Comparative effects of the two endothelin ETA receptor antagonists, BQ-123 and FR139317, on endothelin-1-induced contraction in guinea-pig iliac artery

European Journal of Pharmacology, 241 (1993) 165-169

165

Elsevier Science Publishers B.V.

EJP 53266

Comparative effects of the two endothelin ETA receptor antagonists, BQ-123 and FR139317, on endothelin-l-induced contraction in guinea-pig iliac artery P. S c h o e f f t e r , A. R a n d r i a n t s o a l, B. J o s t a n d K. B r u t t e l Preclinical Research 386/527, Sandoz Pharma Ltd., CH-4002Basel, Switzerland

Received 1 March 1993, revised MS received 19 May 1993, accepted 29 June 1993

The effects of two recently introduced endothelin ETA receptor antagonists, BQ-123 and FR139317, were investigated and compared in guinea-pig isolated iliac artery. Endothelins and sarafotoxins induced contraction of guinea-pig iliac artery with a pharmacological profile characteristic of the ETA receptor. The rank order of agonist potency was (mean ECs0 values, nM): endothelin-1 (11.7) > endothelin-2 (14.9) > vasoactive intestinal contractor (19.5) > sarafotoxin S6b (49.8) _>[Alaa.11]endothelin-1 (55.0) > sarafotoxin S6a (> 100) > endothelin-3 (> 1000). The C-terminal hexapeptide, endothelin-(16-21), sarafotoxin $6c and sarafotoxin S6d were neither agonists nor antagonists at concentrations up to 10, 3 and 1 ~zM, respectively. Both FR139317 (1-10 /zM) and BQ-123 (0.1-1 /zM) surmountably antagonized the effects of endothelin-1. Schild analysis suggested competitive antagonism for FR139317 (Schild slope 1.32 + 0.21, pA 2 5.82 + 0.16, n = 5), but not for BQ-123 (Schild slope 0.28 + 0.08, n = 5), which was however more potent (apparent pK B 6.6-7.2) than FR139317. The potency of FR139317 was particularly low with respect to the reported affinity for ETA receptors, suggesting heterogeneity among ETA receptors. Thus, the endothelin receptor present in guinea-pig iliac artery has the following features: (1) rank order of agonist potencies of the ETA type; (2) low potency of FR139317 and (3) non-competitive antagonism by BQ-123. FR139317; BQ-123; Endothelins; Sarafotoxins; Endothelin receptors; Iliac artery (guinea-pig)

I. Introduction Endothelin-1 is a potent vasoconstrictor peptide first isolated from supernatants of cultured endothelial cells (Yanagisawa et al., 1988). It belongs to a family of isopeptides including endothelin-2 and endothelin-3, with different genes coding for each (Inoue et al., 1989). A peptide called vasoactive intestinal contractor (VIC), initially found in mouse intestine (Saida et al., 1989), is also a m e m b e r of this family. A structurally related class of peptides, the sarafotoxins, isolated from the v e n o m of the burrowing asp (Atractaspis engaddensis), shares common receptors with endothelins (Kloog et al., 1988; Kloog and Sokolovsky, 1989). The complementary D N A s encoding two endothelin receptors have been cloned and sequenced. One recepCorrespondence to: P. Schoeffter, Preclinical Research 368/527, Sandoz Pharma Ltd., CH-4002 Basel, Switzerland. Tel. 41.61.324.92.61, fax 41.61.324.27.33. 1 Present address: Laboratoire de Physiologie-Pharmacologie,Universit6 d'Antananarivo, Antananarivo, Madagascar.

tor, known as ETA, has marked selectivity for endothelin-1 and endothelin-2 vis-h-vis endothelin-3 (Arai et al,, 1990), whereas the other (ET B) does not discriminate between the isopeptides (Sakurai et al., 1990). Endothelin receptors mediating vascular contraction are generally believed to be of the E T A type, in spite of recent reports of receptor heterogeneity in some vessels (Harrison et al., 1992) and of E T B receptors being present in veins (Moreland et al., 1992; Sumner et al., 1992). Clearly, potent and selective antagonists would be of help to characterize further the receptor subtypes mediating the vascular effects of endothelins. Two compounds, BQ-123 (Ihara et al., 1992) and FR139317 (Aramori et al., 1993), have recently been introduced as E T A receptor antagonists. In the present study, we compared the effects of these two compounds on endothelin-l-induced contraction of guineapig iliac artery. This model conforms to an E T A receptor model with respect to the order of agonist potencies. However, FR139317 was much less potent than reported at cloned E T A receptors. In addition, BQ-123 was a non-competitive antagonist of endothelin-1.

166

2. Materials and methods

2.1. Experimental protocol Male albino guinea-pigs (250-300 g) were killed by a blow to the neck. The abdominal aorta, iliac and femoral arteries were exposed, removed and placed in a physiological solution containing (in mM): NaCI 122, KCI 5, NaHCO 3 25, KH2PO 4 1, MgSO 4 1.2, CaC12 1.25 and glucose 11.5. The right and left common iliac arteries were carefully cleaned of loosely adherent tissue, using a microscope. Two ring segments (1.5-2 mm long) were cut from each iliac artery. Endothelial cells were removed by a 30-s perfusion of the vessel with 0.3% CHAPS (3-[(3 chloramidopropyl)-dimethylammonio]-l-propanesulfonate; Tesfamariam et al., 1985). This procedure, followed by rinsing with the physiological solution, was shown to abolish the relaxant effect of carbachol (10/~M; see below). Rings were mounted between L-shaped stainless steel wires in 20-ml organ baths filled with the physiological solution bubbled with a mixture of 95% 02-5% CO 2 at 37°C. An initial tension of 0.5 g was applied and repeatedly readjusted during a 2-h period of washing and equilibration. Isometric changes in tension were recorded. Rings were first contracted with a maximally effective concentration of prostaglandin F2,, (PGF2~, 30 /xM). Carbachol (10 /zM) was added when contraction had plateaued to check for the absence of endothelium. After washing and re-equilibration for 60-90 min, during which period the tension was readjusted to 0.5 g, a cumulative concentration-response curve for contraction to one or the other peptide was constructed. When studied, antagonists were present 30 min before the start of the cumulative concentration-response curve to the agonist. Agonists and antagonists were tested on only one arterial ring from a given animal; hence n values refer to number of animals. The effects of antagonists were investigated in rings adjacent to those used as controls.

2.2. Analysis of data Concentration-response curves for agonists were analyzed using SCTFIT, a non-linear regression computerized program (DeLean et al., 1980). ECs0 and Emax values were derived from this analysis. Antagonist pA 2 values were measured according to Arunlakshana and Schild (1959). The slope was constrained to one when found to be not significantly different from unity (using Student's t-test). When the slope was significantly different from unity, antagonist apparent pK B values were calculated according to the formula: pK B = log (CR 1 ) - log [B], where [B] is the concentration of antagonist used and CR (concentration ratio) is the ratio of concentrations producing responses equal to that of

the EC50 of the control curve, in the presence and in the absence of antagonist. Agonist effects and Em~x values are expressed as percentages of the amplitude of the initial PGF2~ (30/~M)-induced contraction. Emax, pA 2 and pK B values are given as means + S.E.M. EC50 values are expressed as geometric means with 95% confidence intervals.

2.3. Drugs Carbachol (carbamylcholine chloride) was obtained from Sigma Chemical Co (St. Louis, MO, USA) and PGF2~ was purchased from Biokema (CrissierLausanne, Switzerland) in the form of Dinolytic (PGF2~ tromethamine salt). Endothelins and sarafotoxin S6b were from Novabiochem (L~iufelfingen, Switzerland). Sarafotoxins S6a and S6d were purchased from American Peptide Co. (Sunnyvale, CA, USA), sarafotoxin $6c from Bachem (Bubendorf, Switzerland). [Ala3'11]endothelin-1 was from Peninsula Laboratories (Belmont, CA, USA). Stock solutions (0.1 mM or 10 /zM) of endothelins and sarafotoxins were prepared in water, aliquots were kept at -30°C and used within one month. BQ-123 (cyclo-(D-Trp-D-Asp-Pro-D-ValLeu)) and FR139317 ((R)2-[(R)-2-[(S)-2-[[1-(hexahydro- 1H-azepinyl)]carbonyl]amino-4-methylpentanoyl]amino-3-[3-(1-methyl-lH-indolyl)]-propionyl]amino-3(2-pyridyl)propionic acid), both from Neosystem Laboratoire (Strasbourg, France), were prepared daily in distilled water.

3. Results

In the 125 arterial rings used for this study, the contraction induced by PGF2, (30 /xM), used as a

lOOI ~o 10

9

8

7

[Pep.de] (-,og M)

6

5

Fig. 1. Concentration-response curves for endothelins and sarafotoxins for contraction of guinea-pig iliac artery. The curves for endothelin-1 (o), endothelin-2 (C3), endothelin-3 (<>), vasoactive intestinal contractor (zx), [Ala3'll]endothelin-1 ( v ) , sarafotoxin S6a (11), sarafotoxin S6b (e) and sarafotoxin S6d ( • ) are presented. Contractions are expressed as percentages of the contraction induced by PGF2~ (30 p.M). The data are means+S.E.M. (vertical bars) of n values (n indicated in table 1).

167 TABLE 1 Maximal effects (Ema~) and ECso values of endothelins and sarafotoxins for eliciting contraction of guinea-pig iliac artery. Emax values are expressed as percentages of PGF2= (30/zM)-induced contraction. ECs0 values are given as geometric means with 95% confidence intervals. In the cases of endothelin-3 and sarafotoxin S6a, Emax was not reached with 3/~M and 0.3/zM, respectively. The highest concentrations tested for endothelin-(16-21), sarafotoxin $6c and sarafotoxin S6d were 10/.~M, 3/.tM and 1 /~M, respectively. Peptide

Emax (%)

ECs0 (nM)

n

Endothelin-1 Endothelin-2 Endothelin-3 VIC [Ala3'll]endothelin-1 Endothelin-(16-21) Sarafotoxin S6a Sarafotoxin S6b Sarafotoxin $6c Sarafotoxin S6d

119-t-3 112+3 > 28 127+9 131 _+3 0 > 76 124+8 0 6+ 2

11.7 (8.1-16.9) 14.9 (8.4-26.5) > 1000 19.5 (10.5-36.3) 55.0 (20.0-151) > 100 49.8 (20.7-119) -

27 10 11 11 6 2 6 10 5 4

reference, amounted to 1.26 + 0.04 g. Concentrationresponse curves for endothelins and sarafotoxins are shown in fig. 1 and agonist parameters (Emax and ECs0 values) are given in table 1. The curves for endothelin-1, endothelin-2 and vasoactive intestinal contractor (VIC) were almost superimposable, with respective mean ECs0 values of 11.7, 14.9 and 19.5 nM. Sarafotoxin S6b and [Ala3'11]endothelin-1 were 4-5 times less potent than endothelin-1, with similar intrinsic activities. The exact potency of sarafotoxin S6a could not be estimated, the maximal effect not being reached at the highest concentration tested (0.3 /zM). Nevertheless, sarafotoxin S6a appeared to be at least 10 times less potent than endothelin-1. Endothelin-3 was a very weak agonist, with no evidence of the Em~, being reached at 3 ~ M and, consequently, an ECs0 value estimated at > 1/~M. Sarafotoxin S6d was virtually without effect up to 1 /~M. Sarafotoxin $6c and the endothelin Cterminal peptide, endothelin-(16-21), had no contractile effect (at concentrations up to 3/~M and 10/~M, respectively). In the presence of sarafotoxin $6c (3/zM; fig. 2 top) or sarafotoxin S6d (1 /zM; fig. 2 bottom), the concentration-response curve to endothelin-1 was not significantly altered; it was also not affected by the presence of the C-terminal peptide, endothelin-(16-21) (10 ~M, n = 2; not illustrated). FR139317 (up to 10/~M) and BQ-123 (up to 1/~M) did not induce contractions of guinea-pig iliac artery. Neither FR139317 nor BQ-123 (10 /.~M each) antagonized the response of guinea-pig iliac artery to PGF2, (n = 2 each, data not shown). FR139317 (1-10 ~M) and BQ-123 (0.1-1 /zM) produced concentration-dependent rightward shifts of the concentration-response curves to endothelin-1, without depression of the Emax

100

0

0'~100 0

I

[EndotheUn(-loM) gl ,

10

9

8

7

I

6

Fig. 2. Concentration-response curves for endothelin-1 for contraction of guinea-pig iliac artery in the absence of (©) and in the presence (e) of sarafotoxin $6c (3 /zM; top) or sarafotoxin S6d (1 gM; bottom). Contractions are expressed as percentages of the contraction induced by PGF2, (30/x M). The data are means + S.E.M. (vertical bars) of five (top) or four (bottom) values.

(fig. 3). Schild analysis of FR139317 antagonism yielded a slope of 1.32 + 0.21 (which was not significantly different from 1; P = 0.19) and a pA 2 value of 5.82 + 0.16

~oC 1000 ~~I 100

0 I

!

|

I

I

10

9

8

7

6

JEndothe.n-lJ (-,o~ M) Fig. 3. Concentration-response curves for endothelin-I for contraction of guinea-pig iliac artery in the absence and in the presence of increasing concentrations of FR139317 (top) or BQ-123 (bottom). (o) Control curves. FR139317 was used at 1/xM ( n ) , 3/~M (e) and 10 # M ( • ) . BQ-123 was used at 0.1/zM ( n ) , 0.3/~M (e) and 1/.~M ( • ) . Contractions are expressed as percentages of the contraction induced by PGF2,~ (30 gM). Data are means + S.E.M. (vertical bars) of five values.

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(n = 5). For BQ-123 antagonism, the slope was 0.28 + 0.08 (n = 5), which was significantly different from 1 (P < 0.001). Mean concentration ratios in the presence of BQ-123 were 2.6, 3.6 and 5.1 (corresponding to apparent pK B values of 7.20, 6.94 and 6.61) using 0.1, 0.3 and 1/zM, respectively.

4. Discussion The endothelin receptor mediating contraction of guinea-pig iliac artery appears to have the characteristics of the E T A receptor, which has a greater affinity for endothelin-1 and endothelin-2 than for endothelin3. The rank order of potency: endothelin-1 > endothelin-2 > sarafotoxin S6b >> endothelin-3 is similar to that of the E T A receptor cloned in bovine lung and transfected into COS cells (Arai et al., 1990). Based on this rank order of potency, most of the vasoconstrictor effects of endothelins seem to involve E T A receptors (see Randall, 1991), with few exceptions, in particular in veins (Moreland et al., 1992; Sumner et al., 1992). The activity of VIC in guinea-pig iliac artery was not surprising, since VIC probably is the rat and mouse form of endothelin-2, differing by only one amino acid residue from human endothelin-2 (Bloch et al., 1991). Also, the reduced potency of [Ala3'11]endothelin-1 compared to endothelin-1, is compatible with an E T A receptor-mediated effect (Topouzis et al., 1989). The C-terminal peptide, endothelin(16-21), has been proposed as a full agonist at E T B receptors (Maggi et al., 1989) and has virtually no affinity for E T A receptors (Saeki et al., 1991). This is in line with its lack of effect in guinea-pig iliac artery. Results with sarafotoxins also support the possibility that E T A receptors are present in this tissue. Apart from sarafotoxin S6b, other sarafotoxins are very weak agonists or ligands at E T A receptors, especially sarafotoxin $6c (Saeki et al., 1991; Williams et al., 1991) and sarafotoxin S6d (Bdolah et al., 1989). We found a relatively weak agonist activity of sarafotoxin S6a (at 0.3 /zM) and no activity for sarafotoxins $6c and S6d, either as agonists or antagonists (up to 1 or 3 /zM, respectively), in guinea-pig iliac artery. FR139317 and BQ-123 both produced a concentration-dependent and surmountable antagonism of endothelin-l-induced contractions of guinea-pig iliac artery. BQ-123 was more potent than FR139317 since the threshold concentration required to shift the curve to endothelin-1 to the right was lower for BQ-123 (0.1 ~ M ) than for FR139317 (1 /~M). However, increasing the concentrations of BQ-123 did not displace the curve for endothelin-1 as much as would have been expected for a competitive antagonist, yielding a Schild slope significantly less than one. Such was not the case for FR139317, the Schild slope for which was not

significantly different from one, indicative of competitive antagonism in the concentration-range tested. Similar to what we found in guinea-pig iliac artery, FR139317 Was a competitive antagonist of endothelin1-induced contractions in guinea-pig pulmonary arteries (Cardell et al., 1993) and rabbit aorta (Sogabe et al., 1993). However, the pA 2 value for FR139317 in guinea-pig iliac artery (5.82 + 0.16) appears to be significantly lower than those reported in the other two vessels (6.65 and 7.2, respectively). Our value is actually closer to the affinity for cloned E T B receptors (7.3 ~ M ) than to that for cloned E T A receptors (1 nM; Aramori et al., 1993). Thus, although the order of agonist potencies for the endothelin receptor in guinea-pig iliac artery conforms to that of an E T A receptor, the former differs from E T A receptors by the low potency of FR139317. This suggests that FR139317 may discriminate between several subtypes of E T A receptors. Also, endothelin E T A receptors in guinea-pig pulmonary arteries (Cardell et al., 1993) and rabbit aorta (Sogabe et al., 1993) might be different from that in guinea-pig iliac artery. Along this line of reasoning, pharmacological differences between rat and human E T A receptors have been recently reported (Elshourbagy et al., 1993). BQ-123 behaved as a competitive E T A receptor antagonist in several vascular models with pA 2 values in the range of 6.9-7.4 (Ihara et al., 1992; Moreland et al., 1992; Sumner et al., 1992; Schoeffter and Randriantsoa, submitted). However, both its specificity and its competitive nature have been questioned (Webb et al., 1992; Hiley et al., 1992). Our results in guinea-pig iliac artery suggest a non-competitive antagonism of endothelin-1 by BQ-123, although with a potency in the expected range. As discussed above, receptor heterogeneity (in terms of E T A and E T B receptors) is unlikely in view of the profile of agonist activities. Another common reason for a Schild slope less than unity, i.e., agonist uptake or metabolism (Kenakin, 1984), can also be ruled out based on the results obtained with FR139317 in the same model. In summary, we found FR139317 and BQ-123 to be antagonists at endothelin receptors mediating contraction of guinea-pig iliac artery which, according to the profile of agonist activities, can be defined as E T A. FR139317 was a competitive antagonist, but less potent than expected at a typical E T A receptor. BQ-123 was more potent than FR139317 (by about one order of magnitude) but, unlike FR139317, did not behave as a competitive antagonist.

Acknowledgements The authors are indebted to J.R. Fozard and R.C. Miller for helpful comments.

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