European Journal of Pharmacology, 214 (1992) 133-141
133
© 1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$05.00
EJP 52411
Effects of endothelin-3 on the isolated guinea-pig ileum: role of Na + ions and endothelin receptor subtypes N o b u c o Miasiro a n d A n t o n i o C.M. Paiva Department of Biophysics, Escola Paulista de Medicina, C.P. 20.388, Sdo Paulo 04034, SP, Brazil Received 24 September 1991, revised MS received 19 November 1991, accepted 4 February 1992
Endothelin-3 induced a relaxation followed by contraction in the isolated guinea-pig ileum. The contractile but not the relaxant component of the response was concentration-dependent in the dose range studied. Neuronal mechanisms, cyclic GMP and ATP-dependent K ÷ channels are not involved in the relaxing effect since this component was not affected by either tetrodotoxin, methylene blue or glibenclamide. Endothelin-3 induced tachyphylaxis (homologous desensitization) that was not fully reversed after a 3-h resting period. The responses were inhibited in low-Na + medium or after treatment with ouabain. Verapamil affected both the relaxant and the contractile components of the response, and phorbol-12,13-dibutyrate affected mainly the contractile component. Cross-tachyphylaxis studies between endothelin-1 and endothelin-3 suggest the existence of at least two endothelin receptor subtypes (or different ligand-receptor complexes) in the guinea-pig ileum. Endothelin-3; Endothelin-1; Ileum (guinea-pig); Desensitization; Na + ions; Endothelin receptor subtypes
I. Introduction
The endothelin/sarafotoxin family of peptides comprises endothelin-1 (porcine endothelin), originally isolated from porcine endothelium in culture (Yanagisawa et al., 1988), endothelin-2 (or human endothelin), endothelin-3 (or rat endothelin), mouse endothelin (or vasoactive intestinal constrictor polypeptide, VIC) (Yanagisawa and Masaki, 1989; Saida et al., 1989) as well as the sarafotoxins, from the venom of the Israeli burrowing asp Atractaspis engaddensis (Kloog et al., 1988). Endothelin-1 is widely distributed in numerous organs and tissues of mammals, including the central nervous system (Hoyer et al., 1989; Kosaki et al., 1989), and has been reported to elicit a wide spectrum of biological effects including vasoconstriction/smooth muscle contraction, mitogenesis and protooncogene expression in diverse tissues and species (Yanagisawa and Masaki, 1989; Inoue et al., 1989; Vane et al., 1989). However, much less is known about endothelin-3, which differs from endothelin-1 in the amino acid sequence at positions 2, 4, 5, 6, 7 and 14, and shares
Correspondence to: N. Miasiro, Departamento de Biofisica, Escola Paulista de Medicina, C.P. 20.388, 04034 Silo Paulo, SP, Brazil. Tel. 55.11.572 4583, fax 55.11.549 2127.
the carboxy-terminal sequence 16-21 of endothelin-1. Endothelin-3 is not derived from the vascular endothelial cells but seems to be expressed in the central nervous system (Inoue et al., 1989). We have recently investigated the effect of endothelin-1 on the isolated guinea-pig ileum, and observed that the peptide induced a direct unusual biphasic effect (relaxation followed by contraction). We have also observed that the responses to endothelin-1 were characterized by a homologous desensitization, and were markedly dependent on the Na ÷ gradient in smooth muscle cells (Miasiro and Paiva, 1990a). Little is known about the comparative pharmacology of endothelin-1 and endothelin-3. It was shown that endothelin-3 constricts rat aorta strips with less potency than endothelin-1, and that the coronary pressor effect of endothelin-3 is more transient than that of endothelin-1. The two substances have qualitatively similar contractile effects in vitro and vasodilator activity in vivo (for a review see Le Monnier de Gouville et al., 1990a). However, the differences in pharmacological potencies between the isopeptides suggested the possible existence of endothelin receptor subtypes (Inoue et al., 1989; Warner et al., 1989; Kloog et al., 1989). Cross-linking studies with membrane preparations from chick heart (Watanabe et al., 1989), rat lung (Masuda et al., 1989), rat mesangial cells (Sugiura et al., 1989) and human placenta (Nakajo et al., 1989)
134
have identified multiple binding sites that suggest the possibility of multiple classes of endothelin receptors. Saturation and competitive displacement binding studies also suggest that multiple classes of receptors exist, with different affinities for endothelin isopeptides and that the distribution of the receptor subtypes is tissuespecific (Yanagisawa and Masaki, 1989; Kloog et al., 1989). It is interesting to observe that the majority of these studies on heterogeneity of endothelin receptors was based mainly on differences in their affinity for endothelin-3. We have now investigated the action of endothelin-3 on the isolated guinea-pig ileum and compared its contractile properties with those previously described for endothelin-1 (Miasiro and Paiva, 1990a), to test whether or not endothelin-3 and endothelin-1 would interact with the same receptor.
2. Materials and methods
Guinea-pigs of either sex (200-500 g) were stunned by a blow to the head and bled. A 20-cm portion of the terminal ileum was removed and washed with Tyrode solution at room temperature. Segments of the ileum (3,5-4.0 cm) were suspended in a 5-ml organ chamber which contained Tyrode solution maintained at 37°C and was bubbled with a constant stream of 0 2. Recordings of isotonic contractions were made under a 1-g load, on smoked drums, using frontal levers with 6-fold amplification. The isometric contractions were recorded through a Narco Bio-Systems force transducer, model F-60, and an ECB model 102-B recorder. Resting tension was adjusted to 1 g. Unless otherwise noted, the agonists were left in contact with the preparations for 3 min. Before the experiment itself, 60 mM KC1 was always added to elicit maximal control responses. The results are presented as means + S.E.M., and the significance of the differences between the values was assessed by means of Student's t-test. P values less than 0.05 were considered significant.
Synthetic rat endothelin (endothelin-3) and porcine endothelin (endothelin-1) were from the Peptide Research Institute (Osaka, Japan). Angiotensin II and [Sarl,AlaS]angiotensin II were synthesized by the solid phase method as previously described (Paiva et al., 1974). Acetylcholine chloride, tetrodotoxin, verapamil and phorbol-12,13-dibutyrate (PDBu) were from Sigma Chemical Co. (St. Louis, MO, USA); ouabain octahydrate from Aldrich Chemical Co. (Milwaukee, WI, USA); glibenclamide was from Hoechst do Brasil Quimica e Farmac~utica S.A. (Suzano, SP, Brazil); methylene blue and inorganic salts from Merck, Darmstadt (FRG). All agonists and inhibitors (except for glibenclamide and PDBu that were dissolved in dimethyl sulfoxide) were prepared as aqueous concentrated solutions and were diluted with saline solution (9 g/1 NaC1) before addition to the bath.
3. Results
3.1. Isometric responses in normal medium
In general, responses induced by KC1 (fig. 1B), angiotensin II (fig. 1A) and other contractile agents in the
A
t
t
ET-3 B
lg l
All
I 2.5 min
2.1. Solutions and drugs
The Tyrode solution had the following composition (in mM): NaC1 137; KC1 2.7; CaC12 1.36; MgC12 0.49; NaHCO 3 11.9; NaH2PO 4 0.36 and glucose 5.1. The sodium-deficient solution was obtained by isosmotic replacement of the NaCI with D-glucose to give a solution containing 80 mM Na ÷. Glucose was chosen as Na + substitute because, in contrast to sucrose, it did not affect the ileum response to the addition of hyperosmotic KCI (Shimuta et al., 1982). The pH of the solutions, when equilibrated with air, was 8.0 + 0.1.
~15 t KCI
t
ET-3
Fig. 1. Isometric recordings of the responses of the isolated guinea-pig ileum induced by (A) 200 nM endothelin-3 (ET-3) and 100 nM angiotensin II (All); (B) 60 mM KCI followed by 100 nM endothelin-3 after a 15-min interval. Upward arrows indicate additions of the agonists to the bath and downward arrows indicate washout of the preparation with fresh medium.
135 80
"' t~ z 0 o. ¢o uJ n,-
60
~
20
40
IE x
133
© 1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$05.00
EJP 52411
Effects of endothelin-3 on the isolated guinea-pig ileum: role of Na + ions and endothelin receptor subtypes N o b u c o Miasiro a n d A n t o n i o C.M. Paiva Department of Biophysics, Escola Paulista de Medicina, C.P. 20.388, Sdo Paulo 04034, SP, Brazil Received 24 September 1991, revised MS received 19 November 1991, accepted 4 February 1992
Endothelin-3 induced a relaxation followed by contraction in the isolated guinea-pig ileum. The contractile but not the relaxant component of the response was concentration-dependent in the dose range studied. Neuronal mechanisms, cyclic GMP and ATP-dependent K ÷ channels are not involved in the relaxing effect since this component was not affected by either tetrodotoxin, methylene blue or glibenclamide. Endothelin-3 induced tachyphylaxis (homologous desensitization) that was not fully reversed after a 3-h resting period. The responses were inhibited in low-Na + medium or after treatment with ouabain. Verapamil affected both the relaxant and the contractile components of the response, and phorbol-12,13-dibutyrate affected mainly the contractile component. Cross-tachyphylaxis studies between endothelin-1 and endothelin-3 suggest the existence of at least two endothelin receptor subtypes (or different ligand-receptor complexes) in the guinea-pig ileum. Endothelin-3; Endothelin-1; Ileum (guinea-pig); Desensitization; Na + ions; Endothelin receptor subtypes
I. Introduction
The endothelin/sarafotoxin family of peptides comprises endothelin-1 (porcine endothelin), originally isolated from porcine endothelium in culture (Yanagisawa et al., 1988), endothelin-2 (or human endothelin), endothelin-3 (or rat endothelin), mouse endothelin (or vasoactive intestinal constrictor polypeptide, VIC) (Yanagisawa and Masaki, 1989; Saida et al., 1989) as well as the sarafotoxins, from the venom of the Israeli burrowing asp Atractaspis engaddensis (Kloog et al., 1988). Endothelin-1 is widely distributed in numerous organs and tissues of mammals, including the central nervous system (Hoyer et al., 1989; Kosaki et al., 1989), and has been reported to elicit a wide spectrum of biological effects including vasoconstriction/smooth muscle contraction, mitogenesis and protooncogene expression in diverse tissues and species (Yanagisawa and Masaki, 1989; Inoue et al., 1989; Vane et al., 1989). However, much less is known about endothelin-3, which differs from endothelin-1 in the amino acid sequence at positions 2, 4, 5, 6, 7 and 14, and shares
Correspondence to: N. Miasiro, Departamento de Biofisica, Escola Paulista de Medicina, C.P. 20.388, 04034 Silo Paulo, SP, Brazil. Tel. 55.11.572 4583, fax 55.11.549 2127.
the carboxy-terminal sequence 16-21 of endothelin-1. Endothelin-3 is not derived from the vascular endothelial cells but seems to be expressed in the central nervous system (Inoue et al., 1989). We have recently investigated the effect of endothelin-1 on the isolated guinea-pig ileum, and observed that the peptide induced a direct unusual biphasic effect (relaxation followed by contraction). We have also observed that the responses to endothelin-1 were characterized by a homologous desensitization, and were markedly dependent on the Na ÷ gradient in smooth muscle cells (Miasiro and Paiva, 1990a). Little is known about the comparative pharmacology of endothelin-1 and endothelin-3. It was shown that endothelin-3 constricts rat aorta strips with less potency than endothelin-1, and that the coronary pressor effect of endothelin-3 is more transient than that of endothelin-1. The two substances have qualitatively similar contractile effects in vitro and vasodilator activity in vivo (for a review see Le Monnier de Gouville et al., 1990a). However, the differences in pharmacological potencies between the isopeptides suggested the possible existence of endothelin receptor subtypes (Inoue et al., 1989; Warner et al., 1989; Kloog et al., 1989). Cross-linking studies with membrane preparations from chick heart (Watanabe et al., 1989), rat lung (Masuda et al., 1989), rat mesangial cells (Sugiura et al., 1989) and human placenta (Nakajo et al., 1989)
134
have identified multiple binding sites that suggest the possibility of multiple classes of endothelin receptors. Saturation and competitive displacement binding studies also suggest that multiple classes of receptors exist, with different affinities for endothelin isopeptides and that the distribution of the receptor subtypes is tissuespecific (Yanagisawa and Masaki, 1989; Kloog et al., 1989). It is interesting to observe that the majority of these studies on heterogeneity of endothelin receptors was based mainly on differences in their affinity for endothelin-3. We have now investigated the action of endothelin-3 on the isolated guinea-pig ileum and compared its contractile properties with those previously described for endothelin-1 (Miasiro and Paiva, 1990a), to test whether or not endothelin-3 and endothelin-1 would interact with the same receptor.
2. Materials and methods
Guinea-pigs of either sex (200-500 g) were stunned by a blow to the head and bled. A 20-cm portion of the terminal ileum was removed and washed with Tyrode solution at room temperature. Segments of the ileum (3,5-4.0 cm) were suspended in a 5-ml organ chamber which contained Tyrode solution maintained at 37°C and was bubbled with a constant stream of 0 2. Recordings of isotonic contractions were made under a 1-g load, on smoked drums, using frontal levers with 6-fold amplification. The isometric contractions were recorded through a Narco Bio-Systems force transducer, model F-60, and an ECB model 102-B recorder. Resting tension was adjusted to 1 g. Unless otherwise noted, the agonists were left in contact with the preparations for 3 min. Before the experiment itself, 60 mM KC1 was always added to elicit maximal control responses. The results are presented as means + S.E.M., and the significance of the differences between the values was assessed by means of Student's t-test. P values less than 0.05 were considered significant.
Synthetic rat endothelin (endothelin-3) and porcine endothelin (endothelin-1) were from the Peptide Research Institute (Osaka, Japan). Angiotensin II and [Sarl,AlaS]angiotensin II were synthesized by the solid phase method as previously described (Paiva et al., 1974). Acetylcholine chloride, tetrodotoxin, verapamil and phorbol-12,13-dibutyrate (PDBu) were from Sigma Chemical Co. (St. Louis, MO, USA); ouabain octahydrate from Aldrich Chemical Co. (Milwaukee, WI, USA); glibenclamide was from Hoechst do Brasil Quimica e Farmac~utica S.A. (Suzano, SP, Brazil); methylene blue and inorganic salts from Merck, Darmstadt (FRG). All agonists and inhibitors (except for glibenclamide and PDBu that were dissolved in dimethyl sulfoxide) were prepared as aqueous concentrated solutions and were diluted with saline solution (9 g/1 NaC1) before addition to the bath.
3. Results
3.1. Isometric responses in normal medium
In general, responses induced by KC1 (fig. 1B), angiotensin II (fig. 1A) and other contractile agents in the
A
t
t
ET-3 B
lg l
All
I 2.5 min
2.1. Solutions and drugs
The Tyrode solution had the following composition (in mM): NaC1 137; KC1 2.7; CaC12 1.36; MgC12 0.49; NaHCO 3 11.9; NaH2PO 4 0.36 and glucose 5.1. The sodium-deficient solution was obtained by isosmotic replacement of the NaCI with D-glucose to give a solution containing 80 mM Na ÷. Glucose was chosen as Na + substitute because, in contrast to sucrose, it did not affect the ileum response to the addition of hyperosmotic KCI (Shimuta et al., 1982). The pH of the solutions, when equilibrated with air, was 8.0 + 0.1.
~15 t KCI
t
ET-3
Fig. 1. Isometric recordings of the responses of the isolated guinea-pig ileum induced by (A) 200 nM endothelin-3 (ET-3) and 100 nM angiotensin II (All); (B) 60 mM KCI followed by 100 nM endothelin-3 after a 15-min interval. Upward arrows indicate additions of the agonists to the bath and downward arrows indicate washout of the preparation with fresh medium.
135 80
"' t~ z 0 o. ¢o uJ n,-
60
~
20
40
IE x
