β-adrenoceptor blockade in rats enhances the ambulation induced by 5-HT1A receptor agonists

European Journal of Pharmacology, 173 (1989) 121-125

121

Elsevier EJP 51074

fl-Adrenoceptor blockade in rats enhances the ambulation induced by 5-HTIA receptor agonists H a n s O. K a l k m a n Preclinical Research Sandoz A G, Postfach 4002, Basel, Switzerland

Received 12 June 1989, revised MS received 11 September 1989, accepted 26 September 1989

The mixed fl-adrenoceptor and 5-HT~A receptor antagonists, (-)-pindolol and propranolol, enhance rather than inhibit the hyperlocomotion induced in rats by the 5-HT~A receptor agonist, 8-OH-DPAT. The mechanism of this effect was now investigated. The rats were pretreated with the fl-adrenoceptor antagonist or saline and with the agonist 45 rain later. Ambulation was quantified as the number of quadrants entered during a 15 min observation period. (-)-Pindolol, alprenolol, betaxolol, ICI 118,551 and a combination of betaxolol and ICI 118,551 (all at 1 mg/kg) significantly enhanced the locomotion induced by 8-OH-DPAT (0.24 mg/kg). Timolol (1 and 10 mg/kg) given 45 min before 8-OH-DPAT was inactive; however, given at 10 mg/kg 15 min prior to 8-OH-DPAT, the compound enhanced locomotion. (-)-Pindolol (1 mg/kg) also enhanced the locomotion induced by the putative selective 5-HTIA receptor partial agonists, flesinoxan and ipsapirone, but not that induced by 5-OH-DPAT, a DA 2 receptor agonist. These results suggest that fla- or fl2-adrenoceptor antagonism can enhance the locomotion induced by 5-HT1Areceptor agonists. In the case of mixed 5-HTIA and fl-adrenoceptor antagonists, the fl-adrenoceptor-mediated effect may mask the inhibition of locomotion expected from 5-HTIA receptor antagonism. 5-HT~A receptors; Locomotion; fl-Adrenoceptor agonists

1. Introduction The selective 5-HT1A receptor agonist, 8-hyd r o x y - 2 - ( d i - n - p r o p y l a m i n o ) tetralin ( 8 - O H DPAT), induces in rats a behavioural syndrome which includes hyperlocomotion, head weaving, flat body posture and reciprocal forepaw treading (Hjorth et al., 1982; Tricklebank et al., 1984). Clear evidence that hyperlocomotion is triggered by stimulation of 5-HT1A receptors has been difficult to obtain. This is due to non-selectivity of the 5-HT~A receptor antagonists available. At doses which inhibited 8-OH-DPAT-induced locomotion, the 5-HT1A receptor antagonists, spiperone

and methiothepin themselves reduced spontaneous activity of rats (Tricklebank et al., 1984). While propranolol and pindolol are also 5-HT1A receptor antagonists they did not inhibit but potentiated (Tricklebank et al., 1984; own results) the hyperlocomotion induced by 8-OH-DPAT. The purpose of the present investigation was to explore the mechanism of the enhancement of the 8-OH-DPAT-induced hyperlocomotion that follows treatment with fl-adrenoceptor blocking agents.

2. Materials and methods 2.1. A n i m a l s

Correspondence to: H.O. Kalkman, Preclinical Research Sandoz AG, Postfach 4002, Basel, Switzerland.

Male Sprague-Dawley rats weighing 250-350 g were used in all experiments. They were housed in

0014-2999/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)

122

groups of four to five under a 12 h light-dark cycle (lights on 06:30 h). Pellet food and water were available ad libitum. All experiments were carried out between 10:00 and 16:00 h during the light phase of the diurnal cycle of the rats.

2.2. Locomotor studies Experiments were carried out using clear Perspex cages (24 × 40 cm; 16 cm high walls) placed on a white floor. The floor of each cage was marked out into four equal quadrants. Animals were assigned randomly to treatment groups and no food or water was available during the test sessions.

2.5. Statistics Comparisons between treatment groups and the appropriate controls were made as follows: (1) test for normality (Wilk-Shapiro), (2) then, for normally distributed values, the F-test to determine whether the variance of the two populations was equal and when this was so, (3) the two-tailed t-test (Wallenstein et al., 1980) was applied. Where populations were not normally distributed, the (two-tailed) Mann-Whitney test was performed. Significance was accepted at P < 0.05. All calculations were performed on an IBM PC-AT computer, using the software package R S / 1 (BBN Software Products Corporation, Cambridge, MA).

2.3. Test protocol 3. Results

The rats injected (s.c.) with either/3-adrenoceptor antagonist or saline and placed individually in their test cages. Locomotion assessment was started 5 min later and continued for 45 s. The observations were repeated every 3 min over a period of 45 min. This allowed four rats to be tested in parallel. Ambulation was scored by counting the total number of quadrants of the cage entered per five observation periods (i.e. for 15 min). After 45 min, 8 - O H - D P A T (or other agonists or saline) was injected (s.c.) and locomotor activity was again counted for 15 min as described above. In a separate experiment, the effect of timolol on 8-OH-DPAT-induced locomotion was investigated with a pretreatment time reduced to 15 min.

3.1. General Under the conditions of these experiments all exploratory activity ceased within 5 rain; when ambulation counting was started, the animals were quiet and in many instances had begun to sleep. In comparison with saline, ( - )-pindolol (1 m g / k g ) slightly, but significantly ( P = 0 . 0 0 4 5 ; MannWhitney test), enhanced locomotion during the first 15 min of observation. The sample median was 4.5 (n = 32) after ( - ) - p i n d o l o l and 2.0 (n = 48) after saline. None of the other/3-adrenoceptor antagonists significantly altered locomotion.

3.2. Effects of ~-adrenoceptor antagonists on hyperlocomotion induced by 8-OH-DPA T

2.4. Drugs 8-Hydroxy-2-(di-n-propylamino)tetralin • H B r (8-OH-DPAT) was purchased from RBI (Natick, MA, USA). ( - ) - P i n d o l o l , (_+)-flesinoxan and (___)-5-OH-DPAT • HC1 were synthesized at Sandoz (Basel, Switzerland). Alprenolol, timolol, betaxolol, ICI 118,551 (erythro-DL-l-(7-methylindan-4-yloxy)-3-isopropylaminobutan-2-ol • HC1) were kindly donated by Hassle (Mi31ndal, Sweden), MSD (Rahway, N J, USA), Synthrlabo (Paris, France) and ICI (Macclesfield, UK), respectively.

Following pretreatment with saline, 8-OHD P A T (0.06-1 m g / k g ) increased ambulation. Pretreatment with ( - ) - p i n d o l o l (1 m g / k g ) significantly enhanced this response (table 1). A higher dose of ( - ) - p i n d o l o l (10 m g / k g ) significantly reduced the response to 8 - O H - D P A T (0.24 m g / k g ; table 1). At a dose of 1 m g / k g , alprenolol, betaxolol, ICI 118,551 and a combination of betaxolol and ICI 118,551, but not timolol, enhanced the 8-OH-DPAT-induced locomotion. The results are summarized in table 2.

123 TABLE 1 Ambulation induced by 8-OH-DPAT alone and after pretreatment with (-)-pindolol. Ambulation was quantified as the number of line crossings counted during five periods of 45 s over the first 15 min after 8-OH-DPAT. Significance levels for 8-OH-DPAT responses between ( - ) - p i n d o l o l and the respective saline pretreated groups: a p 0.047, b p 0.0047, c p 0.03, d p 0.006 and e p 0.012 (two-tailed Student's t-test for normally distributed samples). Doses are given as m g / k g . The antagonist was given 45 min prior to the agonist. Pretreatment NaC1 NaC1 Pindolol NaCI Pindolol NaCI Pindolol NaC1 Pindolol Pindolol

Dose

1 1 1 1 10

Agonist

Dose

NaC1 8-OH-DPAT 8-OH-DPAT 8-OH-DPAT 8-OH-DPAT 8-OH-DPAT 8-OH-DPAT 8-OH-DPAT 8-OH-DPAT 8-OH-DPAT

0.06 0.06 0.24 0.24 0.5 0.5 1 1 0.24

Ambulation (mean ± S.E.M.)

n

2± 1 17 ± 1 24 ± 2 47 + 3 66 ± 7 50 _+7 75 ± 6 34 ± 4 54 ± 4 31 ± 5

8 8 12 18 8 6 6 6 6 6

a b c d e

TABLE 2 The effects of various fl-adrenoceptor blockers on the ambulation induced by 8-OH-DPAT. Ambulation was quantified as the number of line crossings counted during five periods of 45 s over the first 15 min after 8-OH-DPAT. P indicates the significance levels for 8-OH-DPAT responses between saline- and fl-blocker-pretreated groups (two-tailed Student's t-test for normally distributed samples). The antagonist was given 45 min prior to the agonist. Doses in mg/kg. Pretreatment

Dose

Agonist

Dose

Ambulation (mean ± S.E.M.)

n

P

NaC1 Alprenolol Timolol Timolol

1 1 10

8-OH-DPAT 8-OH-DPAT 8-OH-DPAT 8-OH-DPAT

0.24 0.24 0.24 0.24

47 ± 60 ± 43 ± 44 _

3 4 6 4

18 8 8 12

0.015 0.56 0.57

8-OH-DPAT 8-OH-DPAT 8-OH-DPAT

0.24 0.24 0.24

25 ___2 48 _ 5 58 ± 2

8 8 10

0.004 0.0001

8-OH-DPAT

0.24

59 ± 4

14

0.0001

NaC1 Betaxolol ICI 118,551 ICI 118,551 + betaxolol

1 1 1 1

TABLE 3 Effect of ( - ) - p i n d o l o l (1 m g / k g ) pretreatment on ambulation induced by flesinoxan, ipsaplrone and 5-OH-DPAT. Ambulation was quantified as the number of line crossings counted during five periods of 45 s over the first 15 min following administration of the agonist. Results are given as means_S.E.M. P indicates the significance level between saline- and pindolol-pretreated groups. Pretreatment time was 45 min. Pretreatment

Treatment

Dose

Saline Pindolol Saline Pindolol Saline Pindolol

Flesinoxan Flesinoxan Ipsapirone Ipsapirone 5-OH-DPAT 5-OH-DPAT

3 mg/kg 3 mg/kg 3 mg/kg 3 mg/kg 0.1 m g / k g 0.1 m g / k g

Ambulation (mean + S.E.M.) 15 ± 2 23 ± 3 11 _+2 19 _+3 10 ± 1 9i 1

n 8 10 8 8 8 8

P

0.02 0.03 0.64

124

3.3. Effect of pindolol pretreatment on locomotion induced by flesinoxan, ipsapirone and 5-OH-DPA T ( - ) - P i n d o l o l (1 m g / k g ) also enhanced the ambulation induced by the 5-HTaA-selective compounds, flesinoxan and ipsapirone, but not that induced by the dopamine receptor agonist, 5-OHDPAT. The results are shown in table 3.

3.4. Failure of timolol to enhance 8-OH-DPAT-induced locomotion Since timolol failed to enhance the ambulation induced by 8-OH-DPAT, the possibility of rapid metabolism of the/~-adrenoceptor antagonist was considered. To test this, the pretreatment time for timolol (10 mg/kg) was reduced to 15 min. Under these circumstances timolol was found to enhance the locomotion induced by 8-OH-DPAT (0.24 mg/kg). After pretreatment with saline the number of ambulation counts was 27.4 + 2.1 (mean _+ S.E.M.; n = 8) and after pretreatment with timolol the count was 35.4 _+ 3.0 (n = 8; P = 0.046).

4. Discussion The present results show that /~-adrenoceptor blockade can enhance the 5-HT1A agonist-induced locomotion in rats. Tricklebank at al. (1984) described the enhancement of 8-OH-DPAT-induced locomotion for propranolol but in contrast to the present results, not for pindolol. Both compounds are non-selective with respect to ]~-adrenoceptor subtypes and show a high affinity to 5-HT1A and 5 - H T ~ receptors (Middlemiss et al., 1984; Hoyer, 1989). To determine whether the enhancement of 8-OH-DPAT-induced locomotion involved a 5-HT receptor or a //-adrenoceptor-mediated mechanism, the effect of timolol was investigated. Timolol was considered a CNS-penetrating, nonselective//-adrenoceptor antagonist devoid of partial agonist activity (Shanks, 1985; McDevitt, 1986) and with poor affinity to 5-HT receptors (Hoyer, unpublished). Timolol (1 and 10 m g / k g ; - 4 5 rain) however, did not enhance 8-OH-DPAT-induced hyperlocomotion. Furthermore, the compound did not antagonize the enhancing effect of

pindolol (results not shown). These early results thus suggested that the mechanism of the increase in locomotion did not involve fl-adrenoceptors. One way to mimic timolol pretreatment was to combine the fl2-adrenoceptor antagonist, ICI 118,551 (Bilski et al., 1980) and the/~t-adrenoceptor antagonist, betaxolol (Boudot et al., 1979). These two compounds lack affinity to 5-HTj receptors (Hoyer, 1989) and are devoid of/~-adrenoceptor stimulant activity (Boudot et al., 1979; Bilski et al., 1980). The combination, and the two compounds separately, enhanced 8-OH-DPAT-induced locomotion. Since alprenolol also was found to potentiate the 8-OH-DPAT response, timolol apparently was an exception. The original literature on the pharmacokinetics of timolol may give the explanation for this: Tocco et al. (1975) measured the distribution of labelled timolol (10 m g / k g i.v., - 6 0 min) in rats. The brain had the lowest radioactivity counts of all tissues tested and moreover, 1 h after i.v. administration, the drug had been very extensively metabolized. The failure of timolol to enhance 8-OH-DPAT-induced locomotion may therefore reflect insufficient levels of the ]~-adrenoceptor antagonist in the brain. It was consistent with this suggestion that when the pretreatment time for timolol was reduced to 15 min, timolol produced a small but significant potentiation of the locomotor response to 8-OHDPAT. It is difficult to understand why both a selective /~l-adrenoceptor antagonist and a selective /~z-adrenoceptor antagonist enhance 8-OHDPAT-induced ambulation. Again, the explanation may be found in pharmacokinetics. It is possible that either betaxolol or ICI 118,551 is metabolized to a non-selective /~-adrenoceptor blocking product. The possibility of a fl-adrenoceptor-mediated mechanism was supported by the following observation. The dose-response curve for the locomotor activity of 8-OH-DPAT was bellshaped; increasing the dose of 8-OH-DPAT from 0.5 to 1 m g / k g did not produce a further increase in ambulation counts but, instead, decreased the response. However, even at the maximally effective dose of the agonist (0.5 mg/kg), ( - ) - p i n d o l o l still potentiated the response. This results excludes the possibility that ( - ) - p i n d o l o l enhanced the re-

125 s p o n s e to 8 - O H - D P A T b e c a u s e o f a p a r t i a l a g o n i s t a c t i o n at 5-HT1A r e c e p t o r s . T h e results f r o m t h e p r e s e n t i n v e s t i g a t i o n s h o w t h a t f l - a d r e n o c e p t o r a n t a g o n i s t a c t i v i t y c a n enh a n c e 5-HT1A a g o n i s t - i n d u c e d l o c o m o t i o n , a n effect w h i c h in the c a s e o f m i x e d 5 - H T I A a n d fladrenoceptor antagonists (pindolol, alprenolol or p r o p r a n o l o l ) m a y m a s k the e x p e c t e d i n h i b i t i o n o f locomotion. The observation that a higher dose of p i n d o l o l (10 m g / k g s.c.) s i g n i f i c a n t l y i n h i b i t e d 8-OH-DPAT-induced l o c o m o t i o n was c o n s i s t e n t w i t h this s u g g e s t i o n .

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potent and selective simplified ergot congener with central 5-HT-receptor activity, J. Neural Transm. 55, 169. Hoyer, D., 1989, 5-Hydroxytryptamine receptors and effector coupling mechanisms in peripheral tissues, in: The Peripheral Actions of 5-Hydroxytryptamine, ed. J.R. Fozard (Oxford University Press, Oxford) p. 72. McDevitt, D.G., 1986, Pharmacological characterisation of fl blockers and their role in clinical practice, J. Cardiovasc. Pharmacol. 8, $5. Middlemiss, D.N., J. Neill and M.D. Tricklebank, 1985, Subtypes of the 5-HT receptor involved in hypothermia and forepaw treading induced by 8-OH-DPAT, Br. J. Pharmacol. 85, 251P. Shanks, R.G., 1985, Mechanisms of action of beta-adrenoceptor antagonists in migraine, in: Migraine and Beta-blockade, eds. J.D. Carroll, V. Pfaffenroth and O. Sjaastad (Bohusl~iningens Boktryckeri AB, Udvalla, Sweden) p. 45. Tocco, D.J., A.E.W. Duncan, F.A. Deluna, H.B. Hucker, V.F. Gruber and W.J.A. Vandenheuvel, 1975, Physiological disposition and metabolism of timolol in man and laboratory animals, Drug Metab. Dispos. 3, 361. Tricklebank, M.D., C. Forler and J.R. Fozard, 1984, The involvement of subtypes of the 5-HT1 receptor and of catecholaminergic systems in the behavioural response to 8-hydroxy-2-(di-n-propylamino)tetralin in the rat, European J. Pharmacol. 106, 271. Wallenstein, S., S.L. Zucker and J.L. Fleiss, 1980, Some statistical methods useful in circulation research, Circ. Res. 47, 1.