Br. J. Pharmacol.
(1992), 105, 929-932
'."
Macmillan Press Ltd, 1992
Phosphoramidon potentiates the contractile response to endothelin-3, but not endothelin-1 in isolated airway tissue 'K.O. McKay, J.L. Black & *C.L. Armour Departments of Pharmacology and *Pharmacy, The University of Sydney, Australia 1. Phosphoramidon (10 IAM) markedly increased the contractile response to endothelin-3 in human and rabbit bronchus in vitro. In human tissue the contractile response to 0.3 fLM endothelin-3 was significantly increased from 54 ± 12% to 137 ± 34% (of the response to 1 mm acetylcholine) in the presence of phosphoramidon. Similarly, in rabbit isolated bronchus, the endothelin-3-induced response was increased from 34±5% to 61±7%. 2. In addition, the potency (as measured by EC30 values) of this peptide in human and rabbit airways was significantly augmented in the presence of the enzyme inhibitor. The geometric mean EC30 value was decreased from 53 nm (95% CI: 15, 190) to 8 nm (95% CI:3, 23) in human bronchus and from 150 nm (95% CI:89, 250) to 23 nM (95% CI:ll, 50) in rabbit tissue. 3 Neither the potency nor the response (at 0.3 JLM) to endothelin-3 in canine bronchial rings was altered after incubation of the tissue in phosphoramidon. 4 A previous study carried out in human airways has implied that the difference in potency between endothelin-I and endothelin-3 may be attributed to a heterogeneous endothelin receptor population. The results of our study, while also demonstrating this difference in potency, have shown that this marked difference, as well as that obvious in rabbit airway tissue can be abolished in the presence of phosphoramidon. 5 Phosphoramidon produced no change in the cumulative concentration-response curve for endothelin1 in airway tissue from the three species studied. 6 These results suggest that a phosphoramidon-sensitive enzyme (probably neutral endopeptidase) found in lung, may be responsible for local degradation of endothelin-3, but not endothelin-l in human and rabbit isolated bronchus. Keywords: Endothelin-1; endothelin-3; human bronchus; rabbit bronchus; canine bronchus; phosphoramidon; neutral endopeptidase
Introduction The endothelins are a group of 21 amino acid peptides. Currently, three isoforms of endothelin have been isolated and characterized. Endothelin-1 induces potent and prolonged contraction of airways both in vivo and in vitro. Removal of epithelium and inhibition of neutral endopeptidase increases the contractile effects of endothelin in guinea-pig isolated airways (Maggi et al., 1989; Hay, 1989). In addition, pretreatment of guinea-pigs with phosphoramidon significantly augments the bronchopulmonary response to aerosolized endothelin-l (Boichot et al., 1991). Phosphoramidon increases the contractile response to various neuropeptides including neurokinin A in human and rabbit isolated airways (Black et al., 1988; 1990). Neutral endopeptidase (EC 3.4.24.11) is a phosphoramidon-sensitive enzyme located in the pulmonary epithelium (Johnson et al., 1985; Sekizawa et al., 1987). It is believed that inhibition of this enzyme by phosphoramidon is responsible for the potentiation we and others have previously observed in airways from a variety of species including man. In this study we investigated the possibility that a phosphoramidon-sensitive enzyme may be responsible for the local metabolism of endothelin in human, rabbit and canine airway tissue. Various isoforms of endothelin have significantly different potencies in human bronchus (Advenier et al., 1990; McKay et al., 1991a) and there appear to be marked species differences in the action of endothelin in airway tissue (McKay et al., 1991b). We have therefore examined the effect of phosphoramidon on the cumulative concentration-
'Author for correspondence at Department of Pharmacology, Bosch Building [DO5], The University of Sydney, N.S.W. 2006, Australia.
response curves to both endothelin-l and endothelin-3 in human, rabbit and canine bronchus.
Methods Samples of human lung were obtained from tissue surgically removed from 14 patients with pulmonary carcinoma. Lungs were also removed from New Zealand white rabbits and adult mongrel dogs immediately after they had been killed (cervical disslocation or pentobarbitone sodium overdosage) by methods approved by the Animal Ethical Review Committee of The University of Sydney. The tissue was placed in oxygenated ice-cold Krebs-Henseleit solution (composition in mM: NaCl 118.4, KCI 4.7, CaCl2.2H20 2.5, MgSO4.7H20 1.2, KH2PO4 1.2, NaHCO3 25.0 and D-glucose 11. 1). Bronchi were dissected free from surrounding parenchyma and cut into rings measuring 3-6 mm in internal diameter and 4-5 mm in length. Paired rings of bronchus were mounted and equilibrated against a 1-2 g load in 5 ml water jacketed organ baths containing Krebs-Henseleit solution maintained at 37°C and aerated with 5% CO2 in 02 as previously described (Black et al., 1990). When possible the tissue pairs were studied in duplicate. When stable tone had been established, a supramaximal dose of acetylcholine (1 mM) was added to the baths and the contractile response (reference response) allowed to plateau (.
0) CU
8060-
40 20CE Q._
E
0,t Results Both endothelin-1 and endothelin-3 produced sustained contractions of human, rabbit and canine isolated bronchus. Due to limited availability of the peptides, the maximal bath concentration achieved in all experiments was 0.3 JM and at this concentration responses did not appear to be maximal. The mean values for the tension (expressed as a percentage of the reference contraction to acetylcholine) induced by endothelin-l and endothelin-3 at a concentration of 0.3 JM in rabbit and canine airways were not significantly different from one another (Figure 1, Tables 1 and 2). However, the mean tension generated by 0.3 JM endothelin-l was significantly greater than that by 0.3 JM endothelin-3 in human isolated bronchus (P = 0.05, see Tables 1 and 2 for values). When unpaired Student's t tests were used to analyse differences in potency between endothelin-1 and endothelin-3, the geometric mean EC30 value for endothelin-1 was found to be significantly lower than the value for endothelin-3 in tissue from all species studied. Phosphoramidon significantly increased the response to 0.03, 0.1 and 0.3 JM endothelin-3 in the human and rabbit preparations, but had no effect upon the response elicited by this peptide in canine airways (Table 2, Figure 2). The potency of endothelin-3 in human and rabbit bronchial rings, but not in canine bronchus, was increased by phosphoramidon, in that the geometric mean EC30 value in human bronchus was decreased from 53 nM to 8 nM (n = 7, P = 0.04) and in rabbit airways from 150 nM to 23 nM (n = 7, P = 0.0004) (Table 2). In contrast, neither the potency nor the magnitude of the response to any concentration of endothelin-l (Table 1) in airway tissue from the three species was affected by phosphoramidon. In view of the lack of effect of phosphoramidon on the
-
0-J -10 -9 -8 -7 -6 log concentration Endothelin-1 (M)
-11
Figure 1 Mean cumulative concentration-response curves for endothelin-1 in the presence (Q---Q) and absence (e *) of phosphoramidon in human (a), rabbit (b) and dog (c) bronchus. Mean responses for 7 experiments are expressed as a percentage of the reference response obtained to 1 mm acetylcholine; s.e. values are shown as vertical bars; n = 7 in (a), (b) and (c).
Table 1 The effect of phosphoramidon on the magnitude of the contractile response* and potency (EC30)t of endothelin-1 in human, rabbit and canine bronchus
Human
(n = 7) Rabbit
(n = 7)
Dog (n = 7)
Contractile response (s.e.mean) Control Phosph 89.2 118.8 (10.5) (10.5) 60.7 60.6
(10.9) 98.5 (6.1)
(5.5)
107.9
(7.5)
EC30 (95% CQ) Control Phosph 4.5nM 9.0nM (0.6, 35.1) (4.9, 17.0) 42.2 nM 32.7 nM (20.6, 86.3) (17.0, 63.2) 24.3 nM 22.2 nM (11.8, 50.0) (13.2, 37.5)
Mean values are shown together with s.e. mean or 95% confidence intervals (CI). *Response to 0.3 pM endothelin-I (the highest bath concentration achieved) expressed as a percentage of the reference contraction to acetylcholine (see Methods). tThe concentration of endothelin-I at which a contractile response equal to 30% of that induced by I mM acetylcholine is attained (see Methods).
PHOSPHORAMIDON AND ENDOTHELIN IN AIRWAYS Table 2 The effect of phosphoramidon on the magnitude of the contractile responset and potency (EC30)T of endothelin-3 in human, rabbit and canine bronchus
Human (n = 7) Rabbit (n = 7) Dog (n = 7)
Contractile response (s.e.mean) Control Phosph 136.5* 54.2 (33.7) (12.0) 33.8 61.4** (7.0) (5.0) 79.9 96.5 (8.9) (11.6)
EC30
(95% CI)
Control 53.0 nM (15,1, 185.8) 150.3 nM (89.1, 253.5) 87.7 nM (47.8, 161.1)
Phosph 8.0 nM* (2.8, 22.6) 23.3 nM*** (10.9, 50.1) 49.6 nM (28.7, 85.3)
Mean values are shown together with s.e.mean or 95% confidence intervals (CI). * Indicates significantly different from control value (P. 0.05). **Indicates significantly different from control value (PS0.01). *** Indicates significantly different from control value (P 0.001). tResponse to 0.3gM endothelin-3 (the highest bath concentration achieved) expressed as a percentage of the reference contraction to acetylcholine (see Methods). $The concentration of endothelin-3 at which a contractile response equal to 30% of that induced by 1 mM acetylcholine is attained (see Methods).
responses to endothelin in canine airways we decided to assess the effect of this enzyme inhibitor on the contractions
to the neuropeptides substance P and neurokinin A. There contractile response to either neurokinin A
of these other enzyme inhibitors produced the enhancement to endothelin-l. A previous study carried out in human airways has implied that the difference in potency between endothelin-l and endothelin-3 may be attributed to a heterogeneous endothelin receptor population in this tissue (Advenier et al., 1990). The results of our study, while also demonstrating this difference in potency, have shown that this marked difference, as well as that obvious in rabbit airway tissue, can be abolished in the presence of phosphoramidon. It is possible that neutral endopeptidase has differential affinity for isoforms of the endothelin family and is responsible for the local degradation of endothelin-3 but not endothelin-l. Recently, all three isoforms of endothelin have been shown to be substrates for neutral endopeptidase (Vijayaraghavan et al., 1990). Hydrolysis of the endothelins differs according to the amino acid sequence and conformation of the peptide. As one of the major sites for cleavage of endothelin was shown to be at position 6 of the peptide, and as endothelin-1 and endothelin-3 differ in the amino acid at this position, it is possible
180
I
a
*
.s 150X 90-
E 60 CU30
was however, no or substance P
in the presence or the absence of phosphoramidon (n = 3 dogs) although the mean tension generated in response to mM acetylcholine was 5.8 ± 0.7 g.
Discussion This study has shown that the presence of phosphoramidon at a concentration of 1O gM greatly enhanced the contractile activity of endothelin-3 but not endothelin-l in human and rabbit bronchus. Moreover, phosphoramidon abolished the differences in potency between endothelin-1 and endothelin-3 in these preparations. There was however, no change in the potency nor the magnitude of the contractile response to any concentration (up to 0.3 gM) of either isoform of endothelin in canine airways when the tissue had been incubated in phosphoramidon. Previous studies have also shown increased effects of endothelin in the presence of an inhibitor of neutral endopeptidase. Maggi et al. (1989) reported that, in guinea-pig airways, thiorphan, another inhibitor of neutral endopeptidase, caused a greater enhancement of the action of endothelin-3 than endothelin-1 although the responses to both forms of endothelin were augmented. In another study also carried out in guinea-pig airways, Hay (1989) demonstrated that removal of the epithelium and incubation in phosphoramidon resulted in increased responsiveness to endothelin. We however, found no significant potentiation of the action of endothelin1 by phosphoramidon. This discrepancy in the effect on endothelin-1 between our findings and those previously published could be a reflection of the species difference or variation in experimental protocol. Marked differences between species in the mechanism of action of endothelin have been reported. For example, cyclo-oxygenase product generation in response to endothelin, while involved in the contractile response in guinea-pig airways (Sarria et al., 1990), is not responsible for the endothelin-induced contraction of human bronchus (Advenier et al., 1990; McKay et al., 1991a). Maggi et al. (1989) used captopril, bestatin and indomethacin simultaneously with thiorphan in the study in guinea-pig airways and found significant potentiation but it is possible that one
931
.
-10
-11
.
-8
-9
-6
-7
log concentration Endothelin-3 (M) a1)
b 80,
0
*4.
**1
60 ._ 0)
x
E 0-o
40
-
20
-
0
~
~
-10
-9
B
*
o.
-11
.
-8
.
.
-7
-6
log concentration Endothelin-3 (M) c
Q) 120
-
°100-
> 80-
a)
_ 60E 40-
._
200
-
-11
-10
-9
-8
-7
-6
log concentration Endothelin-3 (M)
Figure 2 Mean cumulative concentration-response curves for ) of endothelin-3 in the presence (O---O) and absence ( phosphoramidon in human (a), rabbit (b) and dog (c) bronchus. Mean responses for 7 experiments are expressed as a percentage of the reference response obtained to 1 mM acetylcholine; s.e. values are shown as vertical bars; n = 7 in (a), (b) and (c). Significant differences between control and treated tissues are indicated (*P
(1992), 105, 929-932
'."
Macmillan Press Ltd, 1992
Phosphoramidon potentiates the contractile response to endothelin-3, but not endothelin-1 in isolated airway tissue 'K.O. McKay, J.L. Black & *C.L. Armour Departments of Pharmacology and *Pharmacy, The University of Sydney, Australia 1. Phosphoramidon (10 IAM) markedly increased the contractile response to endothelin-3 in human and rabbit bronchus in vitro. In human tissue the contractile response to 0.3 fLM endothelin-3 was significantly increased from 54 ± 12% to 137 ± 34% (of the response to 1 mm acetylcholine) in the presence of phosphoramidon. Similarly, in rabbit isolated bronchus, the endothelin-3-induced response was increased from 34±5% to 61±7%. 2. In addition, the potency (as measured by EC30 values) of this peptide in human and rabbit airways was significantly augmented in the presence of the enzyme inhibitor. The geometric mean EC30 value was decreased from 53 nm (95% CI: 15, 190) to 8 nm (95% CI:3, 23) in human bronchus and from 150 nm (95% CI:89, 250) to 23 nM (95% CI:ll, 50) in rabbit tissue. 3 Neither the potency nor the response (at 0.3 JLM) to endothelin-3 in canine bronchial rings was altered after incubation of the tissue in phosphoramidon. 4 A previous study carried out in human airways has implied that the difference in potency between endothelin-I and endothelin-3 may be attributed to a heterogeneous endothelin receptor population. The results of our study, while also demonstrating this difference in potency, have shown that this marked difference, as well as that obvious in rabbit airway tissue can be abolished in the presence of phosphoramidon. 5 Phosphoramidon produced no change in the cumulative concentration-response curve for endothelin1 in airway tissue from the three species studied. 6 These results suggest that a phosphoramidon-sensitive enzyme (probably neutral endopeptidase) found in lung, may be responsible for local degradation of endothelin-3, but not endothelin-l in human and rabbit isolated bronchus. Keywords: Endothelin-1; endothelin-3; human bronchus; rabbit bronchus; canine bronchus; phosphoramidon; neutral endopeptidase
Introduction The endothelins are a group of 21 amino acid peptides. Currently, three isoforms of endothelin have been isolated and characterized. Endothelin-1 induces potent and prolonged contraction of airways both in vivo and in vitro. Removal of epithelium and inhibition of neutral endopeptidase increases the contractile effects of endothelin in guinea-pig isolated airways (Maggi et al., 1989; Hay, 1989). In addition, pretreatment of guinea-pigs with phosphoramidon significantly augments the bronchopulmonary response to aerosolized endothelin-l (Boichot et al., 1991). Phosphoramidon increases the contractile response to various neuropeptides including neurokinin A in human and rabbit isolated airways (Black et al., 1988; 1990). Neutral endopeptidase (EC 3.4.24.11) is a phosphoramidon-sensitive enzyme located in the pulmonary epithelium (Johnson et al., 1985; Sekizawa et al., 1987). It is believed that inhibition of this enzyme by phosphoramidon is responsible for the potentiation we and others have previously observed in airways from a variety of species including man. In this study we investigated the possibility that a phosphoramidon-sensitive enzyme may be responsible for the local metabolism of endothelin in human, rabbit and canine airway tissue. Various isoforms of endothelin have significantly different potencies in human bronchus (Advenier et al., 1990; McKay et al., 1991a) and there appear to be marked species differences in the action of endothelin in airway tissue (McKay et al., 1991b). We have therefore examined the effect of phosphoramidon on the cumulative concentration-
'Author for correspondence at Department of Pharmacology, Bosch Building [DO5], The University of Sydney, N.S.W. 2006, Australia.
response curves to both endothelin-l and endothelin-3 in human, rabbit and canine bronchus.
Methods Samples of human lung were obtained from tissue surgically removed from 14 patients with pulmonary carcinoma. Lungs were also removed from New Zealand white rabbits and adult mongrel dogs immediately after they had been killed (cervical disslocation or pentobarbitone sodium overdosage) by methods approved by the Animal Ethical Review Committee of The University of Sydney. The tissue was placed in oxygenated ice-cold Krebs-Henseleit solution (composition in mM: NaCl 118.4, KCI 4.7, CaCl2.2H20 2.5, MgSO4.7H20 1.2, KH2PO4 1.2, NaHCO3 25.0 and D-glucose 11. 1). Bronchi were dissected free from surrounding parenchyma and cut into rings measuring 3-6 mm in internal diameter and 4-5 mm in length. Paired rings of bronchus were mounted and equilibrated against a 1-2 g load in 5 ml water jacketed organ baths containing Krebs-Henseleit solution maintained at 37°C and aerated with 5% CO2 in 02 as previously described (Black et al., 1990). When possible the tissue pairs were studied in duplicate. When stable tone had been established, a supramaximal dose of acetylcholine (1 mM) was added to the baths and the contractile response (reference response) allowed to plateau (.
0) CU
8060-
40 20CE Q._
E
0,t Results Both endothelin-1 and endothelin-3 produced sustained contractions of human, rabbit and canine isolated bronchus. Due to limited availability of the peptides, the maximal bath concentration achieved in all experiments was 0.3 JM and at this concentration responses did not appear to be maximal. The mean values for the tension (expressed as a percentage of the reference contraction to acetylcholine) induced by endothelin-l and endothelin-3 at a concentration of 0.3 JM in rabbit and canine airways were not significantly different from one another (Figure 1, Tables 1 and 2). However, the mean tension generated by 0.3 JM endothelin-l was significantly greater than that by 0.3 JM endothelin-3 in human isolated bronchus (P = 0.05, see Tables 1 and 2 for values). When unpaired Student's t tests were used to analyse differences in potency between endothelin-1 and endothelin-3, the geometric mean EC30 value for endothelin-1 was found to be significantly lower than the value for endothelin-3 in tissue from all species studied. Phosphoramidon significantly increased the response to 0.03, 0.1 and 0.3 JM endothelin-3 in the human and rabbit preparations, but had no effect upon the response elicited by this peptide in canine airways (Table 2, Figure 2). The potency of endothelin-3 in human and rabbit bronchial rings, but not in canine bronchus, was increased by phosphoramidon, in that the geometric mean EC30 value in human bronchus was decreased from 53 nM to 8 nM (n = 7, P = 0.04) and in rabbit airways from 150 nM to 23 nM (n = 7, P = 0.0004) (Table 2). In contrast, neither the potency nor the magnitude of the response to any concentration of endothelin-l (Table 1) in airway tissue from the three species was affected by phosphoramidon. In view of the lack of effect of phosphoramidon on the
-
0-J -10 -9 -8 -7 -6 log concentration Endothelin-1 (M)
-11
Figure 1 Mean cumulative concentration-response curves for endothelin-1 in the presence (Q---Q) and absence (e *) of phosphoramidon in human (a), rabbit (b) and dog (c) bronchus. Mean responses for 7 experiments are expressed as a percentage of the reference response obtained to 1 mm acetylcholine; s.e. values are shown as vertical bars; n = 7 in (a), (b) and (c).
Table 1 The effect of phosphoramidon on the magnitude of the contractile response* and potency (EC30)t of endothelin-1 in human, rabbit and canine bronchus
Human
(n = 7) Rabbit
(n = 7)
Dog (n = 7)
Contractile response (s.e.mean) Control Phosph 89.2 118.8 (10.5) (10.5) 60.7 60.6
(10.9) 98.5 (6.1)
(5.5)
107.9
(7.5)
EC30 (95% CQ) Control Phosph 4.5nM 9.0nM (0.6, 35.1) (4.9, 17.0) 42.2 nM 32.7 nM (20.6, 86.3) (17.0, 63.2) 24.3 nM 22.2 nM (11.8, 50.0) (13.2, 37.5)
Mean values are shown together with s.e. mean or 95% confidence intervals (CI). *Response to 0.3 pM endothelin-I (the highest bath concentration achieved) expressed as a percentage of the reference contraction to acetylcholine (see Methods). tThe concentration of endothelin-I at which a contractile response equal to 30% of that induced by I mM acetylcholine is attained (see Methods).
PHOSPHORAMIDON AND ENDOTHELIN IN AIRWAYS Table 2 The effect of phosphoramidon on the magnitude of the contractile responset and potency (EC30)T of endothelin-3 in human, rabbit and canine bronchus
Human (n = 7) Rabbit (n = 7) Dog (n = 7)
Contractile response (s.e.mean) Control Phosph 136.5* 54.2 (33.7) (12.0) 33.8 61.4** (7.0) (5.0) 79.9 96.5 (8.9) (11.6)
EC30
(95% CI)
Control 53.0 nM (15,1, 185.8) 150.3 nM (89.1, 253.5) 87.7 nM (47.8, 161.1)
Phosph 8.0 nM* (2.8, 22.6) 23.3 nM*** (10.9, 50.1) 49.6 nM (28.7, 85.3)
Mean values are shown together with s.e.mean or 95% confidence intervals (CI). * Indicates significantly different from control value (P. 0.05). **Indicates significantly different from control value (PS0.01). *** Indicates significantly different from control value (P 0.001). tResponse to 0.3gM endothelin-3 (the highest bath concentration achieved) expressed as a percentage of the reference contraction to acetylcholine (see Methods). $The concentration of endothelin-3 at which a contractile response equal to 30% of that induced by 1 mM acetylcholine is attained (see Methods).
responses to endothelin in canine airways we decided to assess the effect of this enzyme inhibitor on the contractions
to the neuropeptides substance P and neurokinin A. There contractile response to either neurokinin A
of these other enzyme inhibitors produced the enhancement to endothelin-l. A previous study carried out in human airways has implied that the difference in potency between endothelin-l and endothelin-3 may be attributed to a heterogeneous endothelin receptor population in this tissue (Advenier et al., 1990). The results of our study, while also demonstrating this difference in potency, have shown that this marked difference, as well as that obvious in rabbit airway tissue, can be abolished in the presence of phosphoramidon. It is possible that neutral endopeptidase has differential affinity for isoforms of the endothelin family and is responsible for the local degradation of endothelin-3 but not endothelin-l. Recently, all three isoforms of endothelin have been shown to be substrates for neutral endopeptidase (Vijayaraghavan et al., 1990). Hydrolysis of the endothelins differs according to the amino acid sequence and conformation of the peptide. As one of the major sites for cleavage of endothelin was shown to be at position 6 of the peptide, and as endothelin-1 and endothelin-3 differ in the amino acid at this position, it is possible
180
I
a
*
.s 150X 90-
E 60 CU30
was however, no or substance P
in the presence or the absence of phosphoramidon (n = 3 dogs) although the mean tension generated in response to mM acetylcholine was 5.8 ± 0.7 g.
Discussion This study has shown that the presence of phosphoramidon at a concentration of 1O gM greatly enhanced the contractile activity of endothelin-3 but not endothelin-l in human and rabbit bronchus. Moreover, phosphoramidon abolished the differences in potency between endothelin-1 and endothelin-3 in these preparations. There was however, no change in the potency nor the magnitude of the contractile response to any concentration (up to 0.3 gM) of either isoform of endothelin in canine airways when the tissue had been incubated in phosphoramidon. Previous studies have also shown increased effects of endothelin in the presence of an inhibitor of neutral endopeptidase. Maggi et al. (1989) reported that, in guinea-pig airways, thiorphan, another inhibitor of neutral endopeptidase, caused a greater enhancement of the action of endothelin-3 than endothelin-1 although the responses to both forms of endothelin were augmented. In another study also carried out in guinea-pig airways, Hay (1989) demonstrated that removal of the epithelium and incubation in phosphoramidon resulted in increased responsiveness to endothelin. We however, found no significant potentiation of the action of endothelin1 by phosphoramidon. This discrepancy in the effect on endothelin-1 between our findings and those previously published could be a reflection of the species difference or variation in experimental protocol. Marked differences between species in the mechanism of action of endothelin have been reported. For example, cyclo-oxygenase product generation in response to endothelin, while involved in the contractile response in guinea-pig airways (Sarria et al., 1990), is not responsible for the endothelin-induced contraction of human bronchus (Advenier et al., 1990; McKay et al., 1991a). Maggi et al. (1989) used captopril, bestatin and indomethacin simultaneously with thiorphan in the study in guinea-pig airways and found significant potentiation but it is possible that one
931
.
-10
-11
.
-8
-9
-6
-7
log concentration Endothelin-3 (M) a1)
b 80,
0
*4.
**1
60 ._ 0)
x
E 0-o
40
-
20
-
0
~
~
-10
-9
B
*
o.
-11
.
-8
.
.
-7
-6
log concentration Endothelin-3 (M) c
Q) 120
-
°100-
> 80-
a)
_ 60E 40-
._
200
-
-11
-10
-9
-8
-7
-6
log concentration Endothelin-3 (M)
Figure 2 Mean cumulative concentration-response curves for ) of endothelin-3 in the presence (O---O) and absence ( phosphoramidon in human (a), rabbit (b) and dog (c) bronchus. Mean responses for 7 experiments are expressed as a percentage of the reference response obtained to 1 mM acetylcholine; s.e. values are shown as vertical bars; n = 7 in (a), (b) and (c). Significant differences between control and treated tissues are indicated (*P
