European Journal of Pharmacology, 176 (1990) 1-9
1
Elsevier EJP 51146
Further studies on the response of the guinea-pig isolated bronchus to endothelins and sarafotoxin S6b Carlo Alberto Maggi, Sandro Giuliani, Riccardo Patacchini, Paolo Santicioli, Antonio Giachetti and Alberto Meli Pharmacology Department, Smooth Muscle Division, Research Laboratories, A. Menarini Pharmaceuticals, Via Sette Santi 3, Florence 50131, Italy
Received 13 July 1989, revised MS received 14 September 1989, accepted 7 November 1989
Endothelin-1 (ET-1), endothelin-3 (ET-3) and sarafotoxin S6b (SRFTX) produced a concentration-dependent contraction of the guinea-pig isolated bronchus. In untreated preparations, SRFTX was about 10 times more potent than ET-1 or ET-3 and produced a greater maximal effect. Mechanical removal of the bronchial epithelium (rubbing) or the addition of indomethacin (5/~M) increased the potency of all peptides, ET-3 being enhanced more than ET-1 or SRFTX. Further, the activity of ET-3 was enhanced by a mixture of peptidase inhibitors (thiorphan, captopril, bestatin, 1/~M each). When the three potentiating factors were combined (rubbed bronchi in presence of indomethacin and peptidase inhibitors), the following order of potency was found, ET-3 = SRTFX > ET-1. For comparison, the activity of the three peptides was also studied in the rubbed rat isolated aorta in the presence of indomethacin and peptidase inhibitors and the following rank order of potency was found: ET-1 > SRFTX > ET-3. When the activity of the peptides was compared in these two preparations, ET-1 was 4 times more potent in the aorta than in the bronchus, while SRFTX and ET-3 were 8.7 and 133 times more potent in the bronchus than the aorta, respectively. These findings indicate that, in the guinea-pig bronchus, the contractile activity of these peptides is attenuated by the concomitant production of epithelium-derived relaxant factor(s), prostanoid production via the cyclooxygenase pathway and, possibly, enzymatic inactivation of added peptides. Further, the existence of multiple receptors mediating the contractile response to these peptides is suggested. Endothelin; Bronchi (guinea-pig); Airway epithelium; Indomethacin; Aorta (rat); Endothelin receptors
1. Introduction Porcine e n d o t h e l i n (ET) is a 2 1 - a m i n o acid p e p t i d e discovered as a p r o d u c t of c u l t u r e d a o r t i c e n d o t h e l i a l cells ( Y a n a g i s a w a et al., 1988b). This p e p t i d e has b e e n shown to be a very p o t e n t vasoc o n s t r i c t o r in various isolated b l o o d vessels as well as in intact a n i m a l s ( Y a n a g i s a w a et al., 1988a,b; L i p p t o n et al., 1988; T o m o b e et al., 1988; H a n et
Correspondence to: C.A. Maggi, Pharmacology Department, Smooth Muscle Division, Research Laboratories, A. Menarini Pharmaceuticals, Via Sette Santi 3, Florence 50131, Italy.
al., 1989; D ' O r l 6 a n s - J u s t e et al., 1989; W a l d e r et al., 1989). S u b s e q u e n t investigations b y the group at T s u k u b a U n i v e r s i t y led to the discovery of three E T gene p r o d u c t s ( Y a n a g i s a w a et al., 1988a; M a s a k i , 1989; I n o u e et al., 1989) which have b e e n d e s i g n a t e d ET-1, ET-2 a n d ET-3. ET-1 is identical to p o r c i n e E T while ET-3 is the sequence originally f o u n d in the r a t g e n o m e ( Y a n a g i s a w a et al., 1988a). F u r t h e r , the T s u k u b a g r o u p r e p o r t e d that at least s o m e of these p e p t i d e s are p r o d u c e d n o t o n l y in e n d o t h e l i a l cells b u t also in neurons. T h u s , the r e p e r t o i r e of b i o l o g i c a l actions exerted b y ETs e n c o m p a s s e s their p o s s i b l e role as c h e m i c a l messengers b e t w e e n e n d o t h e l i a l cells a n d o t h e r ele-
0014-2999/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
ments in the vascular wall and as neurotransmitter (Vane, 1989). New aspects of ET pharmacology have emerged: beside the potent vasoconstricting activity, ET-1 and especially ET-3 also act as vasodilators in certain vascular beds where they promote the release of endothelium-derived relaxing factor and prostanoids (Wright and Fozard, 1988; Warner et al., 1989; De Nucci et al., 1988). Thus there are indications that ETs may, in some instances, exert indirect actions. ETs are also potent contractile agents on certain non-vascular smooth muscles. As examples, ET-1 induces a contraction of guinea-pig (trachea and bronchus) and human (bronchus) isolated airways (Uchida et al., 1989; Maggi et al., 1989c) and of the human isolated urinary bladder (Maggi et al., 1989b). We report here on a comparison of the biological activity of ET-1 and ET-3 in the guinea-pig isolated bronchi and the modification of the effect by epithelium removal, indomethacin or peptidase inhibitors. Sarafotoxin S6b (SRFTX), a 21-residue peptide isolated from the venom of the snake Atractaspis Engaddensis was also included in the study because of its close structural homology with endothelins and its ability to interact with ET receptors (Kloog et al., 1988; A m b a r et al., 1989; G u et al., 1989). The effect of these three peptides was also investigated in the rat isolated aorta to compare their potency to contract vascular smooth muscle.
et al., 1988; Maggi et al., 1989c). Bronchial rings (attached to stainless steel hooks) were placed in a 5 ml organ bath and connected to an isotonic force transducer under a constant load of 5 mN. After a 120 min equilibration period the preparations were exposed to 40 m M KC1 (added to the bath) and the resultant contraction was used as a reference standard for the response to ET. In other experiments, the effect of peptides was investigated in the presence of indomethacin (5 ~M, 90 min before), of a mixture of peptidase inhibitors (thiorphan 1 /~M, captopril 1 ~M, bestatin 1 ~M, 30 min before) or both. Cumulative concentration-response curves to ET were made, the next concentration being added when the effect of the preceding one had reached a steady state. Only one peptide was administered to each preparation. Experiments with different peptides were conducted in parallel within 30-45 min of KC1 administration. N o attempt was made to assess whether the previous exposure to KC1 might have influenced the subsequent response to the test peptides. Preliminary experiments using isometric force transducers in order to quantitatively compare the tension generated by different bronchial rings showed that neither rubbing nor indomethacin or addition of the mixture of peptidase inhibitors had any significant effect on the contractile response to KC1.
2.2. Rat aorta 2. Materials and methods
2.1. Guinea-pig bronchi Male albino guinea-pigs weighing 220-300 g were stunned and bled. The main bronchi were rapidly excised and placed in gassed (96% 02 and 4% CO2) Krebs solution at 37 ° C. The two main bronchi were excised from each animal and the luminal surface of one was rubbed gently several times with a cotton-tipped applicator in order to remove the epithelial layer. The effectiveness of this maneuver was assessed from the relaxant response to arachidonic acid (0.01-0.1 mM) in the rubbed bronchus and the unrubbed control from the same animal, as described previously (Devillier
Male albino rats of the Wistar strain (weight 300-320 g) were stunned and bled. The thoracic aorta was rapidly removed, freed of adhering tissue, placed in Krebs solution at 37 ° C and pinned flat on a Petri dish. The aorta was opened along its longitudinal axis and its intimal surface rubbed gently several times in order to remove the endothelium. The effectiveness of this maneuver was checked by verifying the lack of a relaxant response to acetylcholine (30 /~M) after a contraction was induced with phenylephrine (0.1 #M). A zigzag strip of aortic muscle was prepared and placed in a 5 ml organ bath containing oxygenated Krebs solution, as described above. Mechanical activity was recorded isotonically through a transducer connected to a Basile 7050 Unirecord under
a resting tension of 10 mN. Contractile responses to peptides of the ET family were expressed as % of the maximal response to KC1 (80 mM, added to the bath). All experiments were performed in presence of indomethacin (5 ~tM, 90 rain before), thiorphan, captopril and bestatin (1 /zM each, 30 min before) and started after a 120 min equilibration period.
indomethacin and arachidonic acid (Sigma), acetylcholine chloride (Merck) and phenylephrine HC1 (Serva). Stock solutions of peptides were made in bidistilled water, kept frozen and diluted just before use. A 10 m M solution of indomethacin was prepared in absolute ethanol and diluted in Krebs solution. A 10 mM solution of arachidonic acid was prepared in bidistilled waer by mixing with Na 2CO3.
2.3. Statistical analysis Statistical analysis of the data was made by means of Student's t-test or analysis of variance followed by Dunnett's test, when applicable. Each value in the text and figures is the mean + S.E.M. In some instances, the relative potency of the peptides to produce a contraction of the guinea-pig bronchus was expressed as EC30, the concentration of each peptide which induced a contraction comparable to 30% of that due to KC1. This was done because the response to ET-1 and ET-3 in unrubbed bronchi (see Results) did not exceed 50% of the maximal response to KC1. Both E C 3 0 and ECs0 values were calculated in other instances. Regression analysis was performed by means of the least square method. EC30 or ECs0 and 95% confidence limits (c.1.) were calculated accordingly.
2.4. Drugs The drugs used were: endothelin 1 (Peninsula), endothelin 3 and sarafotoxin S6b (Novabiochem),
3. Results
3.1. Response of unrubbed bronchi ET-1, ET-3 and SRFTX produced a similar response pattern in the isolated guinea-pig bronchus, i.e. a concentration-dependent, slowly developing tonic contraction. The highest concentration tested for each peptide (0.3 /~M) did not produce maximal responses. Because the response to ET-1 and ET-3 did not exceed 50% of the maximal response to KC1, the EC30 of the various peptides was calculated to allow comparison of their potency. SRFTX was clearly more potent (about 10 times) than ET-1 or ET-3 (table 1); it produced a higher maximal response (about twice as high) than that to ET-1 or ET-3 (fig. 1, table 1) and had a lower threshold concentration (30 pM for SRFTX, 100-300 pM for ET-3 and 1-3 nM for ET-1) (fig. 1).
TABLE 1 Comparison of the contractile activity of endothelins and S R F T X on the guinea-pig isolated bronchus. E m ~ = Maximal effect produced at 0.3 ~tM expressed as % of the maximal response to KC1 (40 raM). Each value is the mean + S.E. from six experiments; EC30 values indicate the concentration of peptides required to produce 30% of the maximal response to KCI (40 mM). N u m b e r s in parentheses are the 95% confidence limits; a significantly different from the S R F T X value; P < 0.05; b significantly different from the effect on unrubbed bronchus. Peptide
ET-1 ET-3 SRFTX
Unrubbed bronchus
Rubbed bronchus
U n r u b b e d bronchus plus indomethacin (5/~M)
EC3o (nM)
E max (% of KC1)
EC3o (nM)
E max (% of KC1)
EC3o (nM)
E max (% of KCI)
72 (57-93) 77 (38-221) 7.7 (6-9)
45 + 3 a 41 -/-3 a 78 :t: 2
10 (7-19) 1.3 (1-2) 1.6 (1.4-1.8)
77 + 4 b 65-t-6 b 76 + 3
23 (15-41) 7 (6-10) 3.6 (2.6-6.8)
67 + 6 b 63+8 b 80 5:6
UNRUBBED
UNRUEIBED plus INDOMETHACIN
RUBBED
100
0
I-'
O ET-1 ET-3
80
1 ~/I
LId
¢/~ 6o Z 0
~ 4o ul 2O
u
O
N
0
11
10
9
8
7
6
11
10
9
8
7
6
11
10
9
8
7
6
- L o g [M] Fig. 1. Contractile response to the guinea-pig isolated bronchi to ET-1, ET-3 and SRFTX in unrubbed (left panel) or rubbed preparations (middle panel) and in unrubbed preparations exposed to indomethacin (fight panel). Each value is the mean + S.E. of six experiments.
3.2. Response of rubbed bronchi
ent from the response to 0.3 # M SRFTX (table 1). In the presence of indomethacin (5 /~M) the response to ET-3 (EC30) was markedly potentiated, although this effect was less (11 times) than that produced by rubbing (59 times) (table 1, fig. 1). Likewise, indomethacin slightly enhanced the response to ET-1 or SRFTX, but this effect was smaller than that produced by rubbing.
Rubbing enhanced the contractile effect of all peptides tested. The response to 0.3/~M ET-1 or ET-3 in rubbed bronchi was not significantly different from the maximal response to 0.3 /~M SRFTX (table 1). The EC30 of ET-3 was about 59 times lower in rubbed than in unrubbed bronchi. In contrast, the EC30 of ET-1 and SRFTX was only decreased 7 and 4.8 times, respectively (table 1). The potentiation of the response to ET-3 by rubbing was particularly evident at low concentrations (30-300 pM, fig. 1).
3.4. Effect of thiorphan, captopril and bestatin on the response to ET-3 The hypothesis had been advanced (Maggi et al., 1989c) that enhancement of the response to ET-1 by rubbing might involve degradation of the peptide by enzyme activity localized in the airway epithelium. As rubbing potentiated ET-3 more than other peptides, we studied the effect of ET-3
3.3. Effect of indomethacin in unrubbed bronchi In presence of indomethacin, the response to 0.3/~M ET-1 or ET-3 was not significantly differET - 1
100
SRFTX
ET - 3
80
!,° ~)
60
2O
10
9
8
7
6
11
10
9
-Log
8
7
6
11
10
9
8
7
6
[M]
Fig. 2. Comparison of the contractile response of the guinea-pig isolated bronchi to ET-1, ET-3 and SRFTX in unrubbed and untreated preparations (ET-1 filled squares, ET-3 empty circles, S R F T X empty triangles) as compared to the response obtained in rubbed preparations and in the presence of indomethacin, thiorphan, captopril and bestatin (ET-1 empty squares, ET-3 filled circles, SRFTX filled triangles). Each value is the mean + S.E. of five to six experiments. * Significantly different from the value obtained for unrubbed and untreated bronchi, P < 0.05.
°°F
00]
o780
N
N o
0 11
10
9
8
7
6
11
10
9
8
7
6
-Log [M] Fig. 3. Comparison of the contractile response of rubbed bronchi to ET-1, ET-3 and SRFTX and in presence of indomethacin, thiorphan, captopril and bestatin expressed as % of the maximal response to KCl (left panel) or as % of the maximal response produced by each peptide. Each value is the mean + S.E. of five experiments.
in unrubbed bronchi in the presence of three known peptidase inhibitors, thiorphan, captopril and bestatin (1/~M of each, added 30 min before). ET-3 was markedly more active under these conditions than in the controls (unrubbed bronchi): its maximal effect (at 0.3 #M) was greater (63 + 4 vs. 41 _ 3% relative to the response to KC1, n = 4 and 6, P < 0.01) than in the controls, and its EC30 was 3.7 nM (2.7-6.7 nM are 95% c.1.) as compared to 77 nM (38-221 nM) in the controls.
3.5. Effect of ETs in rubbed bronchi and in presence of indomethacin and peptidase inhibitors It appeared from the above experiments that rubbing, blockade of cyclooxygenase and inhibition of metabohsm by a mixture of peptidase inhibitors potentiate the response to ETs in the guinea-pig isolated bronchus. Therefore, to compare more directly the potency of these peptides to produce contraction, the effects of ET-1, ET-3 and S R F T X were studied in rubbed bronchi and in presence of indomethacin ( 5 / I M , 90 min before), thiorphan, captopril and bestatin (1 /~M of each, 30 rain before). In fig. 2 the response to the three peptides studied under these conditions is compared to that obtained in unrubbed and untreated bronchi. The
activity of ET-1, ET-3 and S R F T X was potentiated, the effect being particularly marked for ET-3. Figure 3 shows a more direct comparison of the response to the three peptides under conditions (rubbing, indomethacin, peptidase inhibitors) selected to optimize the contractile response. ET-3 and S R F T X were distinctly more potent than ET-1, their threshold concentration being 0.01 and 0.03 nM as compared to 0.3 nM for the latter. The estimated EC30s (95% c.1. in parentheses) were: 3.9 nM (2.8-7.6), 0.19 n M (0.11-0.27) and 0.24 nM (0.07-0.4) for ET-1, ET-3 and SRFTX, respectively. Therefore, the potency of ETs to contract the guinea-pig bronchus increased 18, 405 and 32 times for ET-1, ET-3 and SRFTX, respectively as compared to that observed in unrubbed and untreated bronchi. All peptides produced maximal responses of similar magnitude, that is 78 + 2, 77 + 3 and 82 + 3% of the response to KC1 (n = 5 for each peptide). Further, clearly maximal responses were obtained under these conditions at 0.1, 0.3 and 0.03 # M for ET-1, ET-3 and SRFTX, respectively (fig. 3). The concentration-response curve for ET-1 was Sshaped and spanned three order of concentrations. In contrast, the response to ET-3 or S R F T X was more complex, showing a 'notch' or plateau between 0.1 and 1 nM in both cases.
6
3 : 5
loo-
’
60.
o
ET-1
0
ET-3
A
SRFTX
60.
a E 2
40.
20.
k ap
0. 10
7
6
9 -LOG
6
[M]
Fig. 4. Comparison of the contractile response of rubbed thoracic aorta to ET-l, ET-3 and SRFTX in presence of indomethacin, thiorphan, captopril and bestatin. Each value is the mean f S.E. of five to six experiments.
3.6. Effect of peptides isolated aorta
of the ET family
in the rat
The contractile response to ET-l, ET-3 and SRFTX was also studied on the rat isolated thoracic aorta under conditions (rubbing, indomethacin and peptidase inhibitors in the bath) selected to mirror those shown to increase the activity of these peptides in the guinea-pig bronchus. In the aorta, the three peptides produced a concentration-dependent contraction TABLE
2
EC,, and 95% confidence limits for endothelins and SRFTX to produce a contraction of the rubbed guinea-pig bronchus or rat aorta in the presence of indomethacin and peptidase inhibitors. The values are means of four to six experiments. Numbers in parentheses are 95% confidence limits. R.P. = relative potency. All experiments were performed in the presence of indomethacin (5 FM), thiorphan (1 PM), captopril(1 PM) and bestatin (1 PM). Peptide
Rat aorta
R.P.
(A) EC,, (nM) ET-1 ET-3 SRFTX
1.7 (1.6-1.8) 72.0 (32-194) 6.2 (4.3-11.7)
Guinea-pig bronchus
R.P.
A/B
(B) EGO
1 .oo 0.02 0.27
0-W
6.9 (4-12) 0.54 (0.51-0.57) 0.71 (0.61-0.77)
1.00
0.25
12.11
133.3
9.71
8.7
which gave rise to S-shaped curves (fig. 4), the maximal response approaching 100% of that produced by KC1 (80 mM). ET-1 was the most potent peptide, followed by SRFTX and ET-3, which was 50 times less potent than ET-1 (table 2). When the EC,, values obtained in the rubbed guinea-pig bronchus and rubbed rat aorta (both in presence of indomethacin, thiorphan, captopril and bestatin) were compared, ET-l was about 4 times more potent in the aorta than in the bronchus while the contrary was true for SRFTX which was 8.7 times and ET-3 which was 133 times more potent in the bronchus than the aorta (table 2).
4. Discussion ET-l has been reported to be a potent bronchoconstrictor both in vitro (Uchida et al., 1988; Maggi et al., 1989~) and in vivo (Payne and Whittle, 1988; Lagente et al., 1989). We have shown previously that the contraction produced by ET-l in the guinea-pig isolated trachea is slightly reduced by indomethacin (Maggi et al., 1989c), implying a role for prostanoids in this in guinea-pigs, response. In in vivo experiments ET-l administered either i.v. or by aerosol induced a severe bronchospasm which was abolished or markedly inhibited by indomethacin pretreatment (Payne and Whittle, 1988; Lagente et al., 1989). Indeed, since ET-1 has been shown to induce thromboxane release from the isolated perfused guinea-pig lungs (De Nucci et al., 1988) the bronchoconstrictor response may reflect the in vivo release of these substances. The converse picture emerges from the experiments with guineapig isolated bronchus, where indomethacin potentiated the response to ETs. In view of the proposed role of certain prostanoids, and particularly PGE, as epithelium-derived relaxant factors (see Vanhoutte, 1988 for review) we interpret these findings as an indication that the contractile action of ETs on bronchial isolated smooth muscle was attenuated by the concomitant production of relaxant prostanoids. The potentiating effect of indomethacin in the guinea-pig bronchus was clearly more evident for ET-3 (11 times) than for ET-1 (3 times) or SRFTX
(2 times) suggesting that, of the peptides tested, ET-3 has the strongest ability to activate this mechanism. Warner et al. (1989) found ET-3 more effective than ET-1 to induce the release of endothelium-derived relaxing factor in the rat perfused mesenteric bed and proposed that the endothelial receptor activated by these peptides might be distinct from that present on muscle cells. In view of the above, the interesting hypothesis emerges that a similar recetor, for which ET-3 has a higher affinity than ET-1, might mediate the release of both endothelium- and airway epithelium-derived relaxing factors. The present findings do not show directly a relaxant effect of ETs or SRFTX on the guinea-pig bronchus. The production of airway epithelium-derived relaxant factor(s) was deduced from the enhancement of the response to peptides produced by rubbing or indomethacin. Uchida et al. (1989) recently reported the production of an airway smooth muscle relaxing factor from the guinea-pig isolated trachea. We had reported previously (Maggi et al., 1989c) that the response of the guinea-pig isolated bronchus to ET-1 is significantly enhanced by mechanical removal of the bronchial epithelium (rubbing). This observation was confirmed by the present results and was extended to ET-3 and SRFTX. As also observed for indomethacin, rubbing caused a far greater potentiation of ET-3 than of ET-1 or SRFTX activity. Conceivably, ET-3 is more efficient than ET-1 or SRFTX to stimulate the production of relaxant prostanoids from the bronchial epithelium, but further studies are needed to assess this point. We found that the activity of ET-3 was markedly enhanced by the addition of thiorphan, captopril and bestatin, known inhibitors of endopeptidase 24.11, angiotensin-converting enzyme and aminopeptidase, respectively. The presence of robust metabolic activity in the airways has been evidenced in experiments in which metabolic blockade caused a potentiation of the response to peptides such as tachykinins (Sekizawa et al., 1987; Devillier et al., 1988). Very little or no information is available about possible degradation of ETs by tissue peptidases. It was not the aim of this study to assess a potential role of individual peptidases in the degradation of ETs in the airways. Thus the
potentiating effect of the mixture of peptidase inhibitors that we have used might well depend on the inhibition of one particular enzyme only. Also, we cannot exclude that the response to ETs in the bronchus might involve the release of other peptides, the activity of which could in turn have been potentiated by the peptidase inhibitors. Clearly further studies are needed regarding this point. Irrespective of this, the present data showed that peptidase inhibitors enhance the bronchoconstrictor response to ET-3 and these drugs were therefore added routinely to the medium to minimize this drawback in estimating the contractile activity of test peptides. In view of the above, the potentiating effect of rubbing on the contractile response to ETs might involve the removal of enzymatic systems degrading these peptides, failure to generate an epithelium-derived relaxing factor (including prostanoids) or both mechanisms. Regardless of the mechanism involved, the contractile activity of ETs in the guinea-pig bronchus can be fully determined only after removal of these factors. From our observations, this can be achieved by combining the effect of rubbing, indomethacin and peptidase inhibitors. Complete concentration-response curves, including true maximal responses of similar magnitude, were obtained under these experimental conditions for all peptides. The curves showed ET-3 and SRFTX to be more potent bronchoconstrictors than ET-1. The pattern of the response to ET-1 was strikingly different from that of the response to ET-3 or SRFTX. In fact, while ET-1 gave an S-shaped curve spanning three orders of concentrations, the other two peptides gave more complex curves spanning six orders of concentrations. In contrast to the curves for guinea-pig bronchus, the contractile response curves elicited by the three peptides in the rat aorta under comparable conditions (rubbing, indomethaacin and peptidase inhibitors) were characteristically S-shaped. In this latter preparation ET-1 was slightly more potent than SRFTX and distinctly more potent than ET-3. A hypothesis which may account for the difference in response patterns elicited by ET-3 and SRFTX in aorta and bronchus is that the latter tissue contains two ET receptor populations, one
having high affinity for ET-3 and SRFTX but not for ET-1 and a second population recognizing the three peptides with similar affinity. Little information is yet available about the comparative pharmacology of ETs and sarafotoxins. Yanagisawa et al. (1988a) reported that ET-1 is about 20 times less potent that ET-3 to produce a contraction of the rat isolated aorta. Warner et al. (1989) reported that ET-1 and ET-3 are about equipotent as vasoconstrictors in the rat perfused mesentery, ET-3 being about 10 times more potent than ET-1 to produce an endothelium-dependent vasodilatation. The present findings support the possibility (Inoue et al., 1989; Warner et al., 1989; Maggi et al., 1989a) that multiple receptors mediate the biological effects of ETs in mammalian tissues. Evidence for the existence of multiple receptors was obtained from a recent study in our laboratory. We reported that the C-terminal fragment, ET-(16-21), a sequence common to all mammalian ETs, is a full agonist in the guinea-pig isolated bronchus (33 times less potent than ET-1), while it is weakly active in the rat aorta (at least 10000 times less potent than ET-1) (Maggi et al., 1989a). Studies are in progress to compare the activity of ET-(16-21) and natural ETs in different bioassay. The present findings underscore the importance of careful selection of the experimental conditions for evaluation of the biological activity of ETs in bioassay systems. Recently, Black et al. (1989) presented evidence that cultured epithelial cells from dog trachea produce ET. Further studies are therefore needed to understand the possible pathophysiological significance of ET production and the mechanisms of release as well as of activation of ET receptors in the airways.
References Ambar, I., Y. Kloog, I. Schvartz, E. Hazum and M. Sokolovsky, 1989, Competitive interaction between endothelin and sarafotoxin: binding and phosphoinositides hydrolysis in rat atria and brain, Biochem. Biophys. Res. Commun. 158, 195. Black, P.N., M.A. Ghatei, D. Breterthon-Watt, K. Takahashi, T. Krausz and S.R. Bloom, 1989, Endothelin is formed by
cultured tracheal epithelial cells, Am. Rev. Respir. Dis. 139, A52. De Nucci, G., R. Thomas, P. D'Orleans-Juste, E. Antunes, C. Walder, T.D. Warner and J.R. Vane, 1988, Pressor effect of circulating endothelin are limited by its removal in the pulmonary circulation and by the release of prostacyclin and endothelium-derived relaxating factor, Proc. Natl. Acad. Sci. U.S.A. 85, 9797. Devillier, P., C. Advenier, G. Drapeau, J. Marsac and D. Regoli, 1988, Comparison of the effects of epithelium removal and of an enkephalinase inhibitor on the neurokinin-induced contractions of guinea-pig isolated trachea, Br. J. Pharmacol. 94, 675. D'Orleans-Juste, P., M. Finer, G. De Nucci and J.R. Vane, 1989, Pharmacology of endothelin-1 in isolated vessels: effects of nicardipine, methylene blue, hemoglobin and gossypol, J. Cardiovasc. Pharmacol. 13, $19. Gu, X.H., D.J. Casley and W.G. Nayler, 1989, Sarafotoxin S6b displaces specifically bound 1251-endothelin, European J. Pharmacol. 162, 509. Han, S,P., A.J. Trapani, K.F. Kok, T.C. Westfall and M.M. Knuepfer, 1989, Effect of endothelin on regional hemodynamics in conscious rats, European J. Pharmacol. 159, 303. Inoue, A , M. Yanagisawa, S. Kimura, Y. Kasuya, T. 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. U.S.A. 86, 2863. Kloog, Y., I. Ambar, M. Sokolovsky, E. Kochva, Z. Wollberg and A. Bdolah, 1988, Sarafotoxin, a novel vasoconstrictor peptide" phosphoinositide hydrolysis in rat heart and brain, Science 242, 268. Lagente, V., P.E. Chabrier, J.M. Mencia-Huerta and P. Braquet, 1989, Pharmacological modulation of the bronchopulmonary action of the vasoactive peptide, endothelin, administered by aerosol in guinea-pig, Biochem. Biophys. Res. Commun. 158, 625. Lippton, H., J. Golf and A. Hyman, 1988, Effects of endothelin in the systemic and renal vascular beds in vivo, European J. Pharmacol. 155, 197. Maggi, C.A., S. Giuliani, R. Patacchini, P. Santicioli, P. Rovero, A. Giachetti and A. Meli, 1989a, The C-terminal hexapeptide, endothelin-(16-21) discriminates between different endothelin receptors, European J. Pharmacol. 166, 121. Maggi, C.A., S. Giuliani, R. Patacchini, P. Santicioli, D. Turini, G. Barbanti and A. Meli, 1989b, Potent contractile activity of endothelin on the human isolated urinary bladder, Br. J. Pharmacol. 96, 755. Maggi, C.A., R. Patacchini, S. Giuliani and A. Meli, 1989c, Potent contractile effect of endothelin in isolated guinea-pig airways, European J. Pharrnacol. 160, 179. Masaki, T., 1989, The discovery, the present state and the future prospects of endothelin, J. Cardiovasc. Pharmacol. 13, S1. Payne, A.N. and B.J.R. Whittle, 1988, Potent cyclo-oxygenase mediated bronchoconstrictor effect of endothelin in the guinea-pig in vivo, European J. Pharmacol. 158, 303.
Sekizawa, K., J. Tamaoki, P.D. Graf, C.B. Basbaum, D.B. Borson and J.A. Nadel, 1987, Enkephalinase inhibitor potentiates mammalian tachykinin-induced contraction in ferret trachea, J. Pharmacol. Exp. Ther. 243, 1211. Tomobe, Y., T. Miyauchi, A. Saito, M. Yanagisawa, S. Kimura, K. Goto and T. Masaki, 1988, Effects of endothelin on the renal artery from spontaneously hypertensive and WistarKyoto rats, European J. Pharmacol. 152, 373. Uchida, Y., H. Ninomiya, M. Saotome, A. Nomura, M. Ohtsuka, M. Yanagisawa, K. Goto, T. Masaki and S. Hasegawa, 1988, Endothelin, a novel vasoconstrictor peptide, as potent bronchoconstrictor, European J. Pharmacol. 154, 227. Uchida, Y., A. Nomura, M. Ohtsuka, H. Ninomiya, M. Saotome and S. Hasegawa, 1989, Endothelin released an airway smooth muscle relaxing factor in guinea-pig trachea in vitro, Am. Rev. Respir. Dis. 139, A470. Vane, J.R., 1989, Preface, J. Cardiovasc. Pharmacol. 13, (Suppl. 5), vii. Vanhoutte, P.M., 1988, Epithelium-derived relaxing factor: myth or reality?, Thorax 43, 665.
Walder, C.E., C.R. Thomas, C. Thiemermann and J.R. Vane, 1989, The hemodynamic effects of endothelin-1 in the pithed rat, J. Cardiovasc. Pharmacol. 13, $93. Warner, T.D., G. De Nucci and J.R. Vane, 1989, Rat endothelin is a vasodilator in the isolated perfused mesentery of the rat, European J. Pharmacol. 159, 325. Wright, C.E. and J.R. Fozard, 1988, Regional vasodilation is a prominent feature of the haemodynamic response to endothelin in anaesthetized spontaneously hypertensive rats, European J. Pharmacol. 155, 201. Yanagisawa, M., A. Inoue, T. Ishikawa, Y. Kasuya, S. Kimura, S. Kumagaye, K. Nakajirna, T.X. Watanabe, S. Sakakibara, K. Goto and T. Masaki, 1988a, Primary structure, synthesis and biological activity of rat endothelin an endothelium-derived vasoconstrictor peptide, Proc. Natl. Acad. Sci. U.S.A. 85, 6964. Yanagisawa, M., H. Kurihara, S. Kimura, Y. Tomobe, M. Kobayashi, Y. Mitsui, Y. Yazaki, K. Goto and T. Masaki, 1988b, A novel potent vasoconstrictor peptide produced by vascular endothelial cells, Nature 332, 411.
1
Elsevier EJP 51146
Further studies on the response of the guinea-pig isolated bronchus to endothelins and sarafotoxin S6b Carlo Alberto Maggi, Sandro Giuliani, Riccardo Patacchini, Paolo Santicioli, Antonio Giachetti and Alberto Meli Pharmacology Department, Smooth Muscle Division, Research Laboratories, A. Menarini Pharmaceuticals, Via Sette Santi 3, Florence 50131, Italy
Received 13 July 1989, revised MS received 14 September 1989, accepted 7 November 1989
Endothelin-1 (ET-1), endothelin-3 (ET-3) and sarafotoxin S6b (SRFTX) produced a concentration-dependent contraction of the guinea-pig isolated bronchus. In untreated preparations, SRFTX was about 10 times more potent than ET-1 or ET-3 and produced a greater maximal effect. Mechanical removal of the bronchial epithelium (rubbing) or the addition of indomethacin (5/~M) increased the potency of all peptides, ET-3 being enhanced more than ET-1 or SRFTX. Further, the activity of ET-3 was enhanced by a mixture of peptidase inhibitors (thiorphan, captopril, bestatin, 1/~M each). When the three potentiating factors were combined (rubbed bronchi in presence of indomethacin and peptidase inhibitors), the following order of potency was found, ET-3 = SRTFX > ET-1. For comparison, the activity of the three peptides was also studied in the rubbed rat isolated aorta in the presence of indomethacin and peptidase inhibitors and the following rank order of potency was found: ET-1 > SRFTX > ET-3. When the activity of the peptides was compared in these two preparations, ET-1 was 4 times more potent in the aorta than in the bronchus, while SRFTX and ET-3 were 8.7 and 133 times more potent in the bronchus than the aorta, respectively. These findings indicate that, in the guinea-pig bronchus, the contractile activity of these peptides is attenuated by the concomitant production of epithelium-derived relaxant factor(s), prostanoid production via the cyclooxygenase pathway and, possibly, enzymatic inactivation of added peptides. Further, the existence of multiple receptors mediating the contractile response to these peptides is suggested. Endothelin; Bronchi (guinea-pig); Airway epithelium; Indomethacin; Aorta (rat); Endothelin receptors
1. Introduction Porcine e n d o t h e l i n (ET) is a 2 1 - a m i n o acid p e p t i d e discovered as a p r o d u c t of c u l t u r e d a o r t i c e n d o t h e l i a l cells ( Y a n a g i s a w a et al., 1988b). This p e p t i d e has b e e n shown to be a very p o t e n t vasoc o n s t r i c t o r in various isolated b l o o d vessels as well as in intact a n i m a l s ( Y a n a g i s a w a et al., 1988a,b; L i p p t o n et al., 1988; T o m o b e et al., 1988; H a n et
Correspondence to: C.A. Maggi, Pharmacology Department, Smooth Muscle Division, Research Laboratories, A. Menarini Pharmaceuticals, Via Sette Santi 3, Florence 50131, Italy.
al., 1989; D ' O r l 6 a n s - J u s t e et al., 1989; W a l d e r et al., 1989). S u b s e q u e n t investigations b y the group at T s u k u b a U n i v e r s i t y led to the discovery of three E T gene p r o d u c t s ( Y a n a g i s a w a et al., 1988a; M a s a k i , 1989; I n o u e et al., 1989) which have b e e n d e s i g n a t e d ET-1, ET-2 a n d ET-3. ET-1 is identical to p o r c i n e E T while ET-3 is the sequence originally f o u n d in the r a t g e n o m e ( Y a n a g i s a w a et al., 1988a). F u r t h e r , the T s u k u b a g r o u p r e p o r t e d that at least s o m e of these p e p t i d e s are p r o d u c e d n o t o n l y in e n d o t h e l i a l cells b u t also in neurons. T h u s , the r e p e r t o i r e of b i o l o g i c a l actions exerted b y ETs e n c o m p a s s e s their p o s s i b l e role as c h e m i c a l messengers b e t w e e n e n d o t h e l i a l cells a n d o t h e r ele-
0014-2999/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
ments in the vascular wall and as neurotransmitter (Vane, 1989). New aspects of ET pharmacology have emerged: beside the potent vasoconstricting activity, ET-1 and especially ET-3 also act as vasodilators in certain vascular beds where they promote the release of endothelium-derived relaxing factor and prostanoids (Wright and Fozard, 1988; Warner et al., 1989; De Nucci et al., 1988). Thus there are indications that ETs may, in some instances, exert indirect actions. ETs are also potent contractile agents on certain non-vascular smooth muscles. As examples, ET-1 induces a contraction of guinea-pig (trachea and bronchus) and human (bronchus) isolated airways (Uchida et al., 1989; Maggi et al., 1989c) and of the human isolated urinary bladder (Maggi et al., 1989b). We report here on a comparison of the biological activity of ET-1 and ET-3 in the guinea-pig isolated bronchi and the modification of the effect by epithelium removal, indomethacin or peptidase inhibitors. Sarafotoxin S6b (SRFTX), a 21-residue peptide isolated from the venom of the snake Atractaspis Engaddensis was also included in the study because of its close structural homology with endothelins and its ability to interact with ET receptors (Kloog et al., 1988; A m b a r et al., 1989; G u et al., 1989). The effect of these three peptides was also investigated in the rat isolated aorta to compare their potency to contract vascular smooth muscle.
et al., 1988; Maggi et al., 1989c). Bronchial rings (attached to stainless steel hooks) were placed in a 5 ml organ bath and connected to an isotonic force transducer under a constant load of 5 mN. After a 120 min equilibration period the preparations were exposed to 40 m M KC1 (added to the bath) and the resultant contraction was used as a reference standard for the response to ET. In other experiments, the effect of peptides was investigated in the presence of indomethacin (5 ~M, 90 min before), of a mixture of peptidase inhibitors (thiorphan 1 /~M, captopril 1 ~M, bestatin 1 ~M, 30 min before) or both. Cumulative concentration-response curves to ET were made, the next concentration being added when the effect of the preceding one had reached a steady state. Only one peptide was administered to each preparation. Experiments with different peptides were conducted in parallel within 30-45 min of KC1 administration. N o attempt was made to assess whether the previous exposure to KC1 might have influenced the subsequent response to the test peptides. Preliminary experiments using isometric force transducers in order to quantitatively compare the tension generated by different bronchial rings showed that neither rubbing nor indomethacin or addition of the mixture of peptidase inhibitors had any significant effect on the contractile response to KC1.
2.2. Rat aorta 2. Materials and methods
2.1. Guinea-pig bronchi Male albino guinea-pigs weighing 220-300 g were stunned and bled. The main bronchi were rapidly excised and placed in gassed (96% 02 and 4% CO2) Krebs solution at 37 ° C. The two main bronchi were excised from each animal and the luminal surface of one was rubbed gently several times with a cotton-tipped applicator in order to remove the epithelial layer. The effectiveness of this maneuver was assessed from the relaxant response to arachidonic acid (0.01-0.1 mM) in the rubbed bronchus and the unrubbed control from the same animal, as described previously (Devillier
Male albino rats of the Wistar strain (weight 300-320 g) were stunned and bled. The thoracic aorta was rapidly removed, freed of adhering tissue, placed in Krebs solution at 37 ° C and pinned flat on a Petri dish. The aorta was opened along its longitudinal axis and its intimal surface rubbed gently several times in order to remove the endothelium. The effectiveness of this maneuver was checked by verifying the lack of a relaxant response to acetylcholine (30 /~M) after a contraction was induced with phenylephrine (0.1 #M). A zigzag strip of aortic muscle was prepared and placed in a 5 ml organ bath containing oxygenated Krebs solution, as described above. Mechanical activity was recorded isotonically through a transducer connected to a Basile 7050 Unirecord under
a resting tension of 10 mN. Contractile responses to peptides of the ET family were expressed as % of the maximal response to KC1 (80 mM, added to the bath). All experiments were performed in presence of indomethacin (5 ~tM, 90 rain before), thiorphan, captopril and bestatin (1 /zM each, 30 min before) and started after a 120 min equilibration period.
indomethacin and arachidonic acid (Sigma), acetylcholine chloride (Merck) and phenylephrine HC1 (Serva). Stock solutions of peptides were made in bidistilled water, kept frozen and diluted just before use. A 10 m M solution of indomethacin was prepared in absolute ethanol and diluted in Krebs solution. A 10 mM solution of arachidonic acid was prepared in bidistilled waer by mixing with Na 2CO3.
2.3. Statistical analysis Statistical analysis of the data was made by means of Student's t-test or analysis of variance followed by Dunnett's test, when applicable. Each value in the text and figures is the mean + S.E.M. In some instances, the relative potency of the peptides to produce a contraction of the guinea-pig bronchus was expressed as EC30, the concentration of each peptide which induced a contraction comparable to 30% of that due to KC1. This was done because the response to ET-1 and ET-3 in unrubbed bronchi (see Results) did not exceed 50% of the maximal response to KC1. Both E C 3 0 and ECs0 values were calculated in other instances. Regression analysis was performed by means of the least square method. EC30 or ECs0 and 95% confidence limits (c.1.) were calculated accordingly.
2.4. Drugs The drugs used were: endothelin 1 (Peninsula), endothelin 3 and sarafotoxin S6b (Novabiochem),
3. Results
3.1. Response of unrubbed bronchi ET-1, ET-3 and SRFTX produced a similar response pattern in the isolated guinea-pig bronchus, i.e. a concentration-dependent, slowly developing tonic contraction. The highest concentration tested for each peptide (0.3 /~M) did not produce maximal responses. Because the response to ET-1 and ET-3 did not exceed 50% of the maximal response to KC1, the EC30 of the various peptides was calculated to allow comparison of their potency. SRFTX was clearly more potent (about 10 times) than ET-1 or ET-3 (table 1); it produced a higher maximal response (about twice as high) than that to ET-1 or ET-3 (fig. 1, table 1) and had a lower threshold concentration (30 pM for SRFTX, 100-300 pM for ET-3 and 1-3 nM for ET-1) (fig. 1).
TABLE 1 Comparison of the contractile activity of endothelins and S R F T X on the guinea-pig isolated bronchus. E m ~ = Maximal effect produced at 0.3 ~tM expressed as % of the maximal response to KC1 (40 raM). Each value is the mean + S.E. from six experiments; EC30 values indicate the concentration of peptides required to produce 30% of the maximal response to KCI (40 mM). N u m b e r s in parentheses are the 95% confidence limits; a significantly different from the S R F T X value; P < 0.05; b significantly different from the effect on unrubbed bronchus. Peptide
ET-1 ET-3 SRFTX
Unrubbed bronchus
Rubbed bronchus
U n r u b b e d bronchus plus indomethacin (5/~M)
EC3o (nM)
E max (% of KC1)
EC3o (nM)
E max (% of KC1)
EC3o (nM)
E max (% of KCI)
72 (57-93) 77 (38-221) 7.7 (6-9)
45 + 3 a 41 -/-3 a 78 :t: 2
10 (7-19) 1.3 (1-2) 1.6 (1.4-1.8)
77 + 4 b 65-t-6 b 76 + 3
23 (15-41) 7 (6-10) 3.6 (2.6-6.8)
67 + 6 b 63+8 b 80 5:6
UNRUBBED
UNRUEIBED plus INDOMETHACIN
RUBBED
100
0
I-'
O ET-1 ET-3
80
1 ~/I
LId
¢/~ 6o Z 0
~ 4o ul 2O
u
O
N
0
11
10
9
8
7
6
11
10
9
8
7
6
11
10
9
8
7
6
- L o g [M] Fig. 1. Contractile response to the guinea-pig isolated bronchi to ET-1, ET-3 and SRFTX in unrubbed (left panel) or rubbed preparations (middle panel) and in unrubbed preparations exposed to indomethacin (fight panel). Each value is the mean + S.E. of six experiments.
3.2. Response of rubbed bronchi
ent from the response to 0.3 # M SRFTX (table 1). In the presence of indomethacin (5 /~M) the response to ET-3 (EC30) was markedly potentiated, although this effect was less (11 times) than that produced by rubbing (59 times) (table 1, fig. 1). Likewise, indomethacin slightly enhanced the response to ET-1 or SRFTX, but this effect was smaller than that produced by rubbing.
Rubbing enhanced the contractile effect of all peptides tested. The response to 0.3/~M ET-1 or ET-3 in rubbed bronchi was not significantly different from the maximal response to 0.3 /~M SRFTX (table 1). The EC30 of ET-3 was about 59 times lower in rubbed than in unrubbed bronchi. In contrast, the EC30 of ET-1 and SRFTX was only decreased 7 and 4.8 times, respectively (table 1). The potentiation of the response to ET-3 by rubbing was particularly evident at low concentrations (30-300 pM, fig. 1).
3.4. Effect of thiorphan, captopril and bestatin on the response to ET-3 The hypothesis had been advanced (Maggi et al., 1989c) that enhancement of the response to ET-1 by rubbing might involve degradation of the peptide by enzyme activity localized in the airway epithelium. As rubbing potentiated ET-3 more than other peptides, we studied the effect of ET-3
3.3. Effect of indomethacin in unrubbed bronchi In presence of indomethacin, the response to 0.3/~M ET-1 or ET-3 was not significantly differET - 1
100
SRFTX
ET - 3
80
!,° ~)
60
2O
10
9
8
7
6
11
10
9
-Log
8
7
6
11
10
9
8
7
6
[M]
Fig. 2. Comparison of the contractile response of the guinea-pig isolated bronchi to ET-1, ET-3 and SRFTX in unrubbed and untreated preparations (ET-1 filled squares, ET-3 empty circles, S R F T X empty triangles) as compared to the response obtained in rubbed preparations and in the presence of indomethacin, thiorphan, captopril and bestatin (ET-1 empty squares, ET-3 filled circles, SRFTX filled triangles). Each value is the mean + S.E. of five to six experiments. * Significantly different from the value obtained for unrubbed and untreated bronchi, P < 0.05.
°°F
00]
o780
N
N o
0 11
10
9
8
7
6
11
10
9
8
7
6
-Log [M] Fig. 3. Comparison of the contractile response of rubbed bronchi to ET-1, ET-3 and SRFTX and in presence of indomethacin, thiorphan, captopril and bestatin expressed as % of the maximal response to KCl (left panel) or as % of the maximal response produced by each peptide. Each value is the mean + S.E. of five experiments.
in unrubbed bronchi in the presence of three known peptidase inhibitors, thiorphan, captopril and bestatin (1/~M of each, added 30 min before). ET-3 was markedly more active under these conditions than in the controls (unrubbed bronchi): its maximal effect (at 0.3 #M) was greater (63 + 4 vs. 41 _ 3% relative to the response to KC1, n = 4 and 6, P < 0.01) than in the controls, and its EC30 was 3.7 nM (2.7-6.7 nM are 95% c.1.) as compared to 77 nM (38-221 nM) in the controls.
3.5. Effect of ETs in rubbed bronchi and in presence of indomethacin and peptidase inhibitors It appeared from the above experiments that rubbing, blockade of cyclooxygenase and inhibition of metabohsm by a mixture of peptidase inhibitors potentiate the response to ETs in the guinea-pig isolated bronchus. Therefore, to compare more directly the potency of these peptides to produce contraction, the effects of ET-1, ET-3 and S R F T X were studied in rubbed bronchi and in presence of indomethacin ( 5 / I M , 90 min before), thiorphan, captopril and bestatin (1 /~M of each, 30 rain before). In fig. 2 the response to the three peptides studied under these conditions is compared to that obtained in unrubbed and untreated bronchi. The
activity of ET-1, ET-3 and S R F T X was potentiated, the effect being particularly marked for ET-3. Figure 3 shows a more direct comparison of the response to the three peptides under conditions (rubbing, indomethacin, peptidase inhibitors) selected to optimize the contractile response. ET-3 and S R F T X were distinctly more potent than ET-1, their threshold concentration being 0.01 and 0.03 nM as compared to 0.3 nM for the latter. The estimated EC30s (95% c.1. in parentheses) were: 3.9 nM (2.8-7.6), 0.19 n M (0.11-0.27) and 0.24 nM (0.07-0.4) for ET-1, ET-3 and SRFTX, respectively. Therefore, the potency of ETs to contract the guinea-pig bronchus increased 18, 405 and 32 times for ET-1, ET-3 and SRFTX, respectively as compared to that observed in unrubbed and untreated bronchi. All peptides produced maximal responses of similar magnitude, that is 78 + 2, 77 + 3 and 82 + 3% of the response to KC1 (n = 5 for each peptide). Further, clearly maximal responses were obtained under these conditions at 0.1, 0.3 and 0.03 # M for ET-1, ET-3 and SRFTX, respectively (fig. 3). The concentration-response curve for ET-1 was Sshaped and spanned three order of concentrations. In contrast, the response to ET-3 or S R F T X was more complex, showing a 'notch' or plateau between 0.1 and 1 nM in both cases.
6
3 : 5
loo-
’
60.
o
ET-1
0
ET-3
A
SRFTX
60.
a E 2
40.
20.
k ap
0. 10
7
6
9 -LOG
6
[M]
Fig. 4. Comparison of the contractile response of rubbed thoracic aorta to ET-l, ET-3 and SRFTX in presence of indomethacin, thiorphan, captopril and bestatin. Each value is the mean f S.E. of five to six experiments.
3.6. Effect of peptides isolated aorta
of the ET family
in the rat
The contractile response to ET-l, ET-3 and SRFTX was also studied on the rat isolated thoracic aorta under conditions (rubbing, indomethacin and peptidase inhibitors in the bath) selected to mirror those shown to increase the activity of these peptides in the guinea-pig bronchus. In the aorta, the three peptides produced a concentration-dependent contraction TABLE
2
EC,, and 95% confidence limits for endothelins and SRFTX to produce a contraction of the rubbed guinea-pig bronchus or rat aorta in the presence of indomethacin and peptidase inhibitors. The values are means of four to six experiments. Numbers in parentheses are 95% confidence limits. R.P. = relative potency. All experiments were performed in the presence of indomethacin (5 FM), thiorphan (1 PM), captopril(1 PM) and bestatin (1 PM). Peptide
Rat aorta
R.P.
(A) EC,, (nM) ET-1 ET-3 SRFTX
1.7 (1.6-1.8) 72.0 (32-194) 6.2 (4.3-11.7)
Guinea-pig bronchus
R.P.
A/B
(B) EGO
1 .oo 0.02 0.27
0-W
6.9 (4-12) 0.54 (0.51-0.57) 0.71 (0.61-0.77)
1.00
0.25
12.11
133.3
9.71
8.7
which gave rise to S-shaped curves (fig. 4), the maximal response approaching 100% of that produced by KC1 (80 mM). ET-1 was the most potent peptide, followed by SRFTX and ET-3, which was 50 times less potent than ET-1 (table 2). When the EC,, values obtained in the rubbed guinea-pig bronchus and rubbed rat aorta (both in presence of indomethacin, thiorphan, captopril and bestatin) were compared, ET-l was about 4 times more potent in the aorta than in the bronchus while the contrary was true for SRFTX which was 8.7 times and ET-3 which was 133 times more potent in the bronchus than the aorta (table 2).
4. Discussion ET-l has been reported to be a potent bronchoconstrictor both in vitro (Uchida et al., 1988; Maggi et al., 1989~) and in vivo (Payne and Whittle, 1988; Lagente et al., 1989). We have shown previously that the contraction produced by ET-l in the guinea-pig isolated trachea is slightly reduced by indomethacin (Maggi et al., 1989c), implying a role for prostanoids in this in guinea-pigs, response. In in vivo experiments ET-l administered either i.v. or by aerosol induced a severe bronchospasm which was abolished or markedly inhibited by indomethacin pretreatment (Payne and Whittle, 1988; Lagente et al., 1989). Indeed, since ET-1 has been shown to induce thromboxane release from the isolated perfused guinea-pig lungs (De Nucci et al., 1988) the bronchoconstrictor response may reflect the in vivo release of these substances. The converse picture emerges from the experiments with guineapig isolated bronchus, where indomethacin potentiated the response to ETs. In view of the proposed role of certain prostanoids, and particularly PGE, as epithelium-derived relaxant factors (see Vanhoutte, 1988 for review) we interpret these findings as an indication that the contractile action of ETs on bronchial isolated smooth muscle was attenuated by the concomitant production of relaxant prostanoids. The potentiating effect of indomethacin in the guinea-pig bronchus was clearly more evident for ET-3 (11 times) than for ET-1 (3 times) or SRFTX
(2 times) suggesting that, of the peptides tested, ET-3 has the strongest ability to activate this mechanism. Warner et al. (1989) found ET-3 more effective than ET-1 to induce the release of endothelium-derived relaxing factor in the rat perfused mesenteric bed and proposed that the endothelial receptor activated by these peptides might be distinct from that present on muscle cells. In view of the above, the interesting hypothesis emerges that a similar recetor, for which ET-3 has a higher affinity than ET-1, might mediate the release of both endothelium- and airway epithelium-derived relaxing factors. The present findings do not show directly a relaxant effect of ETs or SRFTX on the guinea-pig bronchus. The production of airway epithelium-derived relaxant factor(s) was deduced from the enhancement of the response to peptides produced by rubbing or indomethacin. Uchida et al. (1989) recently reported the production of an airway smooth muscle relaxing factor from the guinea-pig isolated trachea. We had reported previously (Maggi et al., 1989c) that the response of the guinea-pig isolated bronchus to ET-1 is significantly enhanced by mechanical removal of the bronchial epithelium (rubbing). This observation was confirmed by the present results and was extended to ET-3 and SRFTX. As also observed for indomethacin, rubbing caused a far greater potentiation of ET-3 than of ET-1 or SRFTX activity. Conceivably, ET-3 is more efficient than ET-1 or SRFTX to stimulate the production of relaxant prostanoids from the bronchial epithelium, but further studies are needed to assess this point. We found that the activity of ET-3 was markedly enhanced by the addition of thiorphan, captopril and bestatin, known inhibitors of endopeptidase 24.11, angiotensin-converting enzyme and aminopeptidase, respectively. The presence of robust metabolic activity in the airways has been evidenced in experiments in which metabolic blockade caused a potentiation of the response to peptides such as tachykinins (Sekizawa et al., 1987; Devillier et al., 1988). Very little or no information is available about possible degradation of ETs by tissue peptidases. It was not the aim of this study to assess a potential role of individual peptidases in the degradation of ETs in the airways. Thus the
potentiating effect of the mixture of peptidase inhibitors that we have used might well depend on the inhibition of one particular enzyme only. Also, we cannot exclude that the response to ETs in the bronchus might involve the release of other peptides, the activity of which could in turn have been potentiated by the peptidase inhibitors. Clearly further studies are needed regarding this point. Irrespective of this, the present data showed that peptidase inhibitors enhance the bronchoconstrictor response to ET-3 and these drugs were therefore added routinely to the medium to minimize this drawback in estimating the contractile activity of test peptides. In view of the above, the potentiating effect of rubbing on the contractile response to ETs might involve the removal of enzymatic systems degrading these peptides, failure to generate an epithelium-derived relaxing factor (including prostanoids) or both mechanisms. Regardless of the mechanism involved, the contractile activity of ETs in the guinea-pig bronchus can be fully determined only after removal of these factors. From our observations, this can be achieved by combining the effect of rubbing, indomethacin and peptidase inhibitors. Complete concentration-response curves, including true maximal responses of similar magnitude, were obtained under these experimental conditions for all peptides. The curves showed ET-3 and SRFTX to be more potent bronchoconstrictors than ET-1. The pattern of the response to ET-1 was strikingly different from that of the response to ET-3 or SRFTX. In fact, while ET-1 gave an S-shaped curve spanning three orders of concentrations, the other two peptides gave more complex curves spanning six orders of concentrations. In contrast to the curves for guinea-pig bronchus, the contractile response curves elicited by the three peptides in the rat aorta under comparable conditions (rubbing, indomethaacin and peptidase inhibitors) were characteristically S-shaped. In this latter preparation ET-1 was slightly more potent than SRFTX and distinctly more potent than ET-3. A hypothesis which may account for the difference in response patterns elicited by ET-3 and SRFTX in aorta and bronchus is that the latter tissue contains two ET receptor populations, one
having high affinity for ET-3 and SRFTX but not for ET-1 and a second population recognizing the three peptides with similar affinity. Little information is yet available about the comparative pharmacology of ETs and sarafotoxins. Yanagisawa et al. (1988a) reported that ET-1 is about 20 times less potent that ET-3 to produce a contraction of the rat isolated aorta. Warner et al. (1989) reported that ET-1 and ET-3 are about equipotent as vasoconstrictors in the rat perfused mesentery, ET-3 being about 10 times more potent than ET-1 to produce an endothelium-dependent vasodilatation. The present findings support the possibility (Inoue et al., 1989; Warner et al., 1989; Maggi et al., 1989a) that multiple receptors mediate the biological effects of ETs in mammalian tissues. Evidence for the existence of multiple receptors was obtained from a recent study in our laboratory. We reported that the C-terminal fragment, ET-(16-21), a sequence common to all mammalian ETs, is a full agonist in the guinea-pig isolated bronchus (33 times less potent than ET-1), while it is weakly active in the rat aorta (at least 10000 times less potent than ET-1) (Maggi et al., 1989a). Studies are in progress to compare the activity of ET-(16-21) and natural ETs in different bioassay. The present findings underscore the importance of careful selection of the experimental conditions for evaluation of the biological activity of ETs in bioassay systems. Recently, Black et al. (1989) presented evidence that cultured epithelial cells from dog trachea produce ET. Further studies are therefore needed to understand the possible pathophysiological significance of ET production and the mechanisms of release as well as of activation of ET receptors in the airways.
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