Sumatripan and 5-benzyloxytryptamine: contractility of two 5-HT1D receptor ligands in canine saphenous veins

European Journal of Pharmacology, 211 (1992) 43-46

43

© 1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$05.(1(I

EJP 52255

Sumatriptan and 5-benzyloxytryptamine: contractility of two 5-HTID receptor ligands iff canine saphenous veins M a r l e n e L. C o h e n , K a t h r y n S c h e n c k , D a v i d N e l s o n a n d D a v i d W. R o b e r t s o n Lilly Research Laboratories, Eli l.illy and Company. Lilly Corporate (;enter, IndianapolL~', IN 46285, U.S.A. Received 4 September 1991, revised MS received 30 October 1991, accepted 5 November 1991

Sumatriptan and 5-benzyloxytryptamine arc ligands with high affinity for 5-HT m rcceptors in the caudatc nucleus. Both compounds contracted canine saphenous veins, in vitro. Benzyloxytryptamine was less potent as a contractile agonist than sumatriptan which was less potent than scrotonin. In high concentrations (> 10 -5 M) serotonin-induced contraction resulted, in part, from activation of a-adrenoccptors as determined by blockade of contraction with prazosin (10 -6 M) and idazoxan (10 -6 M). Likewise, benzyloxytryptamine but not sumatriptan also activated contractile a-receptors in the canine saphenous vein. Furthermore, benzyloxytryptamine antagonized contraction to sumatriptan in an apparently non-competitive fashion. Thus, bcnzylox'ytryptamine, although possessing some a-receptor agonist activity, like sumatriptan, can interact with serotonin receptors in canine saphcnous veins. Although effects of sumatriptan and benzyloxytryptaminc quantitatively differed in canine saphenous veins, both agents showed similar affinity and agonist efficacy at 5-HT m receptors in brain. These studies may reflect potential differences between the 5-HT m receptor in brain and the 5-HT~-like receptor in canine saphenous veins. Saphcnous veins (canine); Sumatriptan; 5-HT m receptors; 5-HT (5-hydroxytryptamine, serotonin)

1. Introduction

Sumatriptan was initially identified as a contractile agonist at '5-HTl-like' receptors in canine saphenous veins, an action thought to be related to its clinical effects in the treatment of migraine (Humphrey et al., 1988). More recently, sumatriptan has been reported to have relatively high binding affinity at 5 - H T m receptors in brain cortical m e m b r a n e s (Schoeffter and Hoyer, 19891. Benzyloxytryptamine, like sumatriptan, is an indole derivative with a large substituent at position 5 of the indole ring. Although bcnzyloxytryptamine has high affinity at the 5 - H T m receptor in bovine caudate membranes (Peroutka et al., 1991), little information is available as to whether it is an agonist or antagonist at these receptors, whether it is an agonist or antagonist in canine saphenous veins, or how its pharmacological effects compare to the prototypic compound, sumatriptan. The present study was designed to compare receptor affinities of these two ligands at central 5-HT m

Correspondence to: M.L. Cohen. Cardiovascular Research, MC304, Lilly Research Lalmratories, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, IN 46285, U.S.A. Tel. 1.317.276 4728, fax 1.317.277 0892.

receptors and to compare their potencies as contractile agonists in canine saphenous veins, in vitro.

2. Materials and methods

Mongrel dogs (15-20 kg) were anesthetized with sodium pentobarbital (30 m g / k g i.v.). After tying off collaterals, both lateral saphenous veins (segments of 4 - 6 cm in length) were dissected. Tissues were cleaned and spiral strips devoid of endothelium were mounted in 10 ml isolated tissue baths containing Krebs bicarbonate buffer of the following composition (raM concentration): NaCI 118.2; KCI 4.6; C a C i 2 . 2 H 2 0 1.6; K H 2 P O 4 1.2; MgSO 4 1.2; dextrose 10.0; and N a H C O 3 24.8. Tissue bath solutions were maintained at 37°C and equilibrated with 95% oxygen 5% CO 2. Each tissue was placed under an initial optimum resting force (5.0 g) and allowed to equilibrate for 1-2 h before exposure to compounds. Maximal contraction to 0.67 mM KCI of the canine saphenous veins ranged from 4 to 12 g of force under these conditions. For antagonist experiments, tissues were incubated with vehicle, a combination of prazosin ( 1 0 - 6 M ) and idazoxan (10 -6 M) or benzyloxytryptamine for approximately 1 h prior to challenge with contractile agonist. Isometric contractions were recorded as changes in

44

grams of force on a Beckman Dynograph (Beckman Instruments, Fullerton, CA) with Statham UC-3 transducers (Statham Medical Instruments, Los Angeles, CA). Affinities at central 5-HT:o binding sites were determined using a modification of the binding assay described by Heuring and Peroutka (1987). Membranes were prepared from bovine caudate nuclei as previously described (Xiong and Nelson, 1989). Each tube for the binding assay had a final volume of 800/xl and contained the following: Tris-HCl (50 mM), pargyline (10 ~M), ascorbate (5.7 mM), CaCI z (3 mM), 8-OHDPAT (100 nM to mask 5-HT]A receptors), mesulergine (100 nM to mask 5-HTIc receptors), [3H]5-HT (1.7-1.9 nM), appropriate compound dilutions and membrane resuspension equivalent to 5 mg of original tissue wet weight (final pH = 7.4). Tubes were incubated for 10 min at 37°C, and the contents were then rapidly filtered through G F / B filters (pretreated with 0.5% polyethylenimine), followed by four l-ml washes with ice-cold buffer. Radioactivity trapped by the filters was quantitated by liquid scintillation spectrometry, and specific [3H]5-HT binding to the 5-HT:D sites was defined as the difference between [3H]5-HT bound in the presence and absence of 10 ~ M 5-HT.

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Fig. 1. Contractile concentration-response curves to serotonin, sumatriptan and 5-benzyloxytryptaminein canine saphenous veins. Maximal force to serotonin (3 × 10 6 M) was 6.45× 0.37 g. Points are mean values and vertical bars represent S.E.M. for the number of tissues indicated in parentheses.

tively, blocked contraction to high concentrations of serotonin ( > 10 -5 M) and to benzyloxytryptamine ( > 3 × 10 -5 M), but not to sumatriptan (fig. 2). Thus, the serotonergic component of contraction to benzyloxytryptamine was consistent with its classification as a partial agonist relative to serotonin. m60

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Competition curves for 5-HT, sumatriptan and benzyloxytryptamine at 5-HT]D binding sites yielded pK i values of 8.15 + 0.03, 7.84 _+ 0.25 and 7.75 + 0.02, respectively. The values for sumatriptan and benzyloxytryptamine agree well with those previously found by Peroutka and coworkers: 7.36 for sumatriptan (Peroutka and McCarthy, 1989) and 7.40 for benzyloxytryptamine (Peroutka et al., 1991). These data confirmed that both sumatriptan and benzyloxytryptamine have relatively high and equivalent affinity for central 5-HT,o binding sites. Figure 1 shows the relative potencies of serotonin, sumatriptan and benzyloxytryptamine in contracting canine saphenous veins. Sumatriptan was a partial agonist in canine saphenous veins, producing approximately 80% of the maximum force generated by serotonin. Benzyloxytryptamine was approximately 100-fold less potent than sumatriptan as a contractile agonist in this vascular tissue. Although benzyloxytryptamine was markedly less potent than serotonin, high concentrations produced a maximal contractile response similar to that for serotonin (fig. 1). Since the canine saphenous vein is enriched with a-adrenoceptors (Milnor et al., 1988), we examined the possibility that contraction to these agonists resulted from a-receptor activation. The a~- and ae-receptor antagonists, prazosin and idazoxan, respec-

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Fig. 2. Effect of prazosin (10 -6 M) and idazoxan (10 -~' M) on the contractile response of serotonin (top), sumatriptan (middle) and benzyloxytryptamine (bottom) in canine saphenous veins. Maximal force (100%) to KCI (67 ram) was 6.16×0.68 (12) g in vehicle tissues. Points are mean values and vertical bars represent S.E.M. for the n u m b e r of tissues indicated in parentheses.

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contractile concentration-response curves to sumatriptan in canine saphenous veins. Maximal force to sumatriptan (3:,,: 10 ~' M) was 4.56+0.5(I (11) g in control tissues. Points arc mean values and vertical bars represent S.E.M. for the number of tissues indicated in parentheses.

Since bcnzyloxytryptaminc had relatively high affinity for central 5-HTID receptors based on radioligand binding studies, but was considerably less potent than sumatriptan as an agonist in canine saphenous veins, we examined its ability to antagonize contractile responses to sumatriptan. Benzyloxytryptamine (3 × 10 -~' and 10 -s M) inhibited contractile responscs to sumatriptan in a concentration-dependent fashion (fig. 3). However, the compound did not produce simple dextral shifts in the sumatriptan conccntration-rcsponse curve; maximal response to sumatriptan was markedly reduced in the presence of bcnzyloxytryptamine (10 -5 M). Thus bcnzyloxytryptamine, likc sumatriptan, interacted with a serotonergic contractile receptor in caninc saphenous veins, but it did not act as a simple competitive antagonist.

tonin-induced contractility in canine saphenous veins is identical to the 5-HTIo site identificd in brain binding studies, then the lower agonist potency of benzyloxytryptamine relative to sumatriptan is due to its considerably poorer efficacy (ability to activate the 5-Hq'H) receptor) in this tissue since it has similar affinity at 5-HT~D brain receptors to sumatriptan. However, this divergence in the functional effects of sumatriptan and benzyloxytryptamine at the serotonin rcceptor in canine saphenous veins contrasts to the equal potency of sumatriptan and benzyloxytryptamine as agonists at the 5-HT receptor which modulates [3H]5-HT release from guinea pig cortical synaptosomes (Peroutka et al., 1991). These apparent functional differences may relate to differences in receptor reserve for the two tissucs: it is possible that the number of 5-HTnD receptors in guinea pig brain mediating inhibition of 5-HT ncuronal relcase is higher than the number of rcccptors in canine saphenous veins, permitting grcater apparcnt agonist activity to bc manifested in brain by a weak partial agonist such as benzyloxytryptamine. Alternatively the '5-HTn-like' reccptor mediating contractility in canine saphenous veins may not be identical to the 5-HT~ receptor defined in radioligand binding studies with brain mcmbranes and sumatriptan may have considerably higher affinity a n d / o r efficacy at the serotonin receptor in saphenous veins than bcnzyloxytryptamine. Whatever the explanation, sumatriptan contracts canine saphenous veins more potently than benzyloxytryptamine, yet both agents show similar affinity and agonist cfficacy at 5-HTno receptors in brain. Thus, these studies comparing sumatriptan to benzyloxytryptamine underscore potential differences betwcen activation of putative 5-HT,D receptors in canine saphenous veins and activation of 5-HT,o receptors in brain.

4. Discussion

These data in canine saphenous veins confirm previous studies (Humphrey et al., 1988) documenting the lower potency of sumatriptan relative to serotonin as a contractile agonist in this tissue. Although Humphrey and colleagues (1988) found similar maximal responses to serotonin and sumatriptan, maximal contraction to sumatriptan was lower than maximal response to serotonin in our studies, consistent with the effect of sumatriptan in human saphenous veins (Chester et al., 1990). By comparison, however, benzyloxytryptamine was even less potent as a contractile agonist acting at serotonergic receptors than sumatriptan in canine saphenous veins. Affinity at central 5-HTH~ receptors was comparable for sumatriptan and benzyloxytryptamine. If the assumption is made that the receptor mediating sero-

References Chester, A., M. Bodelsson, B. Arneklo-Nobin, S. Tadjkarimi, K. Tornebrandt and M. Yacoub, 1990, Characterization of 5-HT receptors in the human saphenous vein, implications for patency of bypass grafts, J. Applied Cardiol. 5, 51. Heuring, R.E. and S.J. Peroutka, 1987, Characterization of a novel 3H-5-hydroxytryptamine binding site subtype in bovine brain membrances, J. Neurosci. 7, 894. Humphrey, P.P.A., W. Feniuk, M.J. Perren, H.E. Connor, A.W. Oxford, I.|I. Coates and D. Butina, 1988. GR 43175, a selective agonist for the 5-HTl-likc receptor in dog isolated saphenous vein, Br. J. Pharmacol. 94, 1123. Milnor, W.R., D.N. Stone and A. Sastre, 1988, Contributions of alphas- and alphaz-adrenoeeptors to contractilc responsc in canine blood v e s s l e s , B l o o d V e s s e l s 25, 199. Peroutka, S.J. and B.G. McCarthy, 1989, Sumatriptan (GR 43175) interacts selectively with 5-HT m and 5-tiTlE~ binding sites, Eur. J. Pharmacol. 163, 133.

46 Peroutka. S.J., B.G. McCarthy and X.-M. Guan, 1991, 5-Benzyloxytryptamine: a relatively selective 5-hydroxytryptaminc I D / I B agent, Life Sci. 49, 409. Schoeffter, P. and D. |toyer, 1989, Ilow selective is GR 43175'? Interactions with functional 5-11TIA, 5-11TIB, 5-II]'1(. and 5-HT~t) receptors, Naunyn-Schmiedcb. Arch. Pharmacol. 340, 135.

Xiong, W. and D.I.. Nelson, 1989, Characterization of a [3H}5-hydroxytrptamine binding site in rabbit caudate nucleus that differs from the 5-HTIA, 5-HTtr ~, 5-EITj(. and 5-HTID subtypes, Life Sci. 45, 1433