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sarafotoxin receptors mediating contraction of the human isolated saphenous vein
European Journal of Pharmacology, 239 (1993) 267-268 © 1993 Elsevier Science Publishers B.V. All rights reserved 0014-2999/93/$06.00
267
EJP 21311
Rapid communication
Heterogeneity of endothelin/sarafotoxin receptors mediating contraction of the human isolated saphenous vein W i l l e m A. Bax a, E g b e r t Bos t, a n d P r a m o d R. S a x e n a a a Department of Pharmacology and b Department of Thoracic Surgery, Thorax Center, Faculty of Medicine and Health Sciences, Erasmus University Rotterdam, P.O. Box 1738, 3000 DR Rotterdam, Netherlands Received 28 June 1993, accepted 2 July 1993
We investigated the effect of the ETA receptor antagonist, BQ-123 (0.1 and 1 /~M), on contraction of the human isolated saphenous vein induced by endothelin-1 or sarafotoxin S6b. Contraction in response to endothelin-1 was not affected by BQ-123. In contrast, BQ-123 biphasically attenuated the contractions due to sarafotoxin S6b. These data indicate that (i) endothelin-1 induces contractions of the human saphenous vein via a non-ETA receptor and (ii) contractions in response to sarafotoxin S6b are mediated in part via a receptor different from the receptor mediating contraction due to endothelin-1. Endothelin-1; Sarafotoxin S6b; BQ-123; Endothelin ETA receptor; Saphenous vein (human)
The cloning of two endothelin receptors, denoted E T A and E T B (Arai et al., 1990; Sakurai et al., 1990), has encouraged the characterization of endothelin receptors that mediate contraction in the vascular bed of several species (e.g. Sumner et al., 1992). Characterization was facilitated by the introduction of compounds with selectivity for one of these two receptors. BQ-123 (cyclo[D-Asp-L-Pro-D-Val-L-Leu-D-Trp-]), for instance, has been used extensively as a selective antagonist with high affinity for the E T A receptor (Ihara et al., 1991; Adachi et al., 1992). Although the E T A receptor was originally believed to be the most important in mediating vascular smooth muscle vasoconstriction (Masaki et al., 1991), several investigators concluded that the E T a receptor (Sumner et al., 1992), or a n o n - E TA, n o n - E T B receptor (Harrison et al., 1992) also plays a role in vasoconstriction. For this reason, and because little information is available on human vascular tissues, we investigated the effect of BQ-123 (0.1 and 1 /~M) on contractions of the human saphenous vein induced by endothelin-1 or sarafotoxin S6b. Leftover human saphenous vein was obtained intraoperatively from 9 patients (8 males, 1 female; age: 53 to 75 years), undergoing coronary bypass surgery. The
Correspondence to: Willem A. Bax, Department of Pharmacology, Thorax Center, Faculty of Medicine and Health Sciences, Erasmus University Rotterdam, P.O. Box 1738, 3000 DR Rotterdam, Netherlands. Tel. (+) 31 10 408 7551/47, fax ( +) 31 10 436 6839. Study supported by the Netherlands Heart Foundation, Grant 89.252.
tissue was immediately placed in a cold, oxygenated Krebs bicarbonate solution of the following composition: 118 mM NaCI, 4.7 mM KC1, 2.5 mM CaCI2, 1.2 mM MgSO4, 1.2 mM K H 2 P O 4 , 25 mM NaHCO2, and 8.3 mM glucose; p H 7.4. The vein was cleaned of adhesive fat and connective tissue, and the endothelium was removed using a cotton swab. Ring segments (4 mm) were suspended in 15-ml organ baths to measure isometric tension. The absence of endothelium was verified by observing a lack of relaxation to acetylcholine (1/xM) or bradykinin (1/~M) after precontraction with noradrenaline (0.1/~M). After measuring the contractile response to K ÷ (100 mM), the segments were either incubated with BQ-123 (0.1 or 1 /xM) for 30 min, or remained untreated as a control segment. Subsequently, a concentration-response curve for endothelin-1 or sarafotoxin S6b was obtained in all vessel segments. Contraction was expressed as a percentage of K + (100 mM)-induced contractions. Endothelin-1, sarafotoxin S6b and BQ-123 were purchased from Neosystem S.A. (Strasbourg, France); all other drugs were from Sigma Chemical Co. (St. Louis, MO, USA). Both endothelin-1 and sarafotoxin S6b caused concentration-dependent contractile responses of the vessel segments (PD2:8.06 + 0.10 and 8.13 + 0.05, respectively; E ~ x : 123 + 7% and 135 + 6% of K ÷ (100 mM)-induced contractions, respectively; n = 7-9). BQ123 (0.1 and 1/~M) did not affect endothelin-l-induced contraction. The contractile responses induced by sarafotoxin in S6b in concentrations not higher than 1 nM also remained unaffected by BQ-123. However,
268
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contractile response to sarafotoxin S6b. It appears likely that the BQ-123-insensitive receptor, which mediates the contractile response to low concentrations of sarafotoxin S6b, is similar to the receptor that mediates the contractile response to endothelin-1. The exact nature of this receptor remains to be determined. The BQ-123-sensitive receptor, which mediates the contractile response to higher concentrations of sarafotoxin S6b, appears to be different from the ETA receptor; since endothelin-1 has a higher affinity than sarafotoxin S6b for the ETA receptor (Arai et al., 1990), one would expect the contractile response to endothelin-1 to also be attenuated by BQ-123. Therefore, the present data indicate that BQ-123 may have relatively high affinity for a non-ETA, non-ET B receptor. Interestingly, a previous study using quantitative receptor autoradiography in the human coronary artery also indicated that [lzSI]sarafotoxin S6b but not [~zSI]endothelin-1 labeled a non-ETA, non-ET a receptor with high affinity for BQ-123 (Bax et al., 1993).
Acknowledgements
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This study was supported by the Netherlands Heart Foundation, Grant 89.252. We especially would like to thank the surgeons and staff of the Thorax Center for providing the tissue, and T. Inan and R.W.G. Petterson for skilful technical assistance.
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Fig. 1. Contractions of the human isolated saphenous vein in response to endothelin-1 (top panel, A) or sarafotoxin S6b (lower panel, B), without (<3, control) or in the presence of BQ-123 (0.1 /zM, e; 1 ttM, A). The concentration refers to tool/1. Contractions are expressed as a percentage of K + (100 mM)-induced contractions. n = 7-9.
contractions induced by higher concentrations of sarafotoxin S6b were concentration dependently attenuated by BQ-123 (0.1 and 1 t~M; fig. 1). We conclude that endothelin-1 contracts the human isolated saphenous vein via a non-ETA receptor, since 1 /~M BQ-123 (a potent ETA receptor antagonist; Ihara et al., 1991; Adachi et al., 1992) did not attenuate the contractile response to endothelin-1. On the other hand, BQ-123 caused a biphasic inhibition of sarafotoxin S6b-induced contractions, indicating heterogeneity of the receptor population mediating the
Adachi, M., Y.-Y. Yang, A. Treciak, Y. Furuichi and C. Miyamoto, 1992, Identification of a domain of ET A receptor required for ligand binding, FEBS Lett. 311, 179. Arai, H., S. Hori, I. Aramori, H. Ohkubo and S. Nakanishi, 1990, Cloning and expression of a eDNA encoding an endothelin receptor, Nature 348, 730. Bax, W.A., A.T. Bruinvels, P.R. Saxena and D. Hoyer, 1993, Endothelin receptors in human cardiac tissue; a quantitative autoradiographic analysis, Br. J. Pharmacol. 108, l12P. Harrison, V.J., A. Randriantsoa and P. Schoeffter, 1992, Heterogeneity of endothelin-sarafotoxin receptors mediating contraction of pig coronary artery, Br. J. Pharmacol. 105, 511. Ihara, M., K. Noguehi, T. Saeki, T. Fukuroda, S. Tsudicha, S. Kimura, T. Fukami, K. Ishikawa, M. Nishikibe and M. Yano, 1991, Biological profiles of highly potent novel endothelin antagonists selective for the ETA receptor, Life Sci. 50, 247. Masaki, T., S. Kimura, M. Yanagisawa and K. Goto, 1991, Molecular and cellular mechanism of endothelin regulation; implication for vascular function, Circulation 84, 1457. Sakurai, T., M. Yanagisawa, Y. Takuwa, H. Miyazaki, S. Kimura, IC Goto and T. Masaki, 1990, Cloning of a cDNA encoding a non-isopeptide-selective subtype of the endothelin receptor, Nature 348, 732. Sumner, M.J., T.R. Cannon, J.W. Mundin, D.G. White and I.S. Watts, 1992, Endothelin ETA and ET B receptors mediate vascular smooth muscle contraction, Br. J. Pharmacol. 107, 858.
267
EJP 21311
Rapid communication
Heterogeneity of endothelin/sarafotoxin receptors mediating contraction of the human isolated saphenous vein W i l l e m A. Bax a, E g b e r t Bos t, a n d P r a m o d R. S a x e n a a a Department of Pharmacology and b Department of Thoracic Surgery, Thorax Center, Faculty of Medicine and Health Sciences, Erasmus University Rotterdam, P.O. Box 1738, 3000 DR Rotterdam, Netherlands Received 28 June 1993, accepted 2 July 1993
We investigated the effect of the ETA receptor antagonist, BQ-123 (0.1 and 1 /~M), on contraction of the human isolated saphenous vein induced by endothelin-1 or sarafotoxin S6b. Contraction in response to endothelin-1 was not affected by BQ-123. In contrast, BQ-123 biphasically attenuated the contractions due to sarafotoxin S6b. These data indicate that (i) endothelin-1 induces contractions of the human saphenous vein via a non-ETA receptor and (ii) contractions in response to sarafotoxin S6b are mediated in part via a receptor different from the receptor mediating contraction due to endothelin-1. Endothelin-1; Sarafotoxin S6b; BQ-123; Endothelin ETA receptor; Saphenous vein (human)
The cloning of two endothelin receptors, denoted E T A and E T B (Arai et al., 1990; Sakurai et al., 1990), has encouraged the characterization of endothelin receptors that mediate contraction in the vascular bed of several species (e.g. Sumner et al., 1992). Characterization was facilitated by the introduction of compounds with selectivity for one of these two receptors. BQ-123 (cyclo[D-Asp-L-Pro-D-Val-L-Leu-D-Trp-]), for instance, has been used extensively as a selective antagonist with high affinity for the E T A receptor (Ihara et al., 1991; Adachi et al., 1992). Although the E T A receptor was originally believed to be the most important in mediating vascular smooth muscle vasoconstriction (Masaki et al., 1991), several investigators concluded that the E T a receptor (Sumner et al., 1992), or a n o n - E TA, n o n - E T B receptor (Harrison et al., 1992) also plays a role in vasoconstriction. For this reason, and because little information is available on human vascular tissues, we investigated the effect of BQ-123 (0.1 and 1 /~M) on contractions of the human saphenous vein induced by endothelin-1 or sarafotoxin S6b. Leftover human saphenous vein was obtained intraoperatively from 9 patients (8 males, 1 female; age: 53 to 75 years), undergoing coronary bypass surgery. The
Correspondence to: Willem A. Bax, Department of Pharmacology, Thorax Center, Faculty of Medicine and Health Sciences, Erasmus University Rotterdam, P.O. Box 1738, 3000 DR Rotterdam, Netherlands. Tel. (+) 31 10 408 7551/47, fax ( +) 31 10 436 6839. Study supported by the Netherlands Heart Foundation, Grant 89.252.
tissue was immediately placed in a cold, oxygenated Krebs bicarbonate solution of the following composition: 118 mM NaCI, 4.7 mM KC1, 2.5 mM CaCI2, 1.2 mM MgSO4, 1.2 mM K H 2 P O 4 , 25 mM NaHCO2, and 8.3 mM glucose; p H 7.4. The vein was cleaned of adhesive fat and connective tissue, and the endothelium was removed using a cotton swab. Ring segments (4 mm) were suspended in 15-ml organ baths to measure isometric tension. The absence of endothelium was verified by observing a lack of relaxation to acetylcholine (1/xM) or bradykinin (1/~M) after precontraction with noradrenaline (0.1/~M). After measuring the contractile response to K ÷ (100 mM), the segments were either incubated with BQ-123 (0.1 or 1 /xM) for 30 min, or remained untreated as a control segment. Subsequently, a concentration-response curve for endothelin-1 or sarafotoxin S6b was obtained in all vessel segments. Contraction was expressed as a percentage of K + (100 mM)-induced contractions. Endothelin-1, sarafotoxin S6b and BQ-123 were purchased from Neosystem S.A. (Strasbourg, France); all other drugs were from Sigma Chemical Co. (St. Louis, MO, USA). Both endothelin-1 and sarafotoxin S6b caused concentration-dependent contractile responses of the vessel segments (PD2:8.06 + 0.10 and 8.13 + 0.05, respectively; E ~ x : 123 + 7% and 135 + 6% of K ÷ (100 mM)-induced contractions, respectively; n = 7-9). BQ123 (0.1 and 1/~M) did not affect endothelin-l-induced contraction. The contractile responses induced by sarafotoxin in S6b in concentrations not higher than 1 nM also remained unaffected by BQ-123. However,
268
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contractile response to sarafotoxin S6b. It appears likely that the BQ-123-insensitive receptor, which mediates the contractile response to low concentrations of sarafotoxin S6b, is similar to the receptor that mediates the contractile response to endothelin-1. The exact nature of this receptor remains to be determined. The BQ-123-sensitive receptor, which mediates the contractile response to higher concentrations of sarafotoxin S6b, appears to be different from the ETA receptor; since endothelin-1 has a higher affinity than sarafotoxin S6b for the ETA receptor (Arai et al., 1990), one would expect the contractile response to endothelin-1 to also be attenuated by BQ-123. Therefore, the present data indicate that BQ-123 may have relatively high affinity for a non-ETA, non-ET B receptor. Interestingly, a previous study using quantitative receptor autoradiography in the human coronary artery also indicated that [lzSI]sarafotoxin S6b but not [~zSI]endothelin-1 labeled a non-ETA, non-ET a receptor with high affinity for BQ-123 (Bax et al., 1993).
Acknowledgements
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~:
75
r-
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This study was supported by the Netherlands Heart Foundation, Grant 89.252. We especially would like to thank the surgeons and staff of the Thorax Center for providing the tissue, and T. Inan and R.W.G. Petterson for skilful technical assistance.
o O
2s
References
C
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0
0 ,
.
11
,
10
-
,
9
•
,
8
-Log [sarafot0xin
.
,
7
.
,
6
S6b]
Fig. 1. Contractions of the human isolated saphenous vein in response to endothelin-1 (top panel, A) or sarafotoxin S6b (lower panel, B), without (<3, control) or in the presence of BQ-123 (0.1 /zM, e; 1 ttM, A). The concentration refers to tool/1. Contractions are expressed as a percentage of K + (100 mM)-induced contractions. n = 7-9.
contractions induced by higher concentrations of sarafotoxin S6b were concentration dependently attenuated by BQ-123 (0.1 and 1 t~M; fig. 1). We conclude that endothelin-1 contracts the human isolated saphenous vein via a non-ETA receptor, since 1 /~M BQ-123 (a potent ETA receptor antagonist; Ihara et al., 1991; Adachi et al., 1992) did not attenuate the contractile response to endothelin-1. On the other hand, BQ-123 caused a biphasic inhibition of sarafotoxin S6b-induced contractions, indicating heterogeneity of the receptor population mediating the
Adachi, M., Y.-Y. Yang, A. Treciak, Y. Furuichi and C. Miyamoto, 1992, Identification of a domain of ET A receptor required for ligand binding, FEBS Lett. 311, 179. Arai, H., S. Hori, I. Aramori, H. Ohkubo and S. Nakanishi, 1990, Cloning and expression of a eDNA encoding an endothelin receptor, Nature 348, 730. Bax, W.A., A.T. Bruinvels, P.R. Saxena and D. Hoyer, 1993, Endothelin receptors in human cardiac tissue; a quantitative autoradiographic analysis, Br. J. Pharmacol. 108, l12P. Harrison, V.J., A. Randriantsoa and P. Schoeffter, 1992, Heterogeneity of endothelin-sarafotoxin receptors mediating contraction of pig coronary artery, Br. J. Pharmacol. 105, 511. Ihara, M., K. Noguehi, T. Saeki, T. Fukuroda, S. Tsudicha, S. Kimura, T. Fukami, K. Ishikawa, M. Nishikibe and M. Yano, 1991, Biological profiles of highly potent novel endothelin antagonists selective for the ETA receptor, Life Sci. 50, 247. Masaki, T., S. Kimura, M. Yanagisawa and K. Goto, 1991, Molecular and cellular mechanism of endothelin regulation; implication for vascular function, Circulation 84, 1457. Sakurai, T., M. Yanagisawa, Y. Takuwa, H. Miyazaki, S. Kimura, IC Goto and T. Masaki, 1990, Cloning of a cDNA encoding a non-isopeptide-selective subtype of the endothelin receptor, Nature 348, 732. Sumner, M.J., T.R. Cannon, J.W. Mundin, D.G. White and I.S. Watts, 1992, Endothelin ETA and ET B receptors mediate vascular smooth muscle contraction, Br. J. Pharmacol. 107, 858.