhibition seen after 3.0 t z m o l / k g FR139317 (49 + 7%). Inhibition of the pressor response to a higher dose of endothelin-1 (0.3 n m o l / k g i.v.) peaked after injection of 3.0 / ~ m o l / k g FR-139317 (41 + 3%). Complete inhibition of the pressor response to endothelin-1 was not obtained, even after administration of 30.0 / ~ m o l / k g FR-139317 (19 + 8% inhibition), which was significantly less inhibition (P < 0.01) than after the 3.0 / z m o l / k g dose of FR-139317. Total inhibition of the endothelin-l-induced (1.0 n m o l / k g ) pressor response after FR-139317 treatment was not observed. The maximal inhibition measured
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Fig. 1. T h e effects of i.v. administration of FR-139317 on the pressor response induced by three doses of endothelin-1 in conscious, normotensive rats are summarized below. T h e closed squares represent the response to 0.1 n m o l / k g i.v. endothelin-1; the open squares represent the response to 0.3 n m o l / k g i.v. endothelin-l; and the closed circles represent the response to 1.0 n m o l / k g i.v. endothelin-1. Means_+ S.E.M. are given.
........
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Fig. 2. The peak pressor responses observed after i.v. injection of
sarafotoxin $6c ($6c) and [Ala11'[5]endothelin-l-(8-21) in conscious, normotensive rats are summarized below. Baseline MAP was 102_+ 1.7 mm Hg in the sarafotoxin S6c-treated rats, and 107_+4.4 mm Hg in the animals treated with [Ala11'15]endothelin-l-(8-21). The closed circles represent the response to sarafotoxin $6c and the open circles represent the response to [Ala~AS]endothelin-l-(8-21). Means_+ S.E.M. are given.
was 25 +_ 3, 32 + 12, and 62 + 5% after the doses of 0.3, 3.0, and 30.0 ~ m o l / k g i.v. FR-139317, respectively (fig. 1). It is conceivable that greater inhibition would be achieved at even higher doses of FR-139317 ( > 30 ~ m o l / k g ) , but side effects of higher doses were observed in other animal models. The depressor response to 1.0 n m o l / k g endothelin-1 was not inhibited by FR139317 (vehicle + endothelin-l: - 1 7 + 2%; 30.0 ~zmol/kg FR-139317 + endothelin-l: - 2 1 + 3% decrease MAP). The effects on M A P of sarafotoxin $6c and [Alatl'tS]endothelin-l-(8-21) were measured. Baseline M A P was 102 + 1.7 m m Hg in sarafotoxin S6c-treated rats, and 1 0 7 + 4 . 4 m m Hg in rats treated with [AlatmS]endothelin-l-(8-21). The lowest doses of sarafotoxin $6c tested (0.03, 0.1 n m o l / k g i.v.) induced significant pressor effects (fig. 2), but not depressor effects. The depressor response to sarafotoxin $6c increased in a dose-dependent fashion (0.3 n m o l / k g : - 13 + 1%; 1.0 n m o l / k g : - 2 2 + 2%; 3.0 n m o l / k g : - 2 2 + 2% decrease MAP). Doses of sarafotoxin $6c ranging from 0.3 to 3.0 n m o l / k g produced a biphasic pressure response. The pressor effects of sarafotoxin $6c were less obvious at the highest dose tested, and may have been masked by the vasodilatory response to sarafotoxin $6c. Dose-dependent pressor responses to [Alalt,~5]endothelin-l-(8-21) were observed (fig. 2). T h e r e was no depressor response at doses of [Ala~l,~5]endothelin-l-(8-21) up to 1.0 n m o l / k g . Depressor effects of [Alal~'tS]endothelin-l-(8-21) were less pronounced (3.0 n m o l / k g : - 12 + 2%; 10.0 n m o l / k g : - 14 + 1%; 30.0 n m o l / k g : - 10 + 1%) than observed after sarafotoxin $6c.
297
4. Discussion Relatively low doses (0.1 p, m o l / k g ) of FR-139317 inhibited the endothelin-l-induced pressor response in conscious, normotensive rats. However, we were unable to achieve 100% inhibition of the pressor effects of endothelin-1 after administration of up to 30 /xmol/kg FR-139317. Maximal inhibition reached a plateau at approximately 50-60%. These results are comparable to the 40% inhibition of the endothelin-1induced pressor response observed after infusion of the ETA-selective receptor antagonist, BQ 123 (Waldron and Bird, 1992), and similar to the approximately 75% inhibition observed by Sogabe et al. (1993) after injection of FR-139317. Partial inhibition of the pressor response to endothelin-1 was also present after administration of BQ 153, an analog of BQ 123 (Ihara et al., 1992). Complete inhibition of an endothelin-l-induced pressor response after infusion of BQ 123 in anesthetized rats has been reported (Pollock and Opgenorth, 1993). The depressor effects observed when BQ 123 was administered in the absence of endothelin1, may have contributed to the apparent complete inhibition of the pressor response to endothelin. The mechanism involved in endothelin-l-induced vasoconstriction is a complex one. Different doses of endothelin-1 induce diverse effects, and different vascular beds vary in response to the same dose of endothelin-1. Warner et al. (1989) reported that low doses of porcine endothelin induced primarily vasodilation of isolated rat mesentery, while at higher doses, vasoconstriction was predominant. Ihara et al. (1992) suggested that E T A receptors mediate vasoconstriction. We have shown, as have others, that E T a receptor agonists induced pressor responses in rats, suggesting that a portion of the pressor response to endothelin-1 may be mediated through E T a receptors (Williams et al., 1991; Webb et al., 1992). Effects involving E T a receptors would presumably be resistant to inhibition by an ETA-Selective receptor antagonist. FR-139317 did not inhibit the depressor response to endothelin-1, suggesting a lack of effect of FR-139317 at E T a receptors. FR-139317 is approximately 150000-fold more selective for E T A receptors than E T a receptors in vitro, and our results suggest that it is also selective in vivo. We have reported that the pressor response to big endothelin-1 was almost completely abolished by administration of phosphoramidon, an inhibitor of endothelin-converting enzyme (Bird et al., 1992). Our results suggest that blockade of synthesis of endothelin-1 or administration of a non-selective endothelin-1 receptor antagonist would be necessary for total inhibition of the pressor response to endothelin-1. An interesting finding in this study was the non-linearity of the dose-response curves for inhibition of the pressor response to lower doses of endothelin-1 by
FR-139317. Similar results were obtained with BQ 123 in conscious rats (Waldron and Bird, 1992). Since endothelin-1 binds with similar affinity to E T A and E T a receptors (Aramori and Takanishi, 1992), the determining factors for which receptor endothelin-1 binds to, will be the number and occupancy state of available receptors..As the highest doses of FR-139317 blocked binding of endothelin-1 to E T A receptors, additional endothelin-1 may have become available for stimulation of E T a receptors in the same microenvironment, which mediate pressor effects resistant to inhibition with an E T A receptor antagonist. In summary, a wide range of doses of the potent, selective E T A receptor antagonist, FR-139317, did not completely inhibit the endothelin-l-induced pressor response. Pretreatment with FR-139317 had no significant effect on the depressor response to endothelin-1. Two ETB-selective receptor agonists, sarafotoxin $6c and [Alall'15]endothelin-l-(8-21), exerted pressor responses. The data suggest that E T a receptors mediate vasoconstrictor responses in vivo, and contribute to the lack of total inhibition of the pressor response to endothelin-1 by a potent, ETA-selective receptor antagonist in conscious, normotensive rats.
Acknowledgements We would like to thank Mrs. Mary Giancarli and Mr. Tom Schaeffer for technical assistance, Dr. Maria Webb for helpful discussions, and Dr. Joseph Sundeen and Mr. Alan Fritz for synthesis of FR-139317.
References Arai, H., S. Hori, I. Aramori, H. Ohkubo and S. Nakanishi, 1990, Cloning and expression of a cDNA encoding an endothelin receptor, Nature 348, 730. Aramori, I. and S. Nakanishi, 1992, Coupling of two endothelin receptor subtypes to differing signal transduction in transfected chinese hamster ovary cells, J. Biol. Chem. 267, 12468. Bird, J.E., T.L. Waldron, D.K. Little, M.M. Asaad, C.R. Dorso, G. DiDonato and J.A. Norman, 1992, The effects of novel cathepsin E inhibitors on the big endothelin pressor response in conscious rats, Biochem. Biophys, Res. Commun. 182, 224. Eur. Pat. App., 1991, No. 0 457 195 A2. Ihara, M., K. Noguchi, T. Saeki, T. Fukuroda, S. Tsuchida, S. Kimura, T. Fukami, K. Ishikawa, M. Nishikibe and M. Yano, 1992, Biological profiles of highly potent novel endothelin antagonists selective for the ETa receptor, Life Sci. 50, 247. Inoue, A., M. Yanagisawa, S. Kimura, Y. Kasuya, Y. Miyauchi, K. Goto and T. Masaki, 1989, The human endothelin family: Three structurally and pharmacologically distinct isopeptides predicted by three separate genes, Proc. Natl. Acad. Sci. USA 86, 2863. Pollock, D,M. and T.J. Opgenorth, 1993, Evidence for endothelin-induced renal vasoconstriction independent of ET a receptor activation, Am. J. Physiol. 264, R222.
298 Saeki, T., M. Ihara, T. Fukuroda, M. Yamagiwa and M. Yano, 1991, [Ala 1.3.11.15]Endothelin-1 analogs with ET a agonistic activity, Biochem. Biophys. Res. Commun. 179, 286. Sakurai, T., M. Yanagisawa, Y. Takuwa, H. Miyazaki, S. Kimura, K. Goto and T. Masaki, 1990, Cloning of a cDNA encoding a non-isopeptide-selective subtype of the endothelin receptor, Nature 348, 732. Sogabi, K., H. Nirei, M. Shoubo, A. Nomoto, S. Ao, Y. Notsu and T. Ono, 1993, Pharmacological profile of FR139317, a novel, potent endothelin ET A receptor antagonist, J. Pharmacol. Exp. Ther. 264, 1040. Waldron, T.L. and J.E. Bird, 1992, Effects of the endothelin A receptor (ETAR) antagonist BQ-123 (BQ) on the pressor and depressor responses to varied doses of endothelin (ET)-l in rats, Pharmacologist 34, 160. Warner, T.D., G. De Nucci and J.R. Vane, 1989, Rat endothelin is a
vasodilator in the isolated perfused mesentery of the rat, Eur. J. Pharmacol. 159, 325. Webb, R.L., T. Okada, B.W. Barclay and R.W. Lappe, 1992, Central and peripheral actions of IRL 1620, an ET B selective endothelin agonist in conscious rats, Hypertension 20, 425. Weeks, J.R. and J.A. Jones, 1960, Routine direct measurement of arterial pressure in unanesthetized rats, Proc. Soc. Exp. Biol. Med. 104, 646. Williams, D.L., K.L. Jones, D.J. Pettibone, E.V. Lis and B.C. Clineschmidt, 1991, Sarafotoxin $6c: an agonist which distinguishes between endothelin receptor subtypes, Biochem. Biophys. Res. Commun. 175, 556. Yanigasawa, M., H. Kurihara, S. Kimura, Y. Tomobe, M. Kobayashi, 5". Mitsui, Y. Yazaki, K. Goto and T. Masaki, 1988, A novel potent vasoconstrictor peptide produced by vascular endothelial cells, Nature 332, 411.
295
© 1993 Elsevier Science Publishers B.V. All rights reserved 0014-2999/93/$06.00
EJP 21305
Short communication
Incomplete inhibition of endothelin-1 pressor effects by an endothelin ETa receptor antagonist J. E i l e e n Bird a n d T h o m a s L. W a l d r o n Department of Pharmacology, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ 08543-4000, USA Received 25 March 1993, revised MS received 21 June 1993, accepted 29 June 1993
Incomplete inhibition of endothelin-l-induced pressor effects by FR-139317, a novel, potent, ETA receptor antagonist, was observed in conscious, normotensive rats. Maximum inhibition by FR-139317 of the endothelin-1 pressor response (0.1, 0.3, 1.0 nmol/kg) was 49 + 7, 41 :i: 3, 62 + 5%, respectively. Two ETa-selective receptor ligands induced pressor responses in conscious rats. A portion of the endothelin-1 pressor response may be mediated by ET a receptors, and ETa-mediated vasoconstriction may contribute to incomplete inhibition of the pressor response to endothelin-1 by an ETA-selective receptor antagonist. Endothelin-1; Mean arterial pressure; Endothelin receptor antagonist; Sarafotoxin $6c
1. Introduction
Endothelin, a 21-amino acid peptide, was first described by Yanagisawa et al. (1988) as a potent vasoconstrictor. Three distinct endothelin isopeptides, endothelin-1, endothelin-2, and endothelin-3 have been characterized (Inoue et al., 1989). There are at least two receptor subtypes: E T A receptors originally found in smooth muscle (Arai et al., 1990), and E T a receptors originally described in endothelial cells (Sakurai et al., 1990). Endothelin-1 has similar binding affinity for E T A and E T a receptors (Aramori and Takanishi, 1992). Ihara et al. (1992) suggested that E T A receptors mediate vasoconstriction and E T a receptors mediate vasodilation. Pressor effects of the ETa-selective agonist, sarafotoxin $6c were observed in rats (Williams et al., 1991), suggesting that E T a receptors mediate pressor responses. Whether the response to endothelin-1 is vasoconstrictive or vasodilatory appears to be dependent on the dose and vascular bed studied (Warner et al., 1989). FR-139317, ((R)2-[(R)-2-[(S)-2-1-(hexahydro- 1Hazepinyl)]carbonyl] amino-4-methylpentanoyl]amino-3[-3-(1-methyl- 1H-indoyl)] propionyl]amino-3-(2-pyridyl)propionic acid), is a novel, modified tripeptide, ETA-selective receptor antagonist (Eur. Pat. App., 1991) with E T A / E T a K i values of 0.5 _+ 0.1 n M / 8 8 + 10 ~zM
Correspondence to: E. Bird, Department of Pharmacology, BristolMyers Squibb Pharmaceutical Research Institute, P.O. Box 4000, Princeton, NJ 08543, USA_. Tel. 1-609-252-4803, fax 1-609-252-6609.
(Dr. Maria Webb, Bristol-Myers Squibb). We characterized the effects of FR-139317 on endothelin-l-induced vascular responses in conscious, normotensive rats, and measured the vascular responses to two E T a receptor ligands, sarafotoxin $6c and [Alal~'15]endo thelin-l-(8-21), the truncated linear peptide analog of endothelin-1 (Saeki et al., 1991).
2. Materials and methods
Male, normotensive Sprague-Dawley rats (9 weeks of age) were prepared surgically according to the method of Weeks and Jones (1960). Two weeks later, the rats were prepared for direct measurement of mean arterial pressure (MAP). Vehicle (10% ethanol + 90% 5% N a H C O 3) was administered prior to an injection of endothelin-1 (0.1, 0.3, or 1.0 n m o l / k g ) as a control challenge (n = 4 - 1 0 / g r o u p ) . At 90 min intervals, consecutive i.v. injections of vehicle or FR-139317 ( 0 . 0 3 - 3 0 . 0 / z m o l / k g ) were made prior to endothelin-1 (Peptides International, Louisville, KY) challenges. MAP was allowed to return to baseline prior to the next challenge. In a separate study, the depressor and pressor effects of i.v. administration of 0.03, 0,1, 0.3, 1.0, and 3.0 n m o l / k g sarafotoxin $6c (Peninsula Laboratories, Belmont, CA) and 0.03, 0.1, 0.3, 1.0, 3.0, 10.0, and 30.0 n m o l / k g [Ala~l'lS]endothelin-l-(8-21) (Bristol-Myers Squibb, Princeton, NJ) were measured. MAP was tabulated during the first 6 min and at 10 min intervals after administration of endothelin-1. Re-
296 suits are expressed as means + S.E.M. The mean % inhibition of the pressor response to endothelin-1 after administration of increasing doses of FR-139317 (Bristol-Myers Squibb, Princeton, NJ) were compared, and mean values of pressor and depressor responses to sarafotoxin $6c and [Ala~l'~5]endothelin-l-(8-21) were compared to responses after vehicle administration by one-way analysis of variance.
20
3. Results 0.01
Relatively low doses of FR-139317 inhibited the pressor response to endothelin-1 (0.1 n m o l / k g i.v.) in conscious rats (fig. 1). The calculated dose of FR139317 to inhibit the endothelin-l-induced pressor response by 25% was 0.065 / x m o l / k g i.v. The dose-response curve for inhibition of the endothelin-1 pressor response was linear at doses of FR-139317 from 0.033.0 ~ m o l / k g . However, the extent of inhibition (28 + 11%) noted after the highest dose of FR-139317 administered (30.0 /~mol/kg) was significantly less (P < 0.05) than the inhibition seen after 3.0 t z m o l / k g FR139317 (49 + 7%). Inhibition of the pressor response to a higher dose of endothelin-1 (0.3 n m o l / k g i.v.) peaked after injection of 3.0 / ~ m o l / k g FR-139317 (41 + 3%). Complete inhibition of the pressor response to endothelin-1 was not obtained, even after administration of 30.0 / ~ m o l / k g FR-139317 (19 + 8% inhibition), which was significantly less inhibition (P < 0.01) than after the 3.0 / z m o l / k g dose of FR-139317. Total inhibition of the endothelin-l-induced (1.0 n m o l / k g ) pressor response after FR-139317 treatment was not observed. The maximal inhibition measured
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Fig. 1. T h e effects of i.v. administration of FR-139317 on the pressor response induced by three doses of endothelin-1 in conscious, normotensive rats are summarized below. T h e closed squares represent the response to 0.1 n m o l / k g i.v. endothelin-1; the open squares represent the response to 0.3 n m o l / k g i.v. endothelin-l; and the closed circles represent the response to 1.0 n m o l / k g i.v. endothelin-1. Means_+ S.E.M. are given.
........
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Fig. 2. The peak pressor responses observed after i.v. injection of
sarafotoxin $6c ($6c) and [Ala11'[5]endothelin-l-(8-21) in conscious, normotensive rats are summarized below. Baseline MAP was 102_+ 1.7 mm Hg in the sarafotoxin S6c-treated rats, and 107_+4.4 mm Hg in the animals treated with [Ala11'15]endothelin-l-(8-21). The closed circles represent the response to sarafotoxin $6c and the open circles represent the response to [Ala~AS]endothelin-l-(8-21). Means_+ S.E.M. are given.
was 25 +_ 3, 32 + 12, and 62 + 5% after the doses of 0.3, 3.0, and 30.0 ~ m o l / k g i.v. FR-139317, respectively (fig. 1). It is conceivable that greater inhibition would be achieved at even higher doses of FR-139317 ( > 30 ~ m o l / k g ) , but side effects of higher doses were observed in other animal models. The depressor response to 1.0 n m o l / k g endothelin-1 was not inhibited by FR139317 (vehicle + endothelin-l: - 1 7 + 2%; 30.0 ~zmol/kg FR-139317 + endothelin-l: - 2 1 + 3% decrease MAP). The effects on M A P of sarafotoxin $6c and [Alatl'tS]endothelin-l-(8-21) were measured. Baseline M A P was 102 + 1.7 m m Hg in sarafotoxin S6c-treated rats, and 1 0 7 + 4 . 4 m m Hg in rats treated with [AlatmS]endothelin-l-(8-21). The lowest doses of sarafotoxin $6c tested (0.03, 0.1 n m o l / k g i.v.) induced significant pressor effects (fig. 2), but not depressor effects. The depressor response to sarafotoxin $6c increased in a dose-dependent fashion (0.3 n m o l / k g : - 13 + 1%; 1.0 n m o l / k g : - 2 2 + 2%; 3.0 n m o l / k g : - 2 2 + 2% decrease MAP). Doses of sarafotoxin $6c ranging from 0.3 to 3.0 n m o l / k g produced a biphasic pressure response. The pressor effects of sarafotoxin $6c were less obvious at the highest dose tested, and may have been masked by the vasodilatory response to sarafotoxin $6c. Dose-dependent pressor responses to [Alalt,~5]endothelin-l-(8-21) were observed (fig. 2). T h e r e was no depressor response at doses of [Ala~l,~5]endothelin-l-(8-21) up to 1.0 n m o l / k g . Depressor effects of [Alal~'tS]endothelin-l-(8-21) were less pronounced (3.0 n m o l / k g : - 12 + 2%; 10.0 n m o l / k g : - 14 + 1%; 30.0 n m o l / k g : - 10 + 1%) than observed after sarafotoxin $6c.
297
4. Discussion Relatively low doses (0.1 p, m o l / k g ) of FR-139317 inhibited the endothelin-l-induced pressor response in conscious, normotensive rats. However, we were unable to achieve 100% inhibition of the pressor effects of endothelin-1 after administration of up to 30 /xmol/kg FR-139317. Maximal inhibition reached a plateau at approximately 50-60%. These results are comparable to the 40% inhibition of the endothelin-1induced pressor response observed after infusion of the ETA-selective receptor antagonist, BQ 123 (Waldron and Bird, 1992), and similar to the approximately 75% inhibition observed by Sogabe et al. (1993) after injection of FR-139317. Partial inhibition of the pressor response to endothelin-1 was also present after administration of BQ 153, an analog of BQ 123 (Ihara et al., 1992). Complete inhibition of an endothelin-l-induced pressor response after infusion of BQ 123 in anesthetized rats has been reported (Pollock and Opgenorth, 1993). The depressor effects observed when BQ 123 was administered in the absence of endothelin1, may have contributed to the apparent complete inhibition of the pressor response to endothelin. The mechanism involved in endothelin-l-induced vasoconstriction is a complex one. Different doses of endothelin-1 induce diverse effects, and different vascular beds vary in response to the same dose of endothelin-1. Warner et al. (1989) reported that low doses of porcine endothelin induced primarily vasodilation of isolated rat mesentery, while at higher doses, vasoconstriction was predominant. Ihara et al. (1992) suggested that E T A receptors mediate vasoconstriction. We have shown, as have others, that E T a receptor agonists induced pressor responses in rats, suggesting that a portion of the pressor response to endothelin-1 may be mediated through E T a receptors (Williams et al., 1991; Webb et al., 1992). Effects involving E T a receptors would presumably be resistant to inhibition by an ETA-Selective receptor antagonist. FR-139317 did not inhibit the depressor response to endothelin-1, suggesting a lack of effect of FR-139317 at E T a receptors. FR-139317 is approximately 150000-fold more selective for E T A receptors than E T a receptors in vitro, and our results suggest that it is also selective in vivo. We have reported that the pressor response to big endothelin-1 was almost completely abolished by administration of phosphoramidon, an inhibitor of endothelin-converting enzyme (Bird et al., 1992). Our results suggest that blockade of synthesis of endothelin-1 or administration of a non-selective endothelin-1 receptor antagonist would be necessary for total inhibition of the pressor response to endothelin-1. An interesting finding in this study was the non-linearity of the dose-response curves for inhibition of the pressor response to lower doses of endothelin-1 by
FR-139317. Similar results were obtained with BQ 123 in conscious rats (Waldron and Bird, 1992). Since endothelin-1 binds with similar affinity to E T A and E T a receptors (Aramori and Takanishi, 1992), the determining factors for which receptor endothelin-1 binds to, will be the number and occupancy state of available receptors..As the highest doses of FR-139317 blocked binding of endothelin-1 to E T A receptors, additional endothelin-1 may have become available for stimulation of E T a receptors in the same microenvironment, which mediate pressor effects resistant to inhibition with an E T A receptor antagonist. In summary, a wide range of doses of the potent, selective E T A receptor antagonist, FR-139317, did not completely inhibit the endothelin-l-induced pressor response. Pretreatment with FR-139317 had no significant effect on the depressor response to endothelin-1. Two ETB-selective receptor agonists, sarafotoxin $6c and [Alall'15]endothelin-l-(8-21), exerted pressor responses. The data suggest that E T a receptors mediate vasoconstrictor responses in vivo, and contribute to the lack of total inhibition of the pressor response to endothelin-1 by a potent, ETA-selective receptor antagonist in conscious, normotensive rats.
Acknowledgements We would like to thank Mrs. Mary Giancarli and Mr. Tom Schaeffer for technical assistance, Dr. Maria Webb for helpful discussions, and Dr. Joseph Sundeen and Mr. Alan Fritz for synthesis of FR-139317.
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