5-HT1 receptors mediating contraction in bovine cerebral arteries: a model for human cerebrovascular ‘5-HT1Dβ‘ receptors

European Journal of Pharrnacology, 242 (1993) 75-82

75

Elsevier Science Publishers B.V.

EJP 53310

5-HT 1 receptors mediating contraction in bovine cerebral arteries: a model for human cerebrovascular '5-HT1D receptors Edith Hamel, Lyne Gr6goire and Benjamin Lau Laboratory of Cerebrovascular Research, Montreal Neurological Institute, 3801 University Street, Montreal, Quebec, Canada H3A 2B4 Received 14 January 1993, revised MS received 14 July 1993, accepted 16 July 1993

We report on the pharmacological profile of the 5-HT receptor which induces contraction of the bovine isolated cerebral arteries. Several 5-HT receptor agonists were tested for their ability to induce vasoconstriction in bovine pial arteries and their potencies were compared to that of 5-HT. The rank order of agonist potency can be summarized as 5-carboxamidotryptamine (5-CT) = RU 24969 > 5-HT > sumatriptan > ct-methyi-5-HT > methysergide > 2-methyl-5-HT > ((+)-2-dipropylamino-8hydroxy-l,2,3,4-tetrahydronaphthalene (8-OH-DPAT). Only methysergide induced a contraction which was smaller than that elicited by 5-HT. Antagonists with selective affinity at 5-HTIA/1 a (propranolol), 5-HTjc (mesulergine), 5-HT 2 (ketanserin, mianserin) and 5-HT 3 (MDL 72222) sites were inactive to block the 5-HT-induced contraction. In contrast, the 5-HT1/5-HT2 receptor antagonists methiothepin and metergoline inhibited the 5-HT-induced response with relatively high affinity (pA 2 = 8.16 + 0.26 and 6.73 + 0.05, respectively). Overall, this pharmacological profile indicated clearly that a 5-HT 1 receptor, most closely related to the 5-HT1D subtype, is responsible for the 5-HT-induced contraction of bovine cerebral arteries. Correlation analysis of the potencies of a series of 5-HT receptor agonists and antagonists in bovine and human cerebrovascular preparations showed a highly significant positive correlation (r = 0.94, P = 0.0051). Analyses of the correlation between the agonist and antagonist potencies at bovine cerebrovascular receptors and their published affinities at the cloned human 5-HT1D" and 5-HT1Dt~ (or human 5-HTIB) receptors showed a highly significant correlation only with the 5-HT1Dt3 (r = 0.82; P = 0.006) subtype. We conclude that cerebral vasoconstriction in bovine cerebral arteries is mediated by a receptor homologous to the human cerebrovascular 5-HT1D receptor and that bovine pial arteries appear to be the best available pharmacological model for the human cerebrovascular 5-HT~D receptor. Further, the results suggest that bovine cerebrovascular 5-HTjD receptors resemble the cloned human 5-HT1D~ (or human 5-HTlB) receptor subtype. 5-HT (5-hydroxytryptamine, serotonin); 5-HT1D receptors; Cerebral vasoconstriction; Migraine; 5-HT1 receptor subtypes; Cerebral blood vessels

1. Introduction 5-Hydroxytryptamine (5-HT or serotonin) has been implicated in various neurovascular disorders including dysfunctions such as vasospasm and migraine (for review see Fozard, 1987, 1989 and Raskin, 1990). The efficacy of the novel anti-migraine drug sumatriptan (Doenicke et al., 1988; Peroutka, 1990; Ferrari et al., 1991), which has high affinity for rodent 5 - H T m and h u m a n 5-HTtD (the species equivalent of the rat 5-HT m receptor, see Discussion for more details) receptor subtypes (Peroutka and McCarthy, 1989; Schoeffter and Hoyer, 1989), supports the possibility of involvement of 5 - H T in the manifestation of migraine attacks.

Correspondence to: E. Hamel, Montreal Neurological Institute, 3801 University Street, Montreal, Quebec, Canada H3A 2B4. Tel. (514) 398-8928, fax (514) 398-8106.

The exact site of action for sumatriptan remains unknown but the existence of both a vascular and a neural mechanism has been suggested (Fozard, 1987; Raskin, 1991; Humphrey, 1991; Moskowitz, 1992). Although the triggering factor in migraine headache is still unknown, it has been hypothesized that trigeminal nerve-induced neurogenic inflammation (which results in plasma protein extravasation and vasodilatation) might be involved in the migraine-associated pain (Markowitz et al., 1987; Moskowitz et al., 1989). Despite a lack of unequivocal pharmacological characterization (due either to the lack of specificity or of activity of some antagonists), receptors with a pharmacology similar to that of the 5 - H T I o receptors have been identified at two locations in relation to cerebral blood vessels. Putative 5-HT1D-like heteroreceptors have been found presynaptically on dural trigeminovascular afferents and reportedly mediate the blockade of neuropeptide release from unmyelinated C fibers (Buzzi et al.,

76

1991). In addition, postsynaptic 5-HT1D-like receptors have been shown on the vascular smooth muscle of human cerebral arteries (Hamel and Bouchard, 1991). Whether or not the dural and cerebrovascular smooth muscle 5-HT~o-like receptors are both responsible for the beneficial effect of sumatriptan in migraine is unclear at the present time (see Raskin, 1991) but it is beyond any doubt that this receptor subtype represents an attractive and promising target for migraine therapeutics. In this respect, the possibility of predicting the clinical efficacy of anti-migraine agents on either dural or cerebrovascular human specimens is severely hampered by limited availability of these tissues. Furthermore, the well-known species-related differences in 5-HT receptor subtypes (for review see Hoyer and Schoeffter, 1991) and more specifically differences in those that mediate cerebral vasoconstriction (Young et al., 1989; Parsons, 1991) strongly emphasize the urgent need for the identification of a pharmacologically reliable model for human cerebrovascular 5-HT m receptors. Dural 5-HTIo receptors (Buzzi et al., 1991; Matsubara et al., 1991) have yet to be found in the human while contractile 5-HT 1 (Parsons et al., 1989) and 5HT m (Hamel and Bouchard, 1991) receptors have been identified in human brain vessels. In a search for a readily available, easy to use pharmacological model of human 5-HTlo contractile cerebrovascular receptor, we have characterized pharmacologically the receptor involved in the vasocontractile response to 5-HT in bovine cerebral arteries, the species in which the 5HTID receptor subtype was first identified (Heuring and Peroutka, 1987). The possibility of using this species as a model of human cerebrovascular 5-HT m receptor was evaluated by correlation analysis.

Polygraph Model 7E coupled to a computer for automatic data acquisition and analysis. The vessels were allowed to stabilize (60-90 min) at approximately 0.4 g with washing at 15-min intervals. For determination of maximal contractile capacity, all vessel segments were then contracted with K + (124 mM). For this purpose, NaC1 was replaced by KC1 in equimolar concentration in the Krebs buffer solution. The segments were subsequently washed and allowed to recover for an additional 30- to 45-min period. Further details were given in previous publications (Hamel and Bouchard, 1991; Hamel et al., 1989).

2.2. Responses to agonists Log concentration-response curves were generated for each agonist by cumulative addition of the drug at the concentrations indicated. The order of the compounds was randomized from one experiment to another. Relative potencies of the various agonists were established by comparison of their pD 2 values (negative logarithm of the molar concentration of the agonist which produces 50% of the maximal response, - l o g ECs0) calculated mathematically according to Van den Brink (1977) from the following equation: r EAmax _ 1 ] pD 2 = - log[A] - log [ - - - ~ A

where EAmax is the maximal contraction induced by agonist A and E A the contractile response to a given concentration of agonist [A]. In addition, the maximal response to each agonist (EAma0 was compared to that elicited by 5-HT in the same vessel segments and expressed as a percentage of 5-HT EAmax.

2.3. Effects of 5-HT receptor antagonists 2. Materials and methods

2.1. Functional assays Bovine (young calves) brains were obtained from a local slaughterhouse and transported on ice to the laboratory. Segments (3-4 mm long) of temporal ramifications (outside diameter = 1 mm) of the middle cerebral artery were dissected out and kept in ice-cold Krebs-Ringer buffer (see below). The vessels were cleaned of surrounding blood and tissue under a dissecting microscope. They were then mounted between two L-shaped metal prongs in a temperature-controlled (37°C) tissue bath (volume of 5 ml) containing a Krebs-Ringer solution (pH 7.4) (in raM): NaC1 118; KCI 4.5; MgSO4-7H20 1.0; KH2PO 4 1.0, NaHCO 3 25; CaCIz-2H20 2.5; and glucose 6.0. Changes in muscle tension were measured by a force displacement transducer (Grass FT 103 D) and recorded on a Grass

Cumulative addition of 5-HT in order to obtain a dose-response curve was first done in the absence of any antagonist and then was repeated in the presence of graded concentrations of antagonists. Depending on the antagonist, the concentrations varied from 10 nM to 10 /xM. The antagonists were in contact with the vascular receptors 30 min before repetition of the 5-HT dose-response curve. Antagonist potency was determined for competitive or metactoid interaction as the negative logarithm of the molar antagonist concentration, in the presence of which twice the original agonist concentration is needed to cause an effect that is 'relatively' equal to the original effect (Van den Brink, 1977). These pA 2 values were calculated as: [ [A12 _ 1] p a 2 = -- log[B] + log [

1

in which [B] is the antagonist concentration, [A] 2 the concentration of agonist needed to reach a vasocon-

77 120

striction relatively equivalent to the control half-maximal response, in the presence of [B]; and [A] 1 the concentration of agonist needed to elicit the half-maximal response in the absence of [B] (Van den Brink, 1977).

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All results are means + S.E.M. Linear regression lines and correlation coefficients were calculated in order to detect and quantify any correlation between bovine and human cerebrovascular potencies of 5-HT receptor agonists and antagonists. In addition, correlation analysis was performed between potencies of agonists and antagonists in bovine vessels and at the cloned human 5-HTID . and 5-HTlo 0 (or human 5HT m) receptor subtypes. Statistical significance was assumed when P < 0.05.

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2.5. Drugs

The following drugs were purchased: 5-HT creatinine sulphate, (+)-propranolol hydrochloride (Sigma Chemical Co., St. Louis, MO, USA), ketanserin tartrate, mianserin hydrochloride, and 8-OH-DPAT ((_)2-dipropylamino-8-hydroxy-l,2,3,4-tetrahydro-naphthalene) hydrobromide (RBI, Natick, MA, USA). All other compounds were kindly provided as follows: ( +)a-methyl-5-HT (a-CH3-5-HT) , 2-methyl-5-HT (2-CH 35-HT), methysergide hydrogen maleinate and mesulergine (Sandoz, Basel, Switzerland); 5-methoxy3(1,2,3,6-tetrahydro-4-pyridinyl) 1H indole succinate (RU 24969; Roussel UCLAF, Paris, France); 3-[2(dimethylamino)ethyl]-N-methyl- 1H-indole-5-methanesulphonamide succinate salt (GR 43175C or sumatriptan; Glaxo Group Research, Greenford, UK); l a H,3a,5a H-tropan-3-yl-3,5-dichlorobenzoate (MDL 72222); Centre de Recherche Merrell Dow International (Strasbourg, France) and methiothepin maleate from Hoffmann-LaRoche (Basel, Switzerland). Meter-

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A, lonist [-log M] Fig. 1. Concentration-response curves for 5-hydroxytryptamine (5HT) and various 5-HT receptor agonists on bovine pial arteries under resting tension. The curve for 5-HT ( o ) represents data obtained with all tissues. The responses to 5-carboxamidotryptamine (5-CT, o); R U 24969 (O); sumatriptan ( I ) , a-methyl-5-HT (zx); 2-dipropylamino-8-hydroxy- 1,2,3,4-tet r a h y d r o - n a p h t h a l e n e (8-OHD P A T ) ( • ); 2-CH3-5-HT ( v ) and methysergide ( • ) are shown. The complete information for individual potency, maximal response and n u m b e r of vascular segments is given in table 1. Vertical bars show S.E.M. of n = 10-41.

goline was a generous gift from Dr. REmi Quirion, Douglas Hospital Research Centre (Verdun, QuEbec, Canada).

3. Results

3.1. Response to 5-HT

The overall maximal contraction elicited by 5-HT (10/zM) was 46 + 3% (n = 70 vascular segments) of the maximal contractile capacity of the cerebrovascular smooth muscle (1.70 + 0.10 g), as determined with 124 mM K ÷. Some arterial segments occasionally showed

TABLE 1 Potency of 5-HT receptor agonists for inducing contraction of bovine pial arteries. All values are m e a n s + S.E.M. from n individual segments as indicated. The values used for 5-HT are from pooled data obtained from all agonist experiments. Agonists

n

EAmax (g)

EAmax (% 5-HT EAmax) a

pD 2 ( - log ECs0)

ECs0 agonist ECs0 5-HT

5-HT 5-CT R U 24969 Sumatriptan ot-CH 3-5-HT Methysergide 2-CH 3-5-HT 8-OH-DPAT

41 11 12 12 10 12 12 11

0.84 + 0.11 0.53 _+0.21 0.45 + 0.08 0.67 + 0.19 0.57+0.10 0.48 _+0.18 0.88 + 0.30 0.77 _+0.22

100 92 + 10 75 _+ 10 79 + 9 88-t- 6 42 + 8 77 ___13 101 + 79

6.89 + 0.08 7.01 _+0.21 7.01 _+0.10 6.49 _+0.13 6.14+0.10 5.36 + 0.19 5.06 _ 0.15 4.64 + 0.15

1 0.8 0.8 2 6 33 68 178

The EAmax expressed as a percent (%) of 5-HT EAmax was obtained from comparison with the maximal response to 5-HT in the same vascular segments. For key to abbreviations, see legend to fig. 1. a

78

no vasomotor response to either K ÷ or 5-HT. In responsive vascular segments, however, the pharmacological profile presented here was very reproducible from one experiment to another.

3.2. Agonist potency All 5-HT receptor agonists tested were found to induce concentration-dependent contraction (fig. 1, table 1). Most compounds behaved as full agonists and induced maximal contraction corresponding to 80100% of the 5-HT maximal response. Methysergide was found to induce a contraction which corresponded to only 42% of 5-HT EAmax (table 1, fig. 1). Compounds 5-CT and RU 24969 had slightly higher potencies at the bovine cerebrovascular receptor than 5-HT itself whereas sumatriptan exhibited a lower affinity than 5-HT (table 1). The relative potencies of the other agonists were much less than that of 5-HT (table 1) with the 5-HT]A agent 8-OH-DPAT being by far the least potent compound to induce vasoconstriction (~ 180-fold less potent than 5-HT, table 1).

Antagonists with affinities for the various 5-HT receptor subtypes were tested for their ability to block 5-HT-induced vasoconstriction. Most receptor antagonists, including 5-HT1A/m (propranolol), 5-HTIc (mesulergine), 5-HT 2 (ketanserin) and 5-HT 3 (MDL 72222) receptor ligands, were devoid of significant inhibitory activity (fig. 2C-F). Methiothepin, an antagonist with affinity at 5-HT~/5-HT 2 receptor subtypes potently inhibited the 5-HT-induced constriction albeit producing both a rightward shift of the dose-response curve and a decrease in the maximal contractile effect (fig. 2A). Such characteristics of inhibition conformed to the mixed metactoid and competitive antagonism described by Van den Brink (1977). A pA 2 value of 8.16 _+0.26 was obtained for methiothepin when it was analyzed as a competitive antagonist (table 2). Metergoline, another non-selective 5-HT~/5-HT 2 agent with affinity at the 5-HTlb subtype (see Hoyer and Schoeffter, 1991), was able to induce competitive antagonism of the 5-HT-induced vasocontractile response

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Fig. 2. Concentration-response curves for 5-hydroxytryptamine (5-HT) on bovine pial arteries in the absence (©, control) and presence of 10 nM (e), 100 nM (D), 1 /zM ( l l ) or 10/xM ( A ) methiothepine (A, n = 4); metergoline (B, n = 11); propranolol (C, n = 5); mesulergine (D, n = 12); ketanserin (E, n = 5) and MDL 72222 (F, n = 5). Vertical bars are S.E.M. of n = 4-12.

79 TABLE 2

5-HT1D.

Effects of antagonists on 5-HT-induced contraction of bovine pial arteries. The concentrations of antagonists used are listed and the pA 2 values were obtained according to Van den Brink as described in Materials and methods. The results are the means±S.E.M, from the number (n) of vascular segments indicated. Antagonist

n

Methiothepin Metergoline Mianserin Mesulergine Propranolol Ketanserin MDL 72222

4 11 5 12 5 5 5

Concentration used

pA 2 value

10 nM-1 ~M 10 nM-1/zM 100 nM-10/~M 1/xM 10 nM-10 ~M 10 nM-1/~M 100 nM-1 ~M

8.16_+0.26 6.73±0.05 Inactive Inactive Inactive Inactive Inactive

w h e n u s e d at a high c o n c e n t r a t i o n (fig. 2B a n d t a b l e 2). W i t h t h e r a n g e o f c o n c e n t r a t i o n s u s e d (10 - 8 to 10 - 6 M), a p A 2 v a l u e o f 6.73 + 0.05 was o b t a i n e d for this a n t a g o n i s t . In c o n t r a s t , m i a n s e r i n , w h i c h also exhibits activity at 5 - H T 1 a n d 5-HT2 r e c e p t o r s b u t a low affinity at t h e 5-HTID site ( H o y e r a n d S c h o e f f t e r , 1991), was d e v o i d o f significant a n t a g o n i s t i c effects a l t h o u g h a small d e c r e a s e in t h e EAm~x a n d a r i g h t w a r d shift w e r e n o t e d in t h e 5 - H T - i n d u c e d c o n t r a c t i o n at t h e h i g h e s t c o n c e n t r a t i o n (10 - s M ) o f m i a n s e r i n ( d a t a n o t shown).

3.4. Correlation analyses C o r r e l a t i o n analysis p e r f o r m e d b e t w e e n t h e p o t e n cies o f 5 - H T r e c e p t o r agonists ( p D 2 v a l u e s ) a n d a n t a g onists ( p A z values) at b o v i n e (this study) a n d h u m a n cerebrovascular 5-HT receptors (Hamel and Bouchard,

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Bovine Cerebrovascular Potencies (pD2 or pA2Values ) Fig. 4. Correlation analyses performed between 5-HT receptor agonist and antagonist cerebrovascular potencies (pD 2 and pA 2 values, respectively) in bovine cerebral vessels and their published affinities (pK i) at the cloned human 5-HT~D,~ (Weinshank et al., 1992; Hamblin and Metcalf, 1991) and 5-HT1Dp (human 5-HTIa) (Weinshank et al., 1992; Demchyshyn et al., 1992; Levy et al., 1992; Jin et al., 1992; Oksenberg et al., 1992) receptor subtypes. Correlation coefficients (r) are given in the figure. Details of correlation analysis are as follows: 5-HT1D,, (r = 0.627, P = 0.07, b = 0.585 and a = 4.307), 5HTaDt3 (r = 0.822, P = 0.006, b = 0.623, a = 3.46). The compounds used for correlation analysis were: 5-HT (©), 5-CT (e), RU 24969 ([]) sumatriptan ( • ) , 2-CH3-5-HT ( ~ ), 8-OH-DPAT ( • ), methysergide ( • ) , methiothepin (o), and metergoline (*).

1991) e v i d e n c e d a highly significant positive c o r r e l a t i o n (fig. 3, P = 0 . 0 0 5 1 , r = 0.94). A d d i t i o n a l c o r r e l a t i o n analyses w e r e p e r f o r m e d b e t w e e n bovine c e r e b r o v a s c u lar p o t e n c i e s a n d p u b l i s h e d p K i v a l u e s at t h e c l o n e d h u m a n 5 - H T l o . a n d 5-HT1D ~ (or h u m a n 5 - H T m) rec e p t o r s u b t y p e s (fig. 4). W h i l e t h e c o r r e l a t i o n o f bovine v a s c u l a r p o t e n c i e s with the 5-HT~D ~ r e c e p t o r was n o t significant (r = 0.627; P = 0.07), t h e analysis s h o w e d a positive a n d highly significant c o r r e l a t i o n with t h e hum a n 5-HTtD 9 (r = 0.822; P = 0.006) r e c e p t o r s u b t y p e (fig. 4).

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Fig. 3. Correlation between the 5-HT receptor agonist (pD 2 values) and antagonist (pA 2 values) potencies in bovine pial arteries (from tables 1 and 2) and those published for human pial arterioles (Hamel and Bouchard, 1991). The compounds used for the correlation analyses are 5-HT (©); 5-CT (e); RU 24969 (n); sumatriptan ( • ) ; a-CH3-5-HT ( zx), 8-OH-DPAT ( • ); 2-CH3-5-HT ( v ); methysergide (v); methiothepin (o) and metergoline (,). The correlation coefficient (r) is 0.94 (P = 0.0051), and the terms for the equation of the line are b = 1.06 and a = 6.34.

M i g r a i n e h e a d a c h e is a c o n d i t i o n which has r e c e n t l y b e e n shown to b e efficiently r e l i e v e d in its a c u t e p h a s e by a 5-HT1B/5-HT1D agonist, s u m a t r i p t a n ( D o e n i c k e et al., 1988; P e r o u t k a , 1990), a b e n e f i c i a l effect t h o u g h t to b e r e l a t e d to a n e u r a l a n d / o r a v a s c u l a r m e c h a nism. T h e results o f t h e p r e s e n t study s h o w e d clearly t h a t bovine small pial a r t e r i e s o b t a i n e d as t e m p o r a l ramifications of the middle cerebral artery contain a c o n t r a c t i l e r e c e p t o r p h a r m a c o l o g i c a l l y i d e n t i c a l to its human cerebrovascular 5-HTlo counterpart (Hamel a n d B o u c h a r d , 1991). F u r t h e r m o r e , c o r r e l a t i o n analysis t e n d e d to suggest t h a t t h e c o n t r a c t i l e r e c e p t o r in bovine b r a i n vessels c o r r e s p o n d s closely to t h e h u m a n 5-HT1D p (or 5-HT1B) c l o n e d r e c e p t o r subtype. T h e r e c e p t o r m e d i a t i n g v a s o c o n s t r i c t i o n in small b o v i n e pial a r t e r i e s was i d e n t i f i e d as a 5-HTlo r e c e p -

80 tor, based on both agonist rank order of potency (Hoyer and Schoeffter, 1991; Hamblin and Metcalf, 1991; Jin et al., 1992; Weinshank et al., 1992) and the lack of inhibitory effect of 5-HTIA/m (propranolol), 5-HT~c (mesulergine), 5-HT 2 (ketanserin) and 5-HT 3 (MDL 72 222) receptor antagonists. The participation of a 5-HT~ receptor in cerebral vasoconstriction agrees with resuits of several other studies performed in various species including the cat (Hamel et al., 1989), dog (Peroutka and Kuhar, 1984; Peroutka et al., 1986; Connor et al., 1989), pig (Van Charldorp et al., 1990), non-human (Connor et al., 1989) and human primates (Hamel and Bouchard, 1991; Parsons et al., 1989). Of all compounds tested as 5-HT receptor antagonists, only methiothepin and, in high doses, metergoline, were able to block the 5-HT-induced contraction, as was observed in human pial arteries (Hamel and Bouchard, 1991). Metergoline has been reported to exert partial agonist activity at functional (Waeber et al., 1990) and cloned (Levy et al., 1992) 5-HT1D receptors. This property was not assessed in the present study although the antagonistic effect described here does not exclude the possibility that it also could be a partial agonist. The potencies of agonists and antagonists could be related to neither the pharmacological profile of receptors such as 5-HT4 (Dumuis et al., 1988), 5-HT1E (McAllister et al., 1992) nor to that of the newly cloned 5-HT1 receptors (Erlander et al., 1992; Lovenberg et al., 1992). The suspected heterogeneity (Mahle et al., 1991; Bruinvels et al., 1991; Beer et al., 1992) of the 5-HT1D receptor has now been established by the cloning of two genes which encode two variants of the pharmacological 5-HTID receptor (Weinshank et al., 1992). These two subtypes have been described as 5-HTtD,~ and 5-HT~D~ (Hartig et al., 1992), the latter being also referred to as the human 5-HTIB (Jin et al., 1992). The human 5-HT1D~ (or 5-HT m) is the species homolog of the rodent 5-HT m receptor (Oksenberg et al., 1992) although its pharmacological profile is clearly that of a 5-HT1D site (Hartig et al., 1992; Adham et al., 1991; Hamblin et al., 1992). The high correlation (P --- 0.0051) between bovine and human cerebrovascular 5-HT receptors indicates that the bovine cerebral vasculature contains a contractile 5-HT receptor that can be used as a model for human therapeutics. When bovine vascular potencies were compared with those obtained for the same compounds at the cloned human 5-HTID ~ and 5-HTtD/3 receptors, only the correlation with the 5-HT1Dt3 subtype reached a statistically significant level. Accepting the limitations of such correlation with non-subtype selective compounds, the high level of significance (P --0.006) obtained with the 5-HT1Dt~ suggests strongly that the receptor involved may correspond to a 5-HT~Dt~ (or human 5-HT m) subtype.

At this point, the bovine cerebral artery receptor appears to be the best model for the human cerebrovascular smooth muscle 5-HTID receptor as it fulfills the criteria for both agonists and antagonists. Indeed, the cat cerebrovascular contractile 5-HT1D receptor can only be evidenced by means of agonists (Hamel et al., 1989). The receptors involved in 5-HTinduced contraction in primate basilar artery (Connor et al., 1989) also appear to share pharmacological similarities with that in the human; however, further studies would be needed to establish their degree of similarity. The only and minor caveat with the use of bovine cerebral arteries was the lack of vasomotor responses (K + a n d / o r 5-HT) that we encountered on a few occasions (see Results). Although we did not explore this phenomenon further, it could have been a result of differences in slaughtering or possibly of seasonal (Vinall et al., 1991) or regional (Ayajiki and Toda, 1990) variations in contractility previously documented for bovine cerebral vasculature. Also, the possibility cannot be excluded that the endothelium a n d / o r smooth muscle might have been damaged in these vascular segments. Our results suggest that if the anti-migraine effect of sumatriptan is vascular-related, the 5-HT1D~ cerebrovascular receptor most probably corresponds to the therapeutic site for sumatriptan, providing the drug can reach the cerebral vascular smooth muscle. Recent studies have suggested that intraluminally administered sumatriptan would not reach the underlying vascular smooth muscle (Connor et al., 1992), most probably due to its inability to readily cross the blood-brain barrier (Sleight et al., 1990). However, the effect of sumatriptan to induce cerebral vasoconstriction in migraine sufferers has been shown to occur during migraine attack (Friberg et al., 1991). These observations would suggest that the interaction of sumatriptan with cerebrovaseular receptors is made possible during inflammation and changes in vascular permeability resulting from the neurogenic inflammation accompanying migraine attack (Fozard, 1987, 1989; Moskowitz et al., 1989; Moskowitz, 1992). In conclusion, bovine cerebral arteries contain a vasocontractile 5-HTID receptor which (1) has a pharmacological profile identical to that of the receptor in human brain vessels, (2) pharmacology shows a positive and highly significant correlation only with that of the cloned human 5-HTID/3 (or human 5-HTIB) receptor subtype. These results indicate that bovine cerebral arteries constitute an adequate pharmacological model of human 5-HTID cerebrovascular receptors and that it is most likely that these 5-HTlD receptors correspond to the same molecular entity. It would appear important to obtain 5-HT~Dt3 subtype-specific compounds to verify that the cerebrovascular site is the one that sumatriptan activates to stop migraine attacks.

81

Acknowledgements The following individuals or drug companies are gratefully acknowledged for providing us with compounds that were not available commercially: Dr. Rdmi Quirion, Douglas Hospital Research Centre; Glaxo Group Research Ltd., Hoffmann-LaRoche & Co., Merrell Dow International, Roussel UCLAF and Sandoz. We thank Ms. S. Kaupp and Mr. C. Hodge for expert artwork and photography, respectively and Ms. Linda Michel for preparation of the manuscript. This work was supported by a grant from the 'Fondation des Maladies du Coeur du Qudbec', a Summer Medical Fellowship (B.L.) from the 'Fonds de Recherche en Santd du Qudbec', as well as a grant (MA-9967) and a scholarship (E.H.) from the Medical Research Council of Canada.

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