Characterization of 5-Hydroxytryptamine receptors in goat cerebral arteries

Gem Pharmac.

Vol. 26, No. 6, pp. 1267-1272, 1995 ElsevierScience Ltd. Printed in Great Britain

Pergamon

0306-3623(95)00015-l

Characterization of SHydroxytryptamine Receptors in Goat Cerebral Arteries FRANCISCO J. MIRANDA,’ GERMAN TORREGROSA: JUAN B. SALOM,’ JOSfi A. ALABADf,’ TERESA JOVER,‘** MARfA D. BARBERA’ and ENRIQUE ALBORCH’q2* ‘Departamento de Fisiologia, Universidad de Valencia, and 2Centro de Investigackh, Hospital Universitario “La Fe”, Valencia, Spain /Tel: 63-86-2797; Fax: 63-86-87181 (Received 29 November 1994)

Abstract-l.

In isolated goat middle cerebral artery segments, 5-hydroxytryptamine (5-HT, IO-‘-3 x 10d5 M) elicited concentration-dependent contractions with EC,, = 2.1 (1.9-2.5) x lo-’ M and E max= 64 + 2% of 50 mM KCl-induced contraction. 2. Several 5-HT receptor agonists were used: (a) the agonist of 5-HT, receptors a-methyl-5-hydroxytryptamine (IO-‘-3 x 10m4M) induced strong contraction (51 + 6%); (b) the selective agonists of 5-HT, receptors sumatriptan (10-8-10~s M) and 5-carboxamidotryptamine (10-9-10-4 M) and the agonist of 5-HT,, receptors Ghydroxy-2-(di-n-propylamino)tetralin (lo-‘-3 x 10m5M) induced weak contractions (8, 18 and 14%, respectively); and (c) the agonist of 5HT, receptors 2-methyl-5-hydroxytryptamine (3 x 10-6-10-4 M) induced almost negligible contraction. 3. Pretreatment with the antagonist of 5-HT,, and 5-HT,, receptors cyanopindolol (IO-‘, 1O-6 M), the antagonist of 5-HT,/S-HT, receptors methysergide (10 - ‘I, 10e9M) and the antagonist of 5-HT, receptors ketanserin (IO- ”, 10 -9 M) induced non-competitive inhibition of the concentration-response curve to 5-HT. The antagonist of 5-HT, receptors 3-trophanyl-3,5-dichlorobenzoate (lo-‘, 10m5M) did not inhibit the contractile curve to 5-HT. 4. These results suggest that 5-HT contracts the goat middle cerebral artery by acting mainly on 5-HT, receptors. Key Words: Cerebral arteries, 5-HT, receptors, 5-HT, receptors

INTRODUCTION It is generally accepted that S-hydroxytryptamine (5-HT) participates in the regulation of cerebral blood flow in the physiological state and during several cerebrovascular disorders: (1) 5-HT is present in perivascular nerves in cerebral blood vessels (Saito and Lee, 1987; Chang and Owman, 1990; Moreno et al., 1991); (2) 5-HT is a potent constrictor of cerebrovascular smooth muscle (Parsons, 1991); (3) patients with vasospasm after subarachnoid hemorrhage have elevated concentrations of 5-HT in their cerebrospinal fluid (Chehrazi et al., 1989); and (4) 5-HT

has been

implicated

in the pathogenesis

migraine (Raskin, Characterization

1991). of specific receptors

ate the contractile

action of 5-HT in cerebral

*To whom all correspondence

of

which medi-

should be addressed.

vessels

have resulted in varied conclusions. The discordant results obtained in different studies indicate that 5-HT has a complex action on cerebral vasculature which probably reflects a diversity of 5-HT receptors (Parsons, 1991). 5-HT contracts cerebral arteries by acting on 5-HT,, 5-HT, or a mixed population of both receptor types (Parsons, 1991). We have previously reported the response of goat isolated middle cerebral artery to 5-HT and its modulation by the endothelium (Miranda et al., 1993). The present study has been carried out to characterize the 5-HT receptors which mediate the contractile effect of 5-HT on cerebral arteries, by using the 5-HT, receptor agonists sumatryptan and 5-carboxamidotryptamine (5-CT), the 5-HT,, receptor agonist 8-hydroxy-2(di-n-propylamino)tetralin (8-OHDPAT), the 5-HT, receptor agonist a-methyl-5hydroxytryptamine (cr-methyl-5-HT), and the 5HT, 2-methyl-5-hydroxytryptamine agonist receptor

1267

1268

Francisco J. Miranda et al.

(2-methyl-5-HT). On the other hand, we have used receptor antagonist cyanopinthe S-HT,,/5-HT,, dolol, the S-HT,/5-HT, receptor antagonist methysergide, the 5-HT, receptor antagonist ketanserin and the 5-HT, receptor antagonist 3-tropanyl-3,5dichlorobenzoate (MDL 72222). MATERIALS AND METHODS Tissue preparation

Female goats (30-45 kg), previously anesthetized with i.v. 2% sodium thiopental (Sodium Pentothal@, Abbott), were killed by i.v. injection of 30 mEq of potassium chloride. The whole brain was rapidly removed and the two middle cerebral arteries were dissected free and cut into cylindrical segments 4 mm in length. Each segment was prepared for isometric tension recording in a 5-ml organ bath. Briefly, two L-shaped stainless steel pins, 125 pm in diameter, were introduced through the arterial lumen. One pin was fixed to the organ bath wall, while the other was connected to a strain gauge for isometric tension recording. The vascular preparations were maintained at 37°C in a modified Ringer-Locke solution gassed with 95% O2 and 5% CO* to give a pH of 7.3-7.4. A resting tension of 1 g was applied to the arterial segments and they were allowed to equilibrate during a period of 60-90 min. Tension was readjusted when necessary and the bath fluid was changed every 15 min. After this period of equilibration the reactivity of the arterial segments was checked by depolarization with KC1 50mM; the arteries with a contraction of less than 1500 mg were discarded. Receptor characterization

To examine the effects of 5-HT, and the agonists sumatriptan, 5-CT, 8-OH-DPAT, a-methyl-5-HT and 2-methyl-5-HT on isolated cerebral arteries, concentration-response curves ( 10 - *-3 x 10e4 M) were obtained cumulatively in arteries at resting tension. We also studied the effect of 5-HT in the presence of the different 5-HT receptor antagonists. To do this we obtained a second concentration-response curve to 5-HT in the absence (control) and in the presence (20 min) of cyanopindolol(l0 -8, 10m6), methysergide 10m9M), ketanserin (10-i’, 10m9M) and (lo-“, MDL 72222 (lo-‘, lo-‘M). Statistical

analysis

The contraction values of 5-HT and the different agonists obtained in arteries at resting tension were expressed as a percentage of the previous contraction induced with 50 mM KCl. The contraction values of 5-HT in the absence (control) and in the presence of the different antagonists were expressed as a percent-

age of the maximum effect (E,,) reached in the first concentration-response curve of 5-HT. For each concentration-response curve, the E,,,,, and the concentration of agonist which produced half of the E,,,, (EC,,) were calculated. Mean EC&, and its confidence limits (95% interval) were calculated by obtaining the mean and the confidence limits of the pD2 values (-log EC,,) since they conformed to a normal distribution. From all the contraction values obtained in each experimental situation, the mean, standard deviation and standard error of the mean (SEM) were calculated. Statistical analysis of the different concentration-response curves was done by applying the Student-Newman-Keuls test both to pD2 and E,,,,, values. A probability value of less than 5% was considered significant. Drugs and solutions

The composition of the modified Ringer-Locke solution was (mM): NaCl, 120; KCl, 5.4; CaCl, 2Hz0, 2.2; MgCl, 7H,O, 1.0; NaHCO,, 25; and glucose, 5.6. In KCl-depolarizing solution NaCl was replaced by an equimolar amount of KCl. The following drugs were used: 5-HT (creatinine sulfate complex) was purchased from Sigma; sumatriptan and 5-CT were supplied by Glaxo; 8-OHDPAT, a-methyl-5-HT, 2-methyl-5-HT and MDL 72222 were purchased from Research Biochemicals Inc.; cyanopindolol and methysergide were supplied by Sandoz; and ketanserin were supplied by Janssen. Drugs were dissolved in distilled water and diluted in saline. RESULTS Cumulative addition of 5-HT (lo-*-3 x lo-’ M) elicited concentration-dependent contractions of isolated goat middle cerebral artery, with an EC,, of 2.1 (1.9-2.5) x lo-‘M and an E,,,, of 1982 _+ 1OOmg (64 + 2% of the 50 mM KCl-induced contraction). Several agonists used had different contractile effects: (1) the agonist of 5-HT, receptors cr-methyl-5HT (lo-‘-3 x 10e4 M) induced strong contraction (51 f 6% of the 50 mM KCl-induced contraction); (2) the agonists of 5-HT, receptors sumatriptan (10-8-10-5M) and 5-CT (10-8-10-4M), and 5HT,, receptors 8-OH-DPAT (lo-‘-3 x lo-‘M), induced weak contraction (8,18 and 14% of the 50 mM KCl-induced contraction, respectively); and (3) the receptors 2-methyl-5-HT of 5-HT, agonist (3 x 10d6-3 x 10e4M) induced almost negligible contraction (5.2 f 1% of the 50 mM KCl-induced contraction) of arterial segments (Fig. 1). The EC% and E,,,, values for the concentration-response curves of all the agonists are summarized in Table 1.

5-HT receptors in goat cerebral arteries

TENSION (76 50 mM KCI)

TENSION

75

1269

(% E_)

120

80

40 25

0 I

I

7

8

6

5-HT (-log Ml

a I

I

8

I.

3

7

6

I.,

,

5

,

4

M

-log

Fig. 1. Concentration-response curves for the contractions of isolated goat middle cerebral artery by 5-HT (0, n = 79), sumatriptan (0, n = 14), 5-CT (A, n = 14), 8-OH-DPAT (m, n =21), a:-methyl-5-HT (0, n = 14) and 2-methyl-5HT (A, n = 15). Tension values are expressed as percentage of the maximum effect reached by previous depolarization with KCI (50 mM), and represent mean + SEM.

Fig. 2. Second concentration-response curves for the contractions of isolated goat middle cerebral artery by 5-HT in the absence (0, n = 21) and in the presence of cyanopindolol (A, 10e8M, n = 8; A, 10m6M, n = 16). Tension values are expressed as percentage of the maximum effect reached by the same arterial segment in a first concentration-response curve of 5-HT, represent and mean &-SEM.

abolished ment

The repetition arterial

segment

contraction higher

with

the contractile

response

shows

the

E,,,,, values

a concentration-dependent

E,,,

(116 f 4%)

significantly different

of

lO-6 M), S-HT,/5-HT,

S-HT,,/5-HT,, and

receptors

‘I, 10 -9 M) induced

(lo-‘M)

(Figs

2, 3 and

a noncompetitive

inhibition

contractile Higher

of methysergide

and

curve

to

concentrations

ketanserin

almost

Table I. Values of effective concentration for 50% response (EC,,) and maximum effect expressed as percentage of previous depolarization with KCI 50 mM [E,,,,, (% KCI)] for concentration-response curves to 5-HT, sumatriptan, 5-CT, 8-OH-DPAT, 2-methyl 5-HT and a-methyl 5-HT in goat isolated middle cerebral artery E mar

5-HT Sumatriptan 5-CT 8-OH-DPAT X-Methyl 5-HT 2-Methvl 5-HT

2.1 1.9 1.4 3.3 4.6 2.6

(1.9-2.5) (1.3-2.7) (0.7-3.0) (2.4-4.4) (4X-5.3) (1.8-3.7)

x x x x x x

lo-‘M IO-‘M 10-6M 10-6M 10-6M IO-‘M

of the different

In this work we have examined different

subtypes

Table

2

concen-

antagonists

of the goat

middle

amine is a potent

(% KCI)

n

64+2 8+1 l8&2 14+2 51+6 5+1

79 14 14 21 14 15

Values of EC,, are mean and 95% confidence limits of n experiments. Values of E,,, are mean k SEM of n experiments.

The constrictor in cerebral Sotnikova monkey

the participation

of 5-HT receptors cerebral

vasoconstrictor

have shown in previous

EC.,

of the

DISCUSSION

ketanserin

4).

to 5-HT.

of

did not

curves to 5-HT in the absence (con-

trol) and in the presence used.

methysergide

receptors

of the concentration-response

tration-response

and

IO-‘M)

cyanopindolol

receptors

5-HT2

MDL 72222 (lo-‘,

EC,,

Pretreat-

the antagonist

inhibit

an

(lo-*,

5-HT

of 5-HT.

with

induced

onists

(10

action

segments

5-HT3 receptors

and an EC,, value not significantly

10m9M)

the contractile

of arterial

of a second S-HT curve in the same

with respect to the corresponding values of the first curve. Incubation of cerebral arteries with the antag-

(lo-“,

5

artery

of

in the response to 5-HT.

This

of this vessel as we

work (Miranda

et al., 1993).

effect of 5-HT has been demonstrated

vessels

from

and Gibala, (Connor

dog (Connor

et al., 1989;

1991), cat (Hamel et al., 1989),

et al.,

1989), sheep

(Gaw

et al.,

1990) and man (Jansen et al., 1991; Hamel and Bouchard, 1991; Parsons et al., 1991). The activity of 5-HT in contracting the goat middle cerebral artery is comparable to data reported previously in binding studies in brain tissue (Glennon, 1987). It has been shown that 5-HT displays a high affinity (Ki = l-10 nM) for 5-HT, binding sites but a relatively low affinity (Ki = 0.4-l PM) for 5-HT, binding

1270

Francisco J. Miranda et al. To study the participation

TENSION (% E_)

the contractile

120-

PT

O4 6

5

5-I-IT (-log M) Fig. 3. Second concentration-response curves for the contractions of isolated goat middle cerebral artery by 5-HT in the absence (0, n = 21) and in the presence of methysergide (@,10-r’ M, n = 7; A, 10e9 M, n = 12). Tension values are expressed as percentage of the maximum effect reached by the same arterial segment in a first concentration-response curve of 5-HT, and represent mean f SEM.

sites (Glennon,

1987). The later

binding

affinity

is

to the affinity of 5-HT (0.21 PM) in the

comparable

present study. TENSION

in

cerebral

receptors sumatriptan (Humphrey et al., 1988; Connor et al., 1989) and 5-CT (Bradley et al., 1986) induced contraction of goat middle cerebral artery with a similar (sumatriptan) or lower (5-CT) potency and much lower efficacy than 5-HT. The antagonist of 5-HT,, and 5-HT,a receptors (+) cyanopindolol (Hoyer et af., 1985) inhibited the contractile effect of 5-HT in a noncompetitive manner at relatively low concentration (lo-* M). This drug has a high affinity at both 5-HT,, and 5-HT,, recognition sites (2.29-5.4 nM) (Zifa and Fillion, 1992). This inhibitory action would suggest that 5-HT,, and/or 5-HT,, receptors could be present in the goat middle cerebral artery. Other studies have reported that (k) cyanopindolol does not antagonize the contractile response to 5-HT in human (Parsons et al., 1989) canine and primate (Connor et al., 1989) basilar arteries, thus demonstrating that the 5-HT,like receptor mediating contraction of these vessels is dissimilar to the 5-HT,, and 5-HT,a recognition sites identified in ligand binding studies and is different from the S-HT,, receptor mediating contraction of the canine basilar artery (Peroutka et al., 1986; Taylor et al., 1986). On the other hand, the agonist of the 5-HT,, receptors 8-OH-DPAT (Middlemiss and Fozard, 1983) also contracted the goat middle cerebral artery. The relatively low affinity of 8-OHDPAT in our study (3.3 PM) is comparable to that at 5-HT, binding sites in brain tissue (5.5-7.1 PM) but is much lower than that at 5-HT,* binding sites in brain tissue (2 nM) (Glennon, 1987) and that at 5-HT,, receptors in canine basilar artery (9 nM) (Peroutka et al., 1986). This result is in agreement with those obtained in ovine uterine and umbilical of S-HT,

T

40,

7

of S-HT, receptors

of the goat middle

artery we studied the effects of several agonists and antagonists of these receptors. The selective agonists

80.

1 8

response

(96 E_)

120-

eo-

Table 2. Values of effective concentration for 50% response (EC,,) and maximum effect expressed as percentage of maximum effect reached by the same arterial segment in a first concentrationresponse curve of 5-HT [(I&,, (%)] for a second concentrationresponse curve of 5-HT in the absence (control) and in the presence of different concentrations of the 5-HT receptor antagonists cyanopindolol, methysergide, ketanserin and MDL 72222 in goat isolated middle cerebral artery

40.

E nmi

EC.,

OA

i,

i

@j :

5

54IT (-log M) Fig. 4. Second concentration-response curves for the contractions of isolated goat middle cerebral artery by 5-HT in the absence (0, n = 21) and in the presence of ketanserin (B, IO-” M, n = 7; 0, 10m9M, n = 8). Tension values are expressed as percentage of the maximum effect reached by the same arterial segment in a first concentration-response curve of 5-HT, and represent mean f SEM.

Control Cyanopindolol 10-s M Cyanopindolol 10m6M Methysergide lo- ” M Methysergide 10m9M Ketanserin IO-” M Ketanserin 10 _ 9 M MDL 72222 IO-‘M MDL 72222 10-5M

2.8 (2.4-3.3) x 3.3 (2.2-4.8) x 3.2 (2.3-4.4) x 2.3 (2X1-2.7) x 2.2 (1.9-2.6) x 1.3 (1.1-1.5) x 4.2 (3.2-5.4) x 1.2 (0.8-1.7) x 1.8 (1.2-2.8) x

(%)

n

116+4 92f3’ 86f4* 92f5’ 75 * 5’ 87+7* 8Ort3’ 105+2 109_+2

21 8 16 I 12 7 8 IO 12

j

iv

IO-‘M IO-‘M IO-‘M IO-‘M IO-‘M IO-‘M IO-‘M IO-‘M lo-‘M

I

Values of EC,, are mean and 95% confidence limits of n experiments. Values of _I&.. are mean + SEM of n experiments. *Significantly different from control values, P < 0.01.

1271

S-HT receptors in goat cerebral arteries (Zhang and Dyer, 1990) suggesting that the 8-OH-DPAT-induced contraction in goat middle cerebral artery is mediated by 5-HT, receptors. The lower efficacies of the 5-HT, receptor agonists when compared to S-HT support their classification as partial agonists in the present study, as described in other cerebral arteries from several species (Connor et al., 1989; Parsons et al., 1991). This fact, together with the relatively low affinity of most of these agonists in the goat middle cerebral artery, suggests that their contractile effects could depend on their action on 5-HT, receptors. The agonist of 5-HT, receptors a-methyl-5-HT (Fozard, 1987) strongly contracted the goat middle cerebral artery. On the other hand, the mixed 5HTJS-HT, antagonist methysergide (Bradley et al., 1986) strongly inhibited in a noncompetitive manner the contraction induced by 5-HT. Because methysergide is considered to be more potent on 5-HT2 receptors and it exhibits its antagonistic effect at lo-” M, it could be accepted that this inhibitory action is achieved through its action on 5-HT, receptors. Moreover, in spite of the fact that its antagonism is noncompetitive, the high potency of ketanserin blocking the contractile effect of 5-HT in goat middle cerebral artery suggests the participation of 5-HT2 receptors. Ketanserin possesses a high affinity (K, < 1 nM) for 5-HT, binding sites in brain tissue (Glennon, 1987). Ketanserin is a potent competitive antagonist of 5-HT, receptors in sheep cerebral vasculature (Gaw et al., 1990). Nevertheless, a noncompetitive antagonism by ketanserin has been observed in cerebral arteries from cat (Hamel et al., 1989), rabbit (Edvinsson et al., 1984) dog and monkey (Connor et al., 1989) and ketanserin is totally devoid of inhibitory activity in human cerebral arteries (Parsons et al., 1989; Hamel and Bouchard, 1991), thus suggesting that there would be either a variation in the receptor type within each species or that a mixed 5-HT receptor population may be present (Gaw et al., 1990). The agonist of 5-HT, receptors 2-methyl-5-HT (Bradley et al., 1986) induced almost negligible contraction of the goat middle cerebral artery and the antagonist of this receptors MDL 72222 (Fozard, 1984) did not modify the contractile action of 5-HT. Therefore, it can be excluded from the participation of 5-HT, receptors in this response, in agreement with data obtained in human cerebral arteries (Parsons et al., 1989; Hamel and Bouchard, 1991). In summary, 5-HT contracts goat middle cerebral artery by acting mainly on 5-HT, receptors. The low efficacy of the $-HT, receptor agonists and the relatively low affinity of most of these drugs suggest that their contractile action is achieved by their action on

arteries

5-HT, receptors. Nevertheless, the inhibitory effect of cyanopindolol does not permit to discard the participation of the 5-HT,-like receptors. SUMMARY We examined the 5-HT receptors which mediate the contractile response to this amine in the goat middle cerebral artery by measuring isometric tension in isolated arterial segments. 5-HT (lo-‘-3 x lo-’ M) elicited concentration-dependent contractions of isolated goat middle cerebral artery, with an ECSo of 2.1 (1.9-2.5) x lo-’ M and an E,,,,, of 1982 + 100 mg (64 f 6% of 50 mM KCl-induced contraction). Several 5-HT receptor agonists were used with different contractile effects: (1) the agonist of 5-HT, receptors a-methyl-5-HT (lo-‘-3 x 10m4M) induced strong contraction (51 + 6% of 50 mM KCl-induced contraction); (2) the selective agonists of 5-HT, receptors sumatriptan (10-9-10-5 M) and 5-CT (10-8-10-4M) and the agonist of 5-HT,, receptors 8-OH-DPAT (lo-‘-3 x 10m5M) induced weak contractions (8, 18 and 14% of 50mM KCl-induced contraction, respectively); and (3) the agonist of 5-HT, receptors 2-methyl-5-HT (3 x 10m6-3 x 10m4 M) induced almost negligible contraction. Incubation of cerebral arteries with the antagonist of 5-HT,, and 5-HT,, receptors cyanopindolol (10 - ‘, 10 - 6 M) and the antagonist of 5-HT, receptors ketanserin (lo- ‘I, 10m9M) induced non-competitive inhibition of the concentration-response curve to 5-HT. The antagonist of 5-HT, receptors 3-trophanyl-3,5-dichlorobenzoate (MDL 72222, lo-‘, lo-‘M) did not inhibit the contractile curve to 5-HT. We conclude that 5-HT contracts the goat middle cerebral artery by acting mainly on 5-HT, receptors. The low efficacy of the 5-HT, receptor agonists and the relatively low affinity of most of these drugs suggest that their contractile action is achieved by their action on 5-HT, receptors. Nevertheless, the inhibitory effect of cyanopindolol does not permit to discard the participation of 5-HT,like receptors. Acknowledgements-This study was partially supported by a grant from the Fondo de Investigaciones Sanitarias de la Seguridad Social (Exp. 92/0442). The authors are grateful to Mrs Carmen Tirados and Mrs Carmen Maxiez for technical assistance.

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