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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HG Androgenic influences on
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H et al Involvement of 5-HT1c-receptors in
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AJ Effects of acetylcholine agonists and
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