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Eur J Pharmacol
1987; 139; 79-89
Effects of acetylcholine agonists and antagonists on yawning and analgesia in the rat
Alma J. Gower
Merrell Dow Research Centre, 16 rue d'Ankara, 67084 Strasbourg France


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. The availability of pirenzepine, an acetylcholine antagonist with selective binding characteristics has contributed considerably to the study of these differences. In fact, muscarinic receptors have been designated as either Ml or M2 depending on whether they have high or low affinity for pirenzepine although it is recognised that such a classification is as yet tentative.
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 neurological 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, as well as reports of selective changes in certain receptor subtypes in postmortem brain of SDAT patients.
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, an effect which can be antagonised by scopolamine. Similarly, acetylcholine agonists such as oxotremorine are known to cause analgesia. 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. [...]
Discussion : The present results confirm previous work that pointed to cholinergic involvement in both yawning and analgesia. 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 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. Their inability to cause either yawning or analgesia is consistent with a central site of action and confirms earlier reports.
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 > methyl atropine (ED50 ratios= 1 :11 :16:167:173). The order of potencies for antagonism of analgesia was trihexyphenidyl >_ atropine > methyl atropine > secoverine > dicyclomine (ED50 ratios = 1 : 1 : 14: 37:47). The separation (approximately 15-fold) between the ED50 values of atropine and methyl atropine on both responses reflects the poor penetration of methyl atropine into the brain. The ED50 value for secoverine for yawmng 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 parenteral 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 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 ED50 of 4.6 µg per mouse. It is possible that this discrepancy was due to species differences in sensitivity to the drug since this ED, 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. Secoverme 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. 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. Alternatively1 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.
The finding that physostigmine and pilocarpine induced yawning confirms previous observations, although RS86-induced yawning has not previously been reported. Also, the range of acetylcholine antagonists investigated extends earlier observations with scopolamine and atropine. Surprisingly, in view of the marked, replicated effects of pilocarpine in the present study, appears that pilocarpine-induced yawning is not reproducible. Salamone et al. (1986) failed to obtain significant yawning after pilocarpine.
They suggested that the discrepancy between their results and previous results 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 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 albeit in a different location, fewer yawns were elicited by the maximal effective dose of physostigrnine, 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 handfing 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 physostigrnineinduced 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 whereas lower brain sites are likely to be involved in analgesia. 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 receptors 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.
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. Furthermore, as already mentioned, secoverine has selective actions at the presynaptic autoreceptor and has a profile of action in certain tissues which suggests that it might be an M2 antagonist. However the selectivity of these agents has not been confirmed in all studies nor shown unequivocally .
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.
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