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Effects of acetylcholine agonists and antagonists on yawning and analgesia in the rat
European Journal of Pharmacology, 139 (1987) 79-89
79
Elsevier EJP 00814
Effects of acetylcholine agonists and antagonists on yawning and analgesia in the rat A l m a J. G o w e r * Merrell Dow Research Centre, 16 rue d'Ankara, 67084 Strasbourg-Cedex, France
Received 11 December1986;revisedMS received11 March 1987, accepted 14 April 1987
The ability of acetylcholine muscarinic agonists, injected subcutaneously (s.c.) to elicit yawning and analgesia (tail-flick response) in rats was examined. Yawning was elicited by physostigmine, RS86 and pilocarpine with an inverted 'U'-shaped dose-response relationship; maximal effects occurred at 0.1, 0.5 and 2.0 mg/kg respectively. Neostigmine (0.05-0.2 mg/kg); arecoline (0.5-2.0 mg/kg); bethanecol (0.1-10 mg/kg) and McN-A-343 (5-20 mg/kg) had marginal or no activity. In contrast, dose-related analgesia was obtained following oxotremorine (0.01-0.3 mg/kg) and arecoline (0.5-4.0 mg/kg) and physostigmine (0.1-0.4 mg/kg), RS86 (0.25-2.5 mg/kg) and pilocarpine (0.5-8.0 mg/kg). The effects of acetylcholine antagonists on physostigmine-induced yawning and physostigmine-induced analgesia were also investigated. Following their s.c. injection, trihexyphenidyl, atropine, dicyclomine, secoverine and metliylatropine but not pirenzepine, inhibited both yawning and analgesia; there were clear differences in their potencies on the two responses. Pirenzepine, intracerebroventricularly (i.c.v.), inhibited yawning (EDs0 value 5.7 #g/rat) but not analgesia (3-100/~g/rat). The results are discussed in terms of a possible functional differentiation of central muscarinic receptors. Muscarinic acetylcholine receptor subtypes; Muscarinic agonists; Muscarinic antagonists; Yawning; Analgesia
1. Introduction
The existence of pharmacologically distinct muscarinic receptor subtypes, both centrally and peripherally, is now widely accepted. Support for the subclassification comes from the differential binding affinities of both agonists and antagonists (Hoss and Ellis, 1985; Watson et al., 1986a, b). The availability of pirenzepine, an acetylcholine antagonist with selective binding characteristics has contributed considerably to the study of these differences (Hammer et al., 1980). In fact, muscarinic receptors have been designated as either M1 or M2 depending on whether they have * To whom all correspondenceshould be addressed at present address: UCB, Secteur Pharmaceutique, Chemin du Foriest, 1420 Braine-l'Alleud, Belgium.
high or low affinity for pirenzepine (Hammer and Giachetti, 1982) although it is recognised that such a classification is as yet tentative (Birdsall and Hulme, 1983). Cholinergic mechanisms are implicated in many behavioural phenomena and the possibility of finding selective agonists or antagonists has produced renewed promise for more effective therapy for certain n e u r o l o g i c a l disorders involving cholinergic mechanisms. Senile dementia of the Alzheimer type (SDAT) is of particular current interest in view of the compelling evidence of a cholinergic deficit underlying the primary symptom, i.e. loss of memory (Bartus et al., 1982; Perry and Perry, 1985), as well as reports of selective changes in certain receptor subtypes in postmortem brain of SDAT patients (Nordberg et al., 1983; Mash et al., 1985).
0014-2999/87/$03.50 © 1987 ElsevierSciencePublishers B.V. (BiomedicalDivision)
80 Although binding studies provide one way of characterising potential selective agonists and antagonists, they are usually done in vitro and are not always predictive of what is happening in the whole animal. The ability to distinguish between receptor subtypes on the basis of behavioural responses would therefore be useful in the development of selective muscarinic drugs. For this reason, we investigated in the rat the effects of selected agonists and antagonists on two behaviours which involve central cholinergic mechanisms, namely yawning and analgesia. Both the cholinesterase inhibitor, physostigmine and the acetylcholine agonist, pilocarpine, induce yawning in rats (Urba-Holmgren et al., 1977; Gower et al., 1984), an effect which can be antagonised by scopolamine (Yamada and Furukawa, 1980). Similarly, acetylcholine agonists such as oxotremorine are known to cause analgesia (Harris and Dewey, 1973; Karczmar and Dun, 1978). We determined the effects of various agents in inducing yawning or analgesia and the effects of antagonists in blocking physostigmine-induced yawning and physostigmine-induced analgesia. The drugs tested included the agonists oxotremorine and McN-A343 and the antagonists pirenzepine and dicyclomine, putatively selective for certain receptor subtypes (Birdsall and Hulme, 1983; Potter et al., 1984; Hoss and Ellis, 1985; Marchi and Raiteri, 1985). 2. Materials and methods
2.1. Animals Naive, male Sprague-Dawley rats (Charles River, France), weighing 250-320 g, were used. Prior to use, the rats were housed in groups of 6 in wire cages on a controlled 12 h light-dark cycle (lights on at 6:00 a.m.) and were allowed ad lib access to standard cube diet and tap water. Each rat was used only once. 2.2. Procedure Yawning and analgesia were investigated separately in different rats. All experiments were carried out between 8:30 a.m. and 1:30 p.m.
2.2.1. Assessment of yawning Each rat was placed in a large glass beaker (18 cm high × 14.5 cm diameter) containing a layer of sawdust. A metal grid prevented the rats from escaping. The number of yawns during a 30 rain period was counted by direct observation. The yawning response, defined as a wide stretched opening of the mouth, has been described previously (Berendsen and Gower, 1986). A mirror was positioned immediately behind the beakers to permit 360 ° observation of the rats. Groups of 4 or 5 rats were tested at a time and the doses were tested in random order. The effects of agonists were determined over a 30 rain period immediately following s.c. injection. The effects of antagonists were determined by pretreatment of the rats with the antagonist s.c. followed by return to their home cages; control animals which received saline in place of antagonist were included in each antagonist experiment. Physostigmine, 0.1 mg/kg s.c., was injected 30 rain later and the number of yawns elicited over the following 30 min was then counted. The effects of atropine sulphate (SO4) and pirenzine were also assessed following their i.c.v, administration, 30 min prior to physostigmine, 0.1 mg/kg s.c. 2.2.2. Analgesia Analgesia was measured with an Appelex DS 20 tail-flick apparatus and was quantitated as increases in latency for the tail-flick response to a focussed heat stimulus. The intensity of the heat stimulus was adjusted to produce a latency of 2-4 s in saline-treated control rats. A maximal cut-off time of 12 s was used for rats which did not respond to the heat stimulus. The effects of agonists were determined by measuring the tall-flick latency 20 rain after s.c. injection. The effects of antagonists were determined by pretreatment of the rats with the antagonist s.c. or saline 30 rain prior to the s.c. injection of physostigmine, 0.1 mg/kg. The tailflick latency was measured 20 rain after physostigmine. Control rats which received only saline were included in each antagonist experiment. The effects of atropine SO4 and pirenzepine injected i.c.v, on physostigmine-induced analgesia were also
81
minimal amount of 0.1 M hydrochloric acid, making the solution up to the required volume with 0.9% saline solution then neutralizing to pH 7.0 by adding a few drops of saturated sodium hydrogen carbonate solution. The remaining drugs were all dissolved in 0.9% saline solution. Solutions of dicyclomine were protected from light by wrapping the bottles in aluminium foil. Solutions of apomorphine were kept on ice. A dose volume of 2 ml/kg used for s.c. injection and the solutions were injected into the loose skin at the back of the neck. For i.c.v, administration, the rats were first prepared with a guide cannula unilaterally, directed at the lateral ventricle and positioned 0.2 mm behind the bregma and 2.0 mm lateral to the midline. At least one week post-operative recovery time was allowed. Injections were made by inserting a 28 gauge injection cannula to a depth of 3.5 mm below the skull surface and injecting the drugs in a dose volume of 2 #1 per rat over a period of 30 s. For both s.c. and i.c.v, injections, control rats received equivalent dose volumes of 0.9% saline.
determined by injecting them 30 min before physostigmine and testing 20 min after physostigmine, 0.1 mg/kg s.c. 2.3. Drugs and injections The following drugs were used: apomorphine hydrochloride, arecoline hydrobromide, atropine methyl nitrate, atropine sulphate, bethanecol (Sigma, USA); dicyclomine (Merrell Dow, Strasbourg); McN-A-343 (Research Biochemicals Incorporated, USA); neostigmine hydrochloride, oxotremorine sesquifumarate, physostigmine salicylate, pilocarpine hydrochloride (Sigma USA); pirenzepine (Dr. Karl Thomae, GMBH, Germany); 2-ethyl-8-methyl-2,8-diazospiro-(4,5)-decan1,3-dionhydrobromide, RS86 (Sandoz, Switzerland); secoverine hydrochloride (Duphar, Holland) and trihexyphenidyl hydrochloride (Sigma, USA). All drug solutions were prepared immediately prior to the experiment. Pirenzepine was prepared by dissolving the powder in a
4@ 4~
4~
0. 0
25 ~ pg/kg
PHYSOSTIGMINE
o m g ~ k g 4.0
0
PILOCARPINE
G 5 1.O S.O 0.1 O.5 2.5 mg/kg
mg/kg
RS 8 6
ARECOLINE
0
1,0
4
0
lOO
/Jg/ k g N E O S T I G M I NE
0
0.3 3.0 mg / kg
BETHANECOL
0
1.0 10 pg / kg
OXOTREMORINE
100
0 10 mg/kg MCN.A.343
Fig. 1. Effects of muscarinic agonists in eliciting yawning in rats. Hatched columns refer to saline-treated controls. Dose groups of 9 or 12 rats were used. * Significantly greater than saline-treated controls; P ~< 0.05, Student's t-test.
82
2.4. Statistics
6
The drug-induced changes in each yawning experiment were first assessed with an analysis of variance. When this yielded a significant result, the effects of individual doses were assessed with Student's t-test. Non-parametric methods were used for the analgesic experiments (Siegel, 1956). A Kruskal-Wallis analysis of variance was first carried out and when this showed significance, a Mann-Whitney U-test was used to compare control and test groups.
gl
Z >.
z i11
3. Results S
10
15
20
25
30
35
40
45
MIN. OF TESTING
3.1. Yawning
Fig. 2. Time course of physostigmine (0.1 m g / k g ) and arecoline (1.0 m g / k g ) for eliciting yawning. The n u m b e r of yawns elicited over consecutive 5 min intervals was recorded. Groups of 12 rats were used.
The effects of muscarinic agonists in causing yawning are shown in fig. 1. Physostigmine, pilocarpine and RS86 elicited marked yawning; the number of yawns followed an inverted 'U'-shaped curve with respect to dose, with low and high
,_E
=E
0 o
loo 400 JJg/kg
o
PHYSOSTIGMIN E ~"
(Is 2.0 mg/kg
8.0
0
PILOCARPINE
FS7~7
0.25 I~) m g / kg
o
1,o
4.0
mg/kg ARECOLINE
RS 8 6
At
12
,,J
,s
O
.~O 200 ~191kg
N EOSTIGMINE
FI~7~.07
0
tO 6£) mglkg
BETHANECOL
~ ~
O
30 300 l~91kg
OXOTREMORIN
E
~7
0 20 mglkg M C N . A . 343
Fig. 3. Effects of muscarinic agonists in causing analgesia in rats. Hatched columns refer to saline-treated controls. Dose groups of 9 rats were used. * Significantly greater than saline-treated controls; P ~< 0.05, Mann-Whitney U-test.
83
doses having little effect. Arecoline, neostigmine, bethanecol, oxotremorine and McN-A-343 caused only marginal or no yawning. Arecoline has a short duration of action which could account for the low level of responding since the total number of yawns over 30 min was recorded. However, the time course of the effects of peak doses of physostigmine and arecoline (fig. 2) makes it evident that the maximum effect of arecoline per 5 min interval was considerably less than that of physostigmine. All the agonists except bethanecol induced licking and chewing movements. A1-
though not systematically quantified, this effect was most marked for drugs causing yawning but was not related to the intensity of yawning. Thus, high doses of agonists, following which yawning was reduced or absent, caused the most pronounced chewing and licking. Yawning induced by physostigmine is characteristically accompanied by stretching (Urba-Holmgren et al., 1977). In the present study, this was observed only for physostigmine and not for the other muscarinic agonists. Typical muscarinic effects such as chromodacryorrhea, salivation and increased defaeca-
TABLE 1 Effects of muscarinic antagonists in antagonising physostigmine-induced (0.1 m g / k g ) yawning in rats. Antagonist
Dose (mg/kg)
n/12 ~
Yawning (mean + S.E.)
5g inhibition
Trihexyphenidyl
Saline 0.025 0.05 0.1
12 11 11 10
11.8 ± 1.7 7.2 ± 1.4 * 5.9 5:1.0 * 2.7+0.8 *
39.0 50.0 67.9
Saline 0.3 0.6 1.0 3.0
11 10 10 4 4
12.4 ± 2.8 9.4 ± 2.3 4.5 ± 1.5 * 1.5±0.8 * 0.5 ± 0.3 *
24.2 63.4 87.9 96.0
Saline 0.3 1.0 3.0 10.0
12 12 9 5 2
11.6 ± 2.0 8.3 _+1.9 4.7 _+2.2 1.8 ± 0.7 0.4 _+0.3
28.4 59.5 84.5 96.6
Saline
5.0 7.5 10.0
12 12 11 11 9 9
10.7 ± 2.1 14.8 ± 2.0 16.8±2.4 * 8.1 ± 2 . 2 4.7 ± 1.8 * 1.9±0.6 *
24.3 56.1 82.1
Saline 1.0 3.0 6.0 10.0
12 12 12 10 8
15.3 + 3.7 14.2 4- 2.4 12.8±2.2 8.4 + 3.2 6.2± 2.5 *
7.2 16.3 45.1 59.5
Saline 1.0 3.0 10.0
12 11 11 12
13.3 ± 15.2 + 10.3 ± 13.3 +
22.6 0
Atropine SO 4
Dicyclomine
Secoverine
1.0 3.0
Atropine M e N O 3
Pirenzepine
2.1 3.8 1.8 2.1
* * * *
ED50 (mg/kg) 0.042
0.48
0.67
-
7.0
7.25
inactive up to 10.0
a n / 1 2 = number of rats yawning per group out of possible m a x i m u m of 12. * Significantly different from saline control; P ~< 0.05 Student's t-test. The EDs0 was calculated as the dose of antagonist which reduced the mean number of yawns induced by physostigmine by 50%.
84
tion were observed for physostigmine, pilocarpine, RS86, arecoline, bethanecol and oxotremorine. These effects were dose-related and were marked at the highest doses. Neostigmine produced slight salivation and chromodacryorrhea and McN-A343 slightly increased defaecation only. The effects of muscarinic antagonists injected s.c. on physostigmine-induced yawning are shown in table 1. Trihexyphenidyl, atropine SO4, dicyclomine and atropine MeNO 3 blocked yawning in a dose-related manner. Pirenzepine had no effect up to 10 mg/kg. Secoverine, 5-10 mg/kg, also inhibited physostigmine-induced yawning in a dose-dependent manner, but yawning was increased at 1.0 and 3.0 mg/kg. Secoverine by itself, 3.0 mg/kg injected 30 min before test, slightly but significantly increased yawning (table 2). In contrast to its effects on physostigmine-induced yawning, secoverine did not enhance the effects of arecoline or the effects of pilocarpine (table 2). In fact, secoverine 3.0 mg/kg significantly reduced the mean number of yawns induced by pilocarpine although it had no effect on the number of rats responding. Since dopamine agonists also induce
yawning (Gower et al., 1984) the effect of apomorphine at a dose eliciting only a low level of yawning on physostigmine-induced yawning was included for comparative purposes (table 2). Apomorphine 0.01 mg/kg enhanced physostigmine-induced yawning markedly.
TABLE 3 Effects of muscarinic antagonists in antagonising physostigmine-induced (0.1 m g / k g ) analgesia in rats. Antagonist
Dose (mg/kg)
Atropine SO4
Control a 3.2 + 0.3 * Saline b 4.0 _+0.3 0.03 4.14- 0.3 0.1 3.4-1-0.1 * 0.3 2.9_+0.2 *
0 71.4 100.0
Control a 3.0_+ 0.1 * Saline b 3.9 _+0.3 0.03 4.0_.+ 0.3 0.06 3.5 + 0.2 0.1 3.3-+0.2 * 0.2 3.0+0.3 *
0 44.4 66.7 100.0
Trihexyphenidyl
Atropine MeNO 3 TABLE 2 Effects of various drug combinations on yawning in the rat. Drug treatment (dose m g / k g )
Yawning (mean_+ S.E.)
n/N a
Saline Secoverine (3.0)
0.2 5:0.1 1.3 -+ 0.4 *
2/12 8/12
Saline + arecoline (1.0) Secoverine (3.0) + arecoline (1.0)
1.4 -+0.8 1.3 -+0.5
5/12 5/12
Saline + pilocarpine (2.0) Secoverine (0.3) + pilocarpine (2.0) Secoverine (1.0) + pilocarpine (2.0) Secoverine (3.0) + pilocarpine (2.0)
13.8 -+ 2.2
12/12
11.4 -+ 2.1
12/12
12.1 _+2.5
11/12
6.7 _+1.9 *
11/12
Saline Apomorphine (0.01) Physostigmine (0.1) Apomorphine (0.01) + physostigmine (0.1)
0.3 + 0.3 2.9 _+1.3 13.9 + 2.7 *
1/8 5/8 8/8
22.9 _+3.2 *
8/8
Number of rats yawning (n) divided by number of rats tested (N). * Significantly different from corresponding control, P ~< 0.05, Student's t-test.
Secoverine
Tall-flick % inhibi- EDs0 latency (s) tion (mg/kg) (mean + S.E.)
Control a 3.2-+ 0.2 * Saline b 4.0 4- 0.2 0.3 4.2 -+0.2 0.6 3.8 -+ 0.3 1.0 3.6 _+0.3 3.0 3.3+0.4 *
0.065
0.073
0 25.0 50.0 87.5
1.05
Control ~ 2.7 _+0.2 * Saline b 3.8 _+0.2 0.3 3.9+0.3 1.0 3.6-+0.4 3.0 3.3 4-_0.2 * 10.0 2.8+0.2 *
0 18.2 45.5 90.9
2.7
Dicyclomine Control ~ 3.1 + 0.2 * Saline b 4.5 -+0.3 1.0 4.2 5:0.5 3.0 3.8_+0.3 10.0 3.4_+0.3 *
21.4 50.0 78,6
3.4
14.2 14.2
inactive up to 10.0
Pirenzepine
Control a 3.1+0.2 Saline b 3.8 -+ 0.2 3.0 3.7 + 0.2 10.0 3.7_+0,2
-
a Control group receiving only saline; b group receiving only physostigmine. Dose groups of 9 rats were used. * P ~<0.05; Mann-Whimey U-test with respect to physostigmine only group. The EDs0 was calculated as the dose inhibiting physostigmine-induced analgesia by 50%.
85
induced analgesia whereas the EDs0 value for atropine was 0.2 #g/rat.
3.2. Analgesia The analgesic effects of muscarinic agonists, seen as prolonged tail-flick latency, are shown in fig. 3. Physostigmine, pilocarpine, RS86, arecoline and oxotremorine all caused analgesia which increased with increasing dose. Neostigmine, bethanecol and McN-A-343 were inactive. The effects of muscarinic antagonists, injected s.c., on physostigmine-induced analgesia are shown in table 3. Atropine SO4, trihexyphenidyl, atropine MeNO 3, secoverine and dicyclomine all inhibited physostigmine-induced analgesia in a dose-dependent manner. Pirenzepine was inactive up to 10 mg/kg.
4. Discussion
The present results confirm previous work that pointed to cholinergic involvement in both yawning and analgesia (Harris and Dewey, 1973; Urba-Holmgren et al., 1977; Karczmar and Dun, 1978; Yamada and Furukawa, 1980). However, there were clear differences between the two responses in terms of both agonist and antagonist effects. Yawning was induced by the cholinesterase inhibitor, physostigmine, and the direct agonists, RS86 and pilocarpine, but oxotremorine, arecoline, bethanecol, neostigmine and McN-A-343 had little or no effect. In contrast, oxotremorine and arecoline, as well as physostigmine, RS86 and pilocarpine, induced analgesia although bethanecol, neostigmine and McN-A-343 were again inactive. The dose-response relationship for yawning followed an inverted 'U'-shaped curve whereas the relationship for analgesia was linear in all cases, with maximal effects occurring at doses considerably higher than those maximal for yawning. There was thus not only a difference in the agonists which elicited yawning versus analgesia but there
3.3. Effects of i.c.v, injection of antagonists," yawning and analgesia The effects of i.c.v, injection of atropine SO4 and pirenzepine on physostigmine-induced yawning and analgesia respectively are shown in table 4. Atropine antagonised both yawning and analgesia but was 5-fold more potent in blocking analgesia. Pirenzepine inhibited yawning dose dependently (EDs0 5.7 ttg/rat) and was thus 6 times less potent than atropine. In contrast, pirenzepine up to 100/~g/rat failed to inhibit physostigmine-
TABLE 4 Effects of atropine SO 4 and pirenzepine i.c.v, in antagonising physostigmine-induced yawning and physostigmine-induced analgesia in rats. Antagonist
Atropine SO4
Pirenzepine
Dose
Yawning
( # g / r a t i.c.v.)
Mean ± S.E.
Control Saline 0.3 1.0 3.0
10.7±2.5 8.4±3.5 6.8±1.6" 1.1±0.7"
Control Safine 3.0 10.0 30.0 1~.0
8.8±2.6 5.6±1.5 3.5±0.9* 1.0±0.7" NT
Analgesia (TFL) ED50 (btg/rat)
Mean ± S.E.
EDs0 ( # g / r a t )
1.0
2.9±0.1 4.8±0.5 3.6±0.3 3.5±0.4 3.0±0.2
0.2
5.7
2.8±0.2 4.0±0.2 4.3±0.3 4.4±0.4 4.3±0.1 4.4±0.4
*
inactive up to 100.0
* P ~< 0.05; Student's t-test versus saline group (yawning) or Mann-Whitney U-test (analgesia). T F L = tail-flick latency in s, N T = not tested. Dose groups of 12 or 9 rats were used for the yawning and analgesia experiments respectively.
86 was a characteristic difference in the dose-response relationship for those agonists which elicited both responses. Neostigmine, bethanecol and McN-A-343 are all drugs which have difficulty in penetrating into the brain following their parenteral administration (Watanabe, 1982; Taylor, 1985). Their inability to cause either yawning or analgesia is consistent with a central site of action and confirms earlier reports (Harris and Dewey, 1973; Urba-Holmgren et al., 1977). All the antagonists except pirenzepine were active against both the yawning and the analgesia induced by physostigmine although there were marked differences in the doses required to inhibit each response. The order of potencies for antagonism of yawning was trihexyphenidyl > atropine >/dicyclomine > secoverine >1methyl atropine (EDs0 ratios = 1 : 11 : 16 : 167 : 173). The order of potencies for antagonism of analgesia was trihexyphenidyl >/atropine > methyl atropine > secoverine >/dicyclomine (EDs0 ratios = 1 : 1 : 14 : 37:47). The separation (approximately 15-fold) between the EDs0 values of atropine and methyl atropine on both responses reflects the poor penetration of methyl atropine into the brain (Herz et al., 1965). The EDs0 value for secoverine for yawning was probably artificially elevated in view of its opposing effect at low doses. The same dose (0.1 mg/kg) of physostigmine was used to induce yawning and analgesia; this was a maximal effective dose for yawning but it was submaximal for analgesia. Hence, the differing potencies of both atropine and methyl atropine to inhibit yawning and analgesia respectively might be due to a greater facility to inhibit submaximal than maximal effects. Alternatively, the difference could reflect a different site of action within the brain, with the site for analgesia being more accessible for drug action following parenterai injection. However, neither explanation accounts for the results obtained with trihexyphenidyl or dicyclomine. It is known that pirenzepine penetrates poorly into the brain from the periphery (Eberlein et al., 1977) hence its lack of effect following s.c. injection was not unexpected in view of the evidence that cholinergic-induced yawning and analgesia are centrally mediated. Pirenzepine now has a key
role in the classification of muscarinic receptor subtypes. We therefore compared its effects following i.c.v, injection with those of atropine i.c.v. Pirenzepine inhibited physostigmine-induced yawning but was inactive against analgesia. This latter result differs from that of Caulfield et al. (1983) who found that pirenzepine injected into the third ventricle inhibited oxotremorine-induced analgesia with an EDs0 of 4.6/xg per mouse. It is possible that this discrepancy was due to species differences in sensitivity to the drug since this EDs0 was almost 50 times greater than that for the inhibition of passive avoidance learning in the mouse. An interesting biphasic effect was obtained with secoverine on physostigmine-induced yawning. Secoverine has been shown to have a selective action at the acetylcholine presynaptic receptor inhibiting acetylcholine release in frontal cortex, with less effect on presynaptic acetylcholine receptors mediating dopamine release (Marchi and Raiteri, 1985). The enhancement by secoverine may therefore be attributable to blockade by autoreceptors, leading to increased acetylcholine being available to interact postsynaptically. In this case, the lack of enhancement of arecoline or pilocarpine-induced yawning suggests that they are inactive at these particular receptors. Alternatively, since physostigmine-induced yawning can be enhanced by threshold doses of apomorphine, the effects of secoverine may be independent of its cholinergic effects. However, dopaminergic activity has not been reported for secoverine and no known actions of secoverine would account for its enhancing effect on yawning (Zwagemakers and Claassen, 1980). The finding that physostigmine and pilocarpine induced yawning confirms previous observations (Urba-Holmgren et al., 1977; Yamada and Furukawa, 1980; Gower et al., 1984; Ushijima et al., 1984), although RS86-induced yawning has not previously been reported. Also, the range of acetylcholine antagonists investigated extends earlier observations with scopolamine and atropine (Urba-Holmgren et al., 1977; Gower et al., 1984). Surprisingly, in view of the marked, replicated efefcts of pilocarpine in the present study, it appears that pilocarpine-induced yawning is not
87 always reproducible. Salamone et al. (1986) failed to obtain significant yawning after pilocarpine. They suggested that the discrepancy between their results and previous results (Ushijima et al., 1984) may have been due either to a difference in the strain of rats used or to a difference in the definition of yawning, i.e. gaping as opposed to yawning. Neither explanation is applicable to the present study. Only prolonged wide, stretched opening of the mouth counted as a yawn (Berendsen and Gower, 1986) and there was an inverted 'U'shaped dose-response function in contrast to the increased gapings with increased dose obtained by Salamone et al. (1986). Also, Sprague-Dawley rats were used in both studies. However, the strain of rat can affect the results; in an earlier study using Wistar rats (Gower et al., 1984) albeit in a different location, fewer yawns were elicited by the maximal effective dose of physostigmine, again 0.1 mg/kg. In the author's experience, a major factor affecting yawning seems to be emotionality which can vary according to the strain and also according to the handling and maintenance procedures; for example, rats stressed during injection usually have a low rate of yawning. The effects on yawning and analgesia obtained with both agonists and antagonists point to a difference in the cholinergic mechanism involved in the two responses. Yawning was pirenzepinesensitive but oxotremorine-insensitive whereas the opposite held for analgesia which was pirenzepine-insensitive but oxotremorine-sensitive. Also, secoverine at low doses enhanced physostigmineinduced yawning but did not increase analgesia. These differences may have been due to differing interactions with other transmitter systems or to different sites of action in the brain; for example, yawning appears to be striatally mediated (Dourish and Hutson, 1985; Dourish et al., 1985) whereas lower brain sites are likely to be involved in analgesia (Karczmar and Dun, 1978). Equally, the differences may represent pre- versus postsynaptic sites of action at the same receptor subtype. However it is possible that the differences in the effects of the agonists and antagonists indicate that different muscarinic receptor subtypes are involved in mediation of the responses. Muscarinic recep-
tors have been designated M1 or M2 depending mainly on their affinity for pirenzepine. It is therefore tempting to speculate that, because yawning was pirenzepine-sensitive, an M1 receptor mediates yawning and conversely because analgesia was pirenzepine-insensitive that an M2 receptor mediates analgesia. The results obtained with the other antagonists and the agonists are consistent with such an interpretation. Thus, on the basis of both binding data and selectivity of action on certain tissues, oxotremorine has been proposed as an M2 agonist and pilocarpine as an M1 agonist (Potter et al., 1984; Van Charldorp et al., 1985; Watson et al., 1986a). As with the antagonists, dicyclomine and trihexyphenidyl appear to be M1 selective in that they have a profile of action similar to that of pirenzepine (Potter et al., 1984; Marchi and Raiteri, 1985; Tien and Walace, 1985; Giachetti et al., 1986). Furthermore, as already mentioned, secoverine has selective actions at the presynaptic autoreceptor (Marchi and Raiteri, 1985) and has a profile of action in certain tissues which suggests that it might be an M2 antagonist (Zwagemakers and Claassen, 1981). However the selectivity of these agents has not been confirmed in all studies nor shown unequivocally (Choo and Mitchelson, 1985; Eglen and Whiting, 1985; Brunner et al., 1986). There is therefore insufficient evidence at this stage to allow certain functional responses to be related convincingly to particular muscarinic subtypes. Unfortunately, one lacks agonists and antagonists which can distinguish sufficiently between receptor subtypes but equally, one lacks a functional means of differentiation. It is through the development of selective compounds and through functional studies such as the present one that the uncertainty regarding muscarinic subtypes will be resolved.
Acknowledgements The excellenttechnical assistanceof Mr. C. Forler is gratefully acknowledged.We thank Dr. Karl Thomae, GMBH and Duphar for their generousgifts of drugs.
88
References Bartus, R., R.L. Dean, B. Beer and A.S. Lippa, 1982, The cholinergic hypothesis of geriatric memory dysfunction, Science 217, 408. Berendsen, H.H.G. and A.J. Gower, 1986, Opiate-androgen interactions in drug-induced yawning and penile erections in the rat, Neuroendocrinology 42, 185. Birdsall, N.J.M. and E.C. Hulme, 1983, Muscarinic receptor subclasses, Trends Pharmacol. Sci. 4, 459. Brunner, F., M. Waelbroeck and J. Chfistophe, 1986, Secoverine is a non-selective muscarinic antagonist on rat heart and brain receptors, European J. Pharmacol. 128, 17. Caulfield, M.P., G.A. Higgins and D.W. Straughan, 1983, Central administration of the muscarinic subtype-selective antagonist pirenzepine selectively impairs passive avoidance learning in the mouse, J. Pharm. Pharmacol. 35, 131. Choo, L.K. and F. Mitchelson, 1985, Comparison of the affinity of secoverine for some muscarinic receptors, Clin. Exp. Pharmacol. Physiol. 12, 95. Dourish, C.T., S.J. Cooper and S.R. Philips, 1985, Yawning elicited by systemic and intrastriatal injection of piribedil and apomorphine in the rat, Psychopharmacology 86, 175. Dourish, C.T. and P.H. Hutson, 1985, Bilateral lesions of the striatum induced with 6-hydroxydopamine abolish apomorphine-induced yawning in rats, Neuropharmacology 24, 1051. Eberlein, W., G. Schmidt, A. Reuter and E. Kutter, 1977, Das Ulkustherapeutikum Pirenzepin (L-S 519) - eine tricyclische Verbindung mit besonderen physikalisch-chemischen Eigenschaften, Arzneim. Forsch. 27, 356. Eglen, R.M. and R.L. Whiting, 1985, Muscarinic receptor subtypes: problems of classification, Trends Pharmacol. Sci. 6, 357. Giachetti, A., E. Giraldo, H. Ladinsky and E. Montagna, 1986, Binding and functional profiles of the selective M1 and muscarinic receptor antagonists trihexyphenidyl and dicyclomine, Br. J. Pharmacol. 89, 83. Gower, A.J., H.H.G. Berendsen, M.M. Princen and C.L.E. Broekkamp, 1984, The yawning-penile erection syndrome as a model for putative autoreceptor activity, European J. Pharmacol. 103, 81. Hammer, R., C.P. Berri, N.J.M. Birdsall, A.S.V. Burgen and E.C. Hulme, 1980, Pirenzepine distinguishes between different subclasses of muscarinic receptors, Nature 283, 90. Hammer, R. and A. Giachetti, 1982, Muscarinic subtypes: M1 and M2, Biochemical and functional characterisation, Life Sci. 31, 2991. Harris, L.S. and W.L. Dewey, 1973, Role of cholinergic systems in the central action of narcotic agonists and antagonists, in: Agonist and Antagonist Actions of Narcotic Analgesic Drugs, eds. H.W. Kosterlitz, H.O.J. Collier and J.E, Villerreal (University Park Press, London) p. 198. Herz, A., H. Teschemacher, A. Hoffstetter and H. Kurz, 1965, The importance of lipid solubility for the central action of cholinergic drugs, Int. J. Neuropharmacol. 4, 207. Hoss, W. and J. Ellis, 1985, Muscarinic receptor subtypes in the central nervous system, Int. Rev. Neurobiol. 26, 151.
Karczmar, A.G. and N.J. Dun, 1978, Cholinergic synapses: physiological, pharmacological and behavioural considerations, in: Psychopharmacology; a Generation of Progress, eds. M.A. Lipton, A. DiMascio and K.F. Killam (Raven Press, New York) p. 293. Marchi, M. and M. Raiteri, 1985, Differential antagonism by dicyclomine, pirenzepine and secoverine at muscarinic receptor subtypes in the rat frontal cortex, European. J. Pharmacol. 107, 287. Mash, D.C., D.D. Flynn and L.T. Potter, 1985, Loss of M2 muscarinic receptors in the cerebral cortex in Alzheimer's disease and experimental cholinergic denervation, Science 228, 1115. Nordberg, A., C. Larsson, R. Adolfsson, I. Alafuzoff and B. Winblad, 1983, Muscarinic receptor compensation in hippocampus of Alzheimer patients, J. Neural Transm. 56, 13. Perry, E.K. and R.H. Perry, 1985, A review of neuropathological and neurochernical correlates of Alzheimer's disease, Dan. Med. Bull. 32, 27. Potter, L.T., D.D. Flynn, H.E. Hanchett, D.L. Kalinoski, J. Luber-Narod and D.C. Mash, 1984, Independent M1 and M2 receptors, ligands, autoradiography and functions, Trends Pharmacol. Sci. (Suppl.) 22. Salamone, J.D., M.D. Lalies, S.L. Channell and S.D. Iversen, 1986, Behavioural and pharmacological characterization of the mouth movements induced by muscarinic agonists in the rat, Psychopharmacology 88, 467. Siegel, S., 1956, Non-parametric Statistics for the Behavioural Sciences (McGraw-Hill, London). Taylor, P., 1985, Anticholinesterase agents, in: Goodman and Gilman's The Pharmacological Basis of Therapeutics, 7th edition, eds. A. Goodman Gilman, L.S. Goodman, T.W. Rail and F. Murad (Macmillan Publishing Company, New York) p. 110. Tien, X-Y. and L.J. Wallace, 1985, Trihexyphenidyl - Further evidence for muscarinic receptor subclassification, Biochem. Pharmacol. 34, 588. Urba-Holmgren, R., R.M. Gonzalez and B. Holmgren, 1977, Is yawning a cholinergic response?, Nature 267, 261. Ushijima, I., Y. Mizuki, J. Imaizurni, M. Yamada, Y. Noda, K. Yamada and T. Furukawa, 1985, Characteristics of yawning behaviour induced by apomorphine, physostigmine and pilocarpine, Arch. Int. Pharmacodyn. 273, 196. Van Charldorp, K.J., A. De Jonge, M.J.M.C. Thoolen and P.A. Van Zwieten, 1985, Subclassification of muscarinic receptors in the heart, urinary bladder and sympathetic ganglia in the pithed rat, Naunyn-Schmiedeb. Arch. Pharmacol. 331, 301. Watanabe, A.M., 1982, Cholinergic receptor stimulants, in: Basic and Clinical Pharmacology, ed. B.G. Katzung (Lange Medical Publications, Los Altos, California, USA) p. 52. Watson, M., H.I. Yamamura and W.R. Roeske, 1986a, (3 H)Pirenzipine and (-)-(3 H)quinuclidinyl benzilate binding to rat cerebral cortical and cardiac muscarinic cholinergic sites, I. Characterisation and regulation of agonist binding to putative muscarinic subtypes, J. Pharmacol. Exp. Ther. 237, 411. Watson, M., H.I. Yamamura and W.R. Roeske, 1986b,
89 ( 3 H)Pirenzipine and ( - )-( 3H)quinuclidinyl benzilate bind-
ing to rat cerebral cortical and cardiac muscarinic cholinergic sites, II. Characterisation and regulation of antagonist binding to putative muscarinic subtypes, J. Pharmacol. Exp. Ther. 237, 419. Yamada, K. and T. Furukawa, 1980, Direct evidence for involvement of dopaminergic inhibition and cholinergic activation in yawning, Psychopharmacology 67, 39.
Zwagemakers, J.M.A. and V. Claassen, 1980, Pharmacology of secoverine, a new spasmolytic agent with specific antimuscarinic properties, Arzneim. Forsch. 30, 1517. Zwagemakers, J.M.A. and V. Claassen, 1981, Secoverine selectively antagonises muscarinic effects in various in vivo preparations, European J. Pharmacol. 71, 165.
79
Elsevier EJP 00814
Effects of acetylcholine agonists and antagonists on yawning and analgesia in the rat A l m a J. G o w e r * Merrell Dow Research Centre, 16 rue d'Ankara, 67084 Strasbourg-Cedex, France
Received 11 December1986;revisedMS received11 March 1987, accepted 14 April 1987
The ability of acetylcholine muscarinic agonists, injected subcutaneously (s.c.) to elicit yawning and analgesia (tail-flick response) in rats was examined. Yawning was elicited by physostigmine, RS86 and pilocarpine with an inverted 'U'-shaped dose-response relationship; maximal effects occurred at 0.1, 0.5 and 2.0 mg/kg respectively. Neostigmine (0.05-0.2 mg/kg); arecoline (0.5-2.0 mg/kg); bethanecol (0.1-10 mg/kg) and McN-A-343 (5-20 mg/kg) had marginal or no activity. In contrast, dose-related analgesia was obtained following oxotremorine (0.01-0.3 mg/kg) and arecoline (0.5-4.0 mg/kg) and physostigmine (0.1-0.4 mg/kg), RS86 (0.25-2.5 mg/kg) and pilocarpine (0.5-8.0 mg/kg). The effects of acetylcholine antagonists on physostigmine-induced yawning and physostigmine-induced analgesia were also investigated. Following their s.c. injection, trihexyphenidyl, atropine, dicyclomine, secoverine and metliylatropine but not pirenzepine, inhibited both yawning and analgesia; there were clear differences in their potencies on the two responses. Pirenzepine, intracerebroventricularly (i.c.v.), inhibited yawning (EDs0 value 5.7 #g/rat) but not analgesia (3-100/~g/rat). The results are discussed in terms of a possible functional differentiation of central muscarinic receptors. Muscarinic acetylcholine receptor subtypes; Muscarinic agonists; Muscarinic antagonists; Yawning; Analgesia
1. Introduction
The existence of pharmacologically distinct muscarinic receptor subtypes, both centrally and peripherally, is now widely accepted. Support for the subclassification comes from the differential binding affinities of both agonists and antagonists (Hoss and Ellis, 1985; Watson et al., 1986a, b). The availability of pirenzepine, an acetylcholine antagonist with selective binding characteristics has contributed considerably to the study of these differences (Hammer et al., 1980). In fact, muscarinic receptors have been designated as either M1 or M2 depending on whether they have * To whom all correspondenceshould be addressed at present address: UCB, Secteur Pharmaceutique, Chemin du Foriest, 1420 Braine-l'Alleud, Belgium.
high or low affinity for pirenzepine (Hammer and Giachetti, 1982) although it is recognised that such a classification is as yet tentative (Birdsall and Hulme, 1983). Cholinergic mechanisms are implicated in many behavioural phenomena and the possibility of finding selective agonists or antagonists has produced renewed promise for more effective therapy for certain n e u r o l o g i c a l disorders involving cholinergic mechanisms. Senile dementia of the Alzheimer type (SDAT) is of particular current interest in view of the compelling evidence of a cholinergic deficit underlying the primary symptom, i.e. loss of memory (Bartus et al., 1982; Perry and Perry, 1985), as well as reports of selective changes in certain receptor subtypes in postmortem brain of SDAT patients (Nordberg et al., 1983; Mash et al., 1985).
0014-2999/87/$03.50 © 1987 ElsevierSciencePublishers B.V. (BiomedicalDivision)
80 Although binding studies provide one way of characterising potential selective agonists and antagonists, they are usually done in vitro and are not always predictive of what is happening in the whole animal. The ability to distinguish between receptor subtypes on the basis of behavioural responses would therefore be useful in the development of selective muscarinic drugs. For this reason, we investigated in the rat the effects of selected agonists and antagonists on two behaviours which involve central cholinergic mechanisms, namely yawning and analgesia. Both the cholinesterase inhibitor, physostigmine and the acetylcholine agonist, pilocarpine, induce yawning in rats (Urba-Holmgren et al., 1977; Gower et al., 1984), an effect which can be antagonised by scopolamine (Yamada and Furukawa, 1980). Similarly, acetylcholine agonists such as oxotremorine are known to cause analgesia (Harris and Dewey, 1973; Karczmar and Dun, 1978). We determined the effects of various agents in inducing yawning or analgesia and the effects of antagonists in blocking physostigmine-induced yawning and physostigmine-induced analgesia. The drugs tested included the agonists oxotremorine and McN-A343 and the antagonists pirenzepine and dicyclomine, putatively selective for certain receptor subtypes (Birdsall and Hulme, 1983; Potter et al., 1984; Hoss and Ellis, 1985; Marchi and Raiteri, 1985). 2. Materials and methods
2.1. Animals Naive, male Sprague-Dawley rats (Charles River, France), weighing 250-320 g, were used. Prior to use, the rats were housed in groups of 6 in wire cages on a controlled 12 h light-dark cycle (lights on at 6:00 a.m.) and were allowed ad lib access to standard cube diet and tap water. Each rat was used only once. 2.2. Procedure Yawning and analgesia were investigated separately in different rats. All experiments were carried out between 8:30 a.m. and 1:30 p.m.
2.2.1. Assessment of yawning Each rat was placed in a large glass beaker (18 cm high × 14.5 cm diameter) containing a layer of sawdust. A metal grid prevented the rats from escaping. The number of yawns during a 30 rain period was counted by direct observation. The yawning response, defined as a wide stretched opening of the mouth, has been described previously (Berendsen and Gower, 1986). A mirror was positioned immediately behind the beakers to permit 360 ° observation of the rats. Groups of 4 or 5 rats were tested at a time and the doses were tested in random order. The effects of agonists were determined over a 30 rain period immediately following s.c. injection. The effects of antagonists were determined by pretreatment of the rats with the antagonist s.c. followed by return to their home cages; control animals which received saline in place of antagonist were included in each antagonist experiment. Physostigmine, 0.1 mg/kg s.c., was injected 30 rain later and the number of yawns elicited over the following 30 min was then counted. The effects of atropine sulphate (SO4) and pirenzine were also assessed following their i.c.v, administration, 30 min prior to physostigmine, 0.1 mg/kg s.c. 2.2.2. Analgesia Analgesia was measured with an Appelex DS 20 tail-flick apparatus and was quantitated as increases in latency for the tail-flick response to a focussed heat stimulus. The intensity of the heat stimulus was adjusted to produce a latency of 2-4 s in saline-treated control rats. A maximal cut-off time of 12 s was used for rats which did not respond to the heat stimulus. The effects of agonists were determined by measuring the tall-flick latency 20 rain after s.c. injection. The effects of antagonists were determined by pretreatment of the rats with the antagonist s.c. or saline 30 rain prior to the s.c. injection of physostigmine, 0.1 mg/kg. The tailflick latency was measured 20 rain after physostigmine. Control rats which received only saline were included in each antagonist experiment. The effects of atropine SO4 and pirenzepine injected i.c.v, on physostigmine-induced analgesia were also
81
minimal amount of 0.1 M hydrochloric acid, making the solution up to the required volume with 0.9% saline solution then neutralizing to pH 7.0 by adding a few drops of saturated sodium hydrogen carbonate solution. The remaining drugs were all dissolved in 0.9% saline solution. Solutions of dicyclomine were protected from light by wrapping the bottles in aluminium foil. Solutions of apomorphine were kept on ice. A dose volume of 2 ml/kg used for s.c. injection and the solutions were injected into the loose skin at the back of the neck. For i.c.v, administration, the rats were first prepared with a guide cannula unilaterally, directed at the lateral ventricle and positioned 0.2 mm behind the bregma and 2.0 mm lateral to the midline. At least one week post-operative recovery time was allowed. Injections were made by inserting a 28 gauge injection cannula to a depth of 3.5 mm below the skull surface and injecting the drugs in a dose volume of 2 #1 per rat over a period of 30 s. For both s.c. and i.c.v, injections, control rats received equivalent dose volumes of 0.9% saline.
determined by injecting them 30 min before physostigmine and testing 20 min after physostigmine, 0.1 mg/kg s.c. 2.3. Drugs and injections The following drugs were used: apomorphine hydrochloride, arecoline hydrobromide, atropine methyl nitrate, atropine sulphate, bethanecol (Sigma, USA); dicyclomine (Merrell Dow, Strasbourg); McN-A-343 (Research Biochemicals Incorporated, USA); neostigmine hydrochloride, oxotremorine sesquifumarate, physostigmine salicylate, pilocarpine hydrochloride (Sigma USA); pirenzepine (Dr. Karl Thomae, GMBH, Germany); 2-ethyl-8-methyl-2,8-diazospiro-(4,5)-decan1,3-dionhydrobromide, RS86 (Sandoz, Switzerland); secoverine hydrochloride (Duphar, Holland) and trihexyphenidyl hydrochloride (Sigma, USA). All drug solutions were prepared immediately prior to the experiment. Pirenzepine was prepared by dissolving the powder in a
4@ 4~
4~
0. 0
25 ~ pg/kg
PHYSOSTIGMINE
o m g ~ k g 4.0
0
PILOCARPINE
G 5 1.O S.O 0.1 O.5 2.5 mg/kg
mg/kg
RS 8 6
ARECOLINE
0
1,0
4
0
lOO
/Jg/ k g N E O S T I G M I NE
0
0.3 3.0 mg / kg
BETHANECOL
0
1.0 10 pg / kg
OXOTREMORINE
100
0 10 mg/kg MCN.A.343
Fig. 1. Effects of muscarinic agonists in eliciting yawning in rats. Hatched columns refer to saline-treated controls. Dose groups of 9 or 12 rats were used. * Significantly greater than saline-treated controls; P ~< 0.05, Student's t-test.
82
2.4. Statistics
6
The drug-induced changes in each yawning experiment were first assessed with an analysis of variance. When this yielded a significant result, the effects of individual doses were assessed with Student's t-test. Non-parametric methods were used for the analgesic experiments (Siegel, 1956). A Kruskal-Wallis analysis of variance was first carried out and when this showed significance, a Mann-Whitney U-test was used to compare control and test groups.
gl
Z >.
z i11
3. Results S
10
15
20
25
30
35
40
45
MIN. OF TESTING
3.1. Yawning
Fig. 2. Time course of physostigmine (0.1 m g / k g ) and arecoline (1.0 m g / k g ) for eliciting yawning. The n u m b e r of yawns elicited over consecutive 5 min intervals was recorded. Groups of 12 rats were used.
The effects of muscarinic agonists in causing yawning are shown in fig. 1. Physostigmine, pilocarpine and RS86 elicited marked yawning; the number of yawns followed an inverted 'U'-shaped curve with respect to dose, with low and high
,_E
=E
0 o
loo 400 JJg/kg
o
PHYSOSTIGMIN E ~"
(Is 2.0 mg/kg
8.0
0
PILOCARPINE
FS7~7
0.25 I~) m g / kg
o
1,o
4.0
mg/kg ARECOLINE
RS 8 6
At
12
,,J
,s
O
.~O 200 ~191kg
N EOSTIGMINE
FI~7~.07
0
tO 6£) mglkg
BETHANECOL
~ ~
O
30 300 l~91kg
OXOTREMORIN
E
~7
0 20 mglkg M C N . A . 343
Fig. 3. Effects of muscarinic agonists in causing analgesia in rats. Hatched columns refer to saline-treated controls. Dose groups of 9 rats were used. * Significantly greater than saline-treated controls; P ~< 0.05, Mann-Whitney U-test.
83
doses having little effect. Arecoline, neostigmine, bethanecol, oxotremorine and McN-A-343 caused only marginal or no yawning. Arecoline has a short duration of action which could account for the low level of responding since the total number of yawns over 30 min was recorded. However, the time course of the effects of peak doses of physostigmine and arecoline (fig. 2) makes it evident that the maximum effect of arecoline per 5 min interval was considerably less than that of physostigmine. All the agonists except bethanecol induced licking and chewing movements. A1-
though not systematically quantified, this effect was most marked for drugs causing yawning but was not related to the intensity of yawning. Thus, high doses of agonists, following which yawning was reduced or absent, caused the most pronounced chewing and licking. Yawning induced by physostigmine is characteristically accompanied by stretching (Urba-Holmgren et al., 1977). In the present study, this was observed only for physostigmine and not for the other muscarinic agonists. Typical muscarinic effects such as chromodacryorrhea, salivation and increased defaeca-
TABLE 1 Effects of muscarinic antagonists in antagonising physostigmine-induced (0.1 m g / k g ) yawning in rats. Antagonist
Dose (mg/kg)
n/12 ~
Yawning (mean + S.E.)
5g inhibition
Trihexyphenidyl
Saline 0.025 0.05 0.1
12 11 11 10
11.8 ± 1.7 7.2 ± 1.4 * 5.9 5:1.0 * 2.7+0.8 *
39.0 50.0 67.9
Saline 0.3 0.6 1.0 3.0
11 10 10 4 4
12.4 ± 2.8 9.4 ± 2.3 4.5 ± 1.5 * 1.5±0.8 * 0.5 ± 0.3 *
24.2 63.4 87.9 96.0
Saline 0.3 1.0 3.0 10.0
12 12 9 5 2
11.6 ± 2.0 8.3 _+1.9 4.7 _+2.2 1.8 ± 0.7 0.4 _+0.3
28.4 59.5 84.5 96.6
Saline
5.0 7.5 10.0
12 12 11 11 9 9
10.7 ± 2.1 14.8 ± 2.0 16.8±2.4 * 8.1 ± 2 . 2 4.7 ± 1.8 * 1.9±0.6 *
24.3 56.1 82.1
Saline 1.0 3.0 6.0 10.0
12 12 12 10 8
15.3 + 3.7 14.2 4- 2.4 12.8±2.2 8.4 + 3.2 6.2± 2.5 *
7.2 16.3 45.1 59.5
Saline 1.0 3.0 10.0
12 11 11 12
13.3 ± 15.2 + 10.3 ± 13.3 +
22.6 0
Atropine SO 4
Dicyclomine
Secoverine
1.0 3.0
Atropine M e N O 3
Pirenzepine
2.1 3.8 1.8 2.1
* * * *
ED50 (mg/kg) 0.042
0.48
0.67
-
7.0
7.25
inactive up to 10.0
a n / 1 2 = number of rats yawning per group out of possible m a x i m u m of 12. * Significantly different from saline control; P ~< 0.05 Student's t-test. The EDs0 was calculated as the dose of antagonist which reduced the mean number of yawns induced by physostigmine by 50%.
84
tion were observed for physostigmine, pilocarpine, RS86, arecoline, bethanecol and oxotremorine. These effects were dose-related and were marked at the highest doses. Neostigmine produced slight salivation and chromodacryorrhea and McN-A343 slightly increased defaecation only. The effects of muscarinic antagonists injected s.c. on physostigmine-induced yawning are shown in table 1. Trihexyphenidyl, atropine SO4, dicyclomine and atropine MeNO 3 blocked yawning in a dose-related manner. Pirenzepine had no effect up to 10 mg/kg. Secoverine, 5-10 mg/kg, also inhibited physostigmine-induced yawning in a dose-dependent manner, but yawning was increased at 1.0 and 3.0 mg/kg. Secoverine by itself, 3.0 mg/kg injected 30 min before test, slightly but significantly increased yawning (table 2). In contrast to its effects on physostigmine-induced yawning, secoverine did not enhance the effects of arecoline or the effects of pilocarpine (table 2). In fact, secoverine 3.0 mg/kg significantly reduced the mean number of yawns induced by pilocarpine although it had no effect on the number of rats responding. Since dopamine agonists also induce
yawning (Gower et al., 1984) the effect of apomorphine at a dose eliciting only a low level of yawning on physostigmine-induced yawning was included for comparative purposes (table 2). Apomorphine 0.01 mg/kg enhanced physostigmine-induced yawning markedly.
TABLE 3 Effects of muscarinic antagonists in antagonising physostigmine-induced (0.1 m g / k g ) analgesia in rats. Antagonist
Dose (mg/kg)
Atropine SO4
Control a 3.2 + 0.3 * Saline b 4.0 _+0.3 0.03 4.14- 0.3 0.1 3.4-1-0.1 * 0.3 2.9_+0.2 *
0 71.4 100.0
Control a 3.0_+ 0.1 * Saline b 3.9 _+0.3 0.03 4.0_.+ 0.3 0.06 3.5 + 0.2 0.1 3.3-+0.2 * 0.2 3.0+0.3 *
0 44.4 66.7 100.0
Trihexyphenidyl
Atropine MeNO 3 TABLE 2 Effects of various drug combinations on yawning in the rat. Drug treatment (dose m g / k g )
Yawning (mean_+ S.E.)
n/N a
Saline Secoverine (3.0)
0.2 5:0.1 1.3 -+ 0.4 *
2/12 8/12
Saline + arecoline (1.0) Secoverine (3.0) + arecoline (1.0)
1.4 -+0.8 1.3 -+0.5
5/12 5/12
Saline + pilocarpine (2.0) Secoverine (0.3) + pilocarpine (2.0) Secoverine (1.0) + pilocarpine (2.0) Secoverine (3.0) + pilocarpine (2.0)
13.8 -+ 2.2
12/12
11.4 -+ 2.1
12/12
12.1 _+2.5
11/12
6.7 _+1.9 *
11/12
Saline Apomorphine (0.01) Physostigmine (0.1) Apomorphine (0.01) + physostigmine (0.1)
0.3 + 0.3 2.9 _+1.3 13.9 + 2.7 *
1/8 5/8 8/8
22.9 _+3.2 *
8/8
Number of rats yawning (n) divided by number of rats tested (N). * Significantly different from corresponding control, P ~< 0.05, Student's t-test.
Secoverine
Tall-flick % inhibi- EDs0 latency (s) tion (mg/kg) (mean + S.E.)
Control a 3.2-+ 0.2 * Saline b 4.0 4- 0.2 0.3 4.2 -+0.2 0.6 3.8 -+ 0.3 1.0 3.6 _+0.3 3.0 3.3+0.4 *
0.065
0.073
0 25.0 50.0 87.5
1.05
Control ~ 2.7 _+0.2 * Saline b 3.8 _+0.2 0.3 3.9+0.3 1.0 3.6-+0.4 3.0 3.3 4-_0.2 * 10.0 2.8+0.2 *
0 18.2 45.5 90.9
2.7
Dicyclomine Control ~ 3.1 + 0.2 * Saline b 4.5 -+0.3 1.0 4.2 5:0.5 3.0 3.8_+0.3 10.0 3.4_+0.3 *
21.4 50.0 78,6
3.4
14.2 14.2
inactive up to 10.0
Pirenzepine
Control a 3.1+0.2 Saline b 3.8 -+ 0.2 3.0 3.7 + 0.2 10.0 3.7_+0,2
-
a Control group receiving only saline; b group receiving only physostigmine. Dose groups of 9 rats were used. * P ~<0.05; Mann-Whimey U-test with respect to physostigmine only group. The EDs0 was calculated as the dose inhibiting physostigmine-induced analgesia by 50%.
85
induced analgesia whereas the EDs0 value for atropine was 0.2 #g/rat.
3.2. Analgesia The analgesic effects of muscarinic agonists, seen as prolonged tail-flick latency, are shown in fig. 3. Physostigmine, pilocarpine, RS86, arecoline and oxotremorine all caused analgesia which increased with increasing dose. Neostigmine, bethanecol and McN-A-343 were inactive. The effects of muscarinic antagonists, injected s.c., on physostigmine-induced analgesia are shown in table 3. Atropine SO4, trihexyphenidyl, atropine MeNO 3, secoverine and dicyclomine all inhibited physostigmine-induced analgesia in a dose-dependent manner. Pirenzepine was inactive up to 10 mg/kg.
4. Discussion
The present results confirm previous work that pointed to cholinergic involvement in both yawning and analgesia (Harris and Dewey, 1973; Urba-Holmgren et al., 1977; Karczmar and Dun, 1978; Yamada and Furukawa, 1980). However, there were clear differences between the two responses in terms of both agonist and antagonist effects. Yawning was induced by the cholinesterase inhibitor, physostigmine, and the direct agonists, RS86 and pilocarpine, but oxotremorine, arecoline, bethanecol, neostigmine and McN-A-343 had little or no effect. In contrast, oxotremorine and arecoline, as well as physostigmine, RS86 and pilocarpine, induced analgesia although bethanecol, neostigmine and McN-A-343 were again inactive. The dose-response relationship for yawning followed an inverted 'U'-shaped curve whereas the relationship for analgesia was linear in all cases, with maximal effects occurring at doses considerably higher than those maximal for yawning. There was thus not only a difference in the agonists which elicited yawning versus analgesia but there
3.3. Effects of i.c.v, injection of antagonists," yawning and analgesia The effects of i.c.v, injection of atropine SO4 and pirenzepine on physostigmine-induced yawning and analgesia respectively are shown in table 4. Atropine antagonised both yawning and analgesia but was 5-fold more potent in blocking analgesia. Pirenzepine inhibited yawning dose dependently (EDs0 5.7 ttg/rat) and was thus 6 times less potent than atropine. In contrast, pirenzepine up to 100/~g/rat failed to inhibit physostigmine-
TABLE 4 Effects of atropine SO 4 and pirenzepine i.c.v, in antagonising physostigmine-induced yawning and physostigmine-induced analgesia in rats. Antagonist
Atropine SO4
Pirenzepine
Dose
Yawning
( # g / r a t i.c.v.)
Mean ± S.E.
Control Saline 0.3 1.0 3.0
10.7±2.5 8.4±3.5 6.8±1.6" 1.1±0.7"
Control Safine 3.0 10.0 30.0 1~.0
8.8±2.6 5.6±1.5 3.5±0.9* 1.0±0.7" NT
Analgesia (TFL) ED50 (btg/rat)
Mean ± S.E.
EDs0 ( # g / r a t )
1.0
2.9±0.1 4.8±0.5 3.6±0.3 3.5±0.4 3.0±0.2
0.2
5.7
2.8±0.2 4.0±0.2 4.3±0.3 4.4±0.4 4.3±0.1 4.4±0.4
*
inactive up to 100.0
* P ~< 0.05; Student's t-test versus saline group (yawning) or Mann-Whitney U-test (analgesia). T F L = tail-flick latency in s, N T = not tested. Dose groups of 12 or 9 rats were used for the yawning and analgesia experiments respectively.
86 was a characteristic difference in the dose-response relationship for those agonists which elicited both responses. Neostigmine, bethanecol and McN-A-343 are all drugs which have difficulty in penetrating into the brain following their parenteral administration (Watanabe, 1982; Taylor, 1985). Their inability to cause either yawning or analgesia is consistent with a central site of action and confirms earlier reports (Harris and Dewey, 1973; Urba-Holmgren et al., 1977). All the antagonists except pirenzepine were active against both the yawning and the analgesia induced by physostigmine although there were marked differences in the doses required to inhibit each response. The order of potencies for antagonism of yawning was trihexyphenidyl > atropine >/dicyclomine > secoverine >1methyl atropine (EDs0 ratios = 1 : 11 : 16 : 167 : 173). The order of potencies for antagonism of analgesia was trihexyphenidyl >/atropine > methyl atropine > secoverine >/dicyclomine (EDs0 ratios = 1 : 1 : 14 : 37:47). The separation (approximately 15-fold) between the EDs0 values of atropine and methyl atropine on both responses reflects the poor penetration of methyl atropine into the brain (Herz et al., 1965). The EDs0 value for secoverine for yawning was probably artificially elevated in view of its opposing effect at low doses. The same dose (0.1 mg/kg) of physostigmine was used to induce yawning and analgesia; this was a maximal effective dose for yawning but it was submaximal for analgesia. Hence, the differing potencies of both atropine and methyl atropine to inhibit yawning and analgesia respectively might be due to a greater facility to inhibit submaximal than maximal effects. Alternatively, the difference could reflect a different site of action within the brain, with the site for analgesia being more accessible for drug action following parenterai injection. However, neither explanation accounts for the results obtained with trihexyphenidyl or dicyclomine. It is known that pirenzepine penetrates poorly into the brain from the periphery (Eberlein et al., 1977) hence its lack of effect following s.c. injection was not unexpected in view of the evidence that cholinergic-induced yawning and analgesia are centrally mediated. Pirenzepine now has a key
role in the classification of muscarinic receptor subtypes. We therefore compared its effects following i.c.v, injection with those of atropine i.c.v. Pirenzepine inhibited physostigmine-induced yawning but was inactive against analgesia. This latter result differs from that of Caulfield et al. (1983) who found that pirenzepine injected into the third ventricle inhibited oxotremorine-induced analgesia with an EDs0 of 4.6/xg per mouse. It is possible that this discrepancy was due to species differences in sensitivity to the drug since this EDs0 was almost 50 times greater than that for the inhibition of passive avoidance learning in the mouse. An interesting biphasic effect was obtained with secoverine on physostigmine-induced yawning. Secoverine has been shown to have a selective action at the acetylcholine presynaptic receptor inhibiting acetylcholine release in frontal cortex, with less effect on presynaptic acetylcholine receptors mediating dopamine release (Marchi and Raiteri, 1985). The enhancement by secoverine may therefore be attributable to blockade by autoreceptors, leading to increased acetylcholine being available to interact postsynaptically. In this case, the lack of enhancement of arecoline or pilocarpine-induced yawning suggests that they are inactive at these particular receptors. Alternatively, since physostigmine-induced yawning can be enhanced by threshold doses of apomorphine, the effects of secoverine may be independent of its cholinergic effects. However, dopaminergic activity has not been reported for secoverine and no known actions of secoverine would account for its enhancing effect on yawning (Zwagemakers and Claassen, 1980). The finding that physostigmine and pilocarpine induced yawning confirms previous observations (Urba-Holmgren et al., 1977; Yamada and Furukawa, 1980; Gower et al., 1984; Ushijima et al., 1984), although RS86-induced yawning has not previously been reported. Also, the range of acetylcholine antagonists investigated extends earlier observations with scopolamine and atropine (Urba-Holmgren et al., 1977; Gower et al., 1984). Surprisingly, in view of the marked, replicated efefcts of pilocarpine in the present study, it appears that pilocarpine-induced yawning is not
87 always reproducible. Salamone et al. (1986) failed to obtain significant yawning after pilocarpine. They suggested that the discrepancy between their results and previous results (Ushijima et al., 1984) may have been due either to a difference in the strain of rats used or to a difference in the definition of yawning, i.e. gaping as opposed to yawning. Neither explanation is applicable to the present study. Only prolonged wide, stretched opening of the mouth counted as a yawn (Berendsen and Gower, 1986) and there was an inverted 'U'shaped dose-response function in contrast to the increased gapings with increased dose obtained by Salamone et al. (1986). Also, Sprague-Dawley rats were used in both studies. However, the strain of rat can affect the results; in an earlier study using Wistar rats (Gower et al., 1984) albeit in a different location, fewer yawns were elicited by the maximal effective dose of physostigmine, again 0.1 mg/kg. In the author's experience, a major factor affecting yawning seems to be emotionality which can vary according to the strain and also according to the handling and maintenance procedures; for example, rats stressed during injection usually have a low rate of yawning. The effects on yawning and analgesia obtained with both agonists and antagonists point to a difference in the cholinergic mechanism involved in the two responses. Yawning was pirenzepinesensitive but oxotremorine-insensitive whereas the opposite held for analgesia which was pirenzepine-insensitive but oxotremorine-sensitive. Also, secoverine at low doses enhanced physostigmineinduced yawning but did not increase analgesia. These differences may have been due to differing interactions with other transmitter systems or to different sites of action in the brain; for example, yawning appears to be striatally mediated (Dourish and Hutson, 1985; Dourish et al., 1985) whereas lower brain sites are likely to be involved in analgesia (Karczmar and Dun, 1978). Equally, the differences may represent pre- versus postsynaptic sites of action at the same receptor subtype. However it is possible that the differences in the effects of the agonists and antagonists indicate that different muscarinic receptor subtypes are involved in mediation of the responses. Muscarinic recep-
tors have been designated M1 or M2 depending mainly on their affinity for pirenzepine. It is therefore tempting to speculate that, because yawning was pirenzepine-sensitive, an M1 receptor mediates yawning and conversely because analgesia was pirenzepine-insensitive that an M2 receptor mediates analgesia. The results obtained with the other antagonists and the agonists are consistent with such an interpretation. Thus, on the basis of both binding data and selectivity of action on certain tissues, oxotremorine has been proposed as an M2 agonist and pilocarpine as an M1 agonist (Potter et al., 1984; Van Charldorp et al., 1985; Watson et al., 1986a). As with the antagonists, dicyclomine and trihexyphenidyl appear to be M1 selective in that they have a profile of action similar to that of pirenzepine (Potter et al., 1984; Marchi and Raiteri, 1985; Tien and Walace, 1985; Giachetti et al., 1986). Furthermore, as already mentioned, secoverine has selective actions at the presynaptic autoreceptor (Marchi and Raiteri, 1985) and has a profile of action in certain tissues which suggests that it might be an M2 antagonist (Zwagemakers and Claassen, 1981). However the selectivity of these agents has not been confirmed in all studies nor shown unequivocally (Choo and Mitchelson, 1985; Eglen and Whiting, 1985; Brunner et al., 1986). There is therefore insufficient evidence at this stage to allow certain functional responses to be related convincingly to particular muscarinic subtypes. Unfortunately, one lacks agonists and antagonists which can distinguish sufficiently between receptor subtypes but equally, one lacks a functional means of differentiation. It is through the development of selective compounds and through functional studies such as the present one that the uncertainty regarding muscarinic subtypes will be resolved.
Acknowledgements The excellenttechnical assistanceof Mr. C. Forler is gratefully acknowledged.We thank Dr. Karl Thomae, GMBH and Duphar for their generousgifts of drugs.
88
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