Pulmonary Pharmacology (1996) 9, 349–356

PULMONARY
PHARMACOLOGY

Role of Opioidergic and Serotonergic Mechanisms in Cough

and Antitussives

J. Kamei
Department of Pathophysiology & Therapeutics, Faculty of Pharmaceutical Sciences, Hoshi University,
4-41 Ebara 2-chome, Shinagawa-ku, Tokyo 142, Japan

SUMMARY: This paper provides an overview of our current understanding of the serotonergic and opioidergic
mechanisms of cough and antitussives.
Systemic administration of 8-OH-DPAT, at doses of 0.1 and 0.3mg/kg, ip, markedly reduced the number of
coughs in rats in a dose-dependent manner. The antitussive effects of 8-OH-DPAT, dihydrocodeine and dex-
tromethorphan were significantly reduced by pretreatment with methysergide, but not with ketanserin. Therefore
it is possible to speculate that 5-HT1 receptors, in particular the 5-HT1A receptors, may be more important than
others with respect to the effect of antitussive drugs.
DAMGO, a selective l-opioid receptor agonist, and U-50,488H, a highly selective j-opioid receptor agonist,
have potent antitussive effects when administered either icv or ip. However, we did not observe a cough-depressant
effect of DPDPE, a selective d-opioid receptor agonist. These results indicate that the antitussive effects of opioids
are mediated predominantly by l- and j-opioid receptors. On the other hand, naloxonazine, a selective l1-opioid
receptor antagonist, had no effect on the antitussive effects associated with icv DAMGO. These results indicate
that l2- rather than l1-opioid receptors are involved in l-opioid receptor-induced antitussive effects.
Antitussive effects of dextromethorphan and noscapine were significantly and dose-dependently reduced by
pretreatment with rimcazole, a specific antagonist of r sites. However, rimcazole did not have a significant effect
on the antitussive effect of morphine. These results suggest that r sites may be involved in the antitussive mechanism
of non-narcotic antitussive drugs.
 1996 Academic Press Limited

KEY WORDS: Serotonin; l-Opioid receptor; j-Opioid receptor; r Sites; Antitussives

INTRODUCTION are neither stable nor reproducible.1 These methodo-

Although there are numerous studies of screening
antitussive agents, very few data are available regard-
ing the mechanisms of the control of the cough reflex,
despite monumental efforts to define their sites of
action. Furthermore, the importance of neuro-
transmitter systems in the cough reflex has received
limited attention.
Rodents are frequently used in pharmacological
investigations and the rat and the mouse, in particular,
are very well suited for neuropharmacological studies.
The evaluation of antitussive drugs in experimental
animals has been carried out by a variety of methods.
However, the experimental methods for inducing the
cough reflex in the rat and the mouse are less well
documented, because of the disadvantage of these
species as coughing animals: only chemical stimulation
can induce the cough reflex and the resultant coughs
0952–0600/96/5/60349+08 $25.00/0
logical disadvantages may arrest the advance of the
neuropharmacological studies of the antitussive drugs.
We recently carried forward neuropharmaco-
logical studies of the mechanisms of the control of
coughs for central acting antitussive drugs, using new
screening methods for antitussive drugs in rat and
mouse.2–4 This paper provides an overview of our
current understanding of the mechanisms of the con-
trol of coughs for centrally acting antitussive drugs.
ROLE OF SEROTONERGIC MECHANISMS IN
ANTITUSSIVE ACTIONS
It is well known that the monoamine neuro-
transmitters, noradrenaline (NA), dopamine (DA)
and serotonin (5-HT), can be identified in the nerve
terminals in the regions of the central respiratory
349  1996 Academic Press Limited
350 J. Kamei

Table 1 Effect of various pretreatments on the ED50 of the whole brain by over 70%, while levels NA and
antitussive drugs. DA were unchanged. The PCPA-treated rats also had
Pretreatment Morphine Dihydrocodeine Dextrometorphan reduced antitussive effects of morphine, dihydro-
codeine and dextromethorphan (Table 1). There was
Control 1.22 1.44 6.06 no difference between the AtD50 of the antitussive
(0.77–1.95) (1.11–1.87) (4.33–8.47)
1.00 1.00 1.00 drug in reserpinized and PCPA-treated rats. a-Methyl-
Reserpine 2.85 3.75 23.57 p-tyrosine (a-MT) produced a reduction of more than
(1.55–5.24) (2.25–6.26) (13.59–40.86) 50% in levels of NA and DA in whole brains. None-
2.34 2.60 3.89
PCPA 2.56 2.84 26.18 theless, treatment with a-MT hardly altered the cough-
(1.16–5.67) (1.91–4.24) (12.98–52.81) suppressant effects of morphine, dihydrocodeine and
2.10 1.98 4.32 dextromethorphan (Table 1).
a-MT 1.30 1.32 5.14
(1.04–1.62) (0.81–2.16) (2.92–9.04) These results indicated that the reduction in levels
1.07 0.92 0.85 of 5-HT in the whole brain decreased the potency of
the antitussive drugs which act via the central nervous
Values are 50% antitussive dose (AtD50, mg/kg), 95% confidence
limits of AtD50 (in parentheses), and AtD50 ratio (pretreated/ system. There is evidence that the presence of 5-HT
control). is needed in order for antitussive drugs to be fully
active at the respiratory centre, and particularly at
the cough centre.2
network. Furthermore, several studies have dem- Rats pretreated at birth with 5,7-dihydroxy-
onstrated that catecholaminergic5–9 and seroto- tryptamine (5,7-DHT), administered intracisternally,
nergic10–13 neuronal pathways may be involved in show specific damage to central serotonergic neurons
respiratory control. In rats anesthetized with halo- and supersensitivity to serotonin receptor agonists.
thane, augmenting serotonergic activity by giving 5- Neonatal treatment with 5,7-DHT, sufficient to reduce
hydroxytryptophan (5-HTP) and 5-methoxy-N,N-di- whole brain levels of 5-HT to 19 % of control levels,
methyltryptamine produced a dose-dependent de- resulted in supersensitivity to the cough-depressant
crease in basal and CO2-stimulated respiration.13 By effect of dihydrocodeine.15 Furthermore, this po-
contrast, iv administration of the a2-adrenoceptor tentiation of the antitussive effect of dihydrocodeine
agonist clonidine depressed phrenic neuronal dis- observed in 5,7-DHT-treated rats was abolished by
charges.12 Florez et al.7 reported that reserpine ant- pretreatment with methysergide, a 5-HT receptor ant-
agonized the morphine-induced depression of agonist. One therefore could argue that the marked
respiration. There is also evidence that monoamine increase of the antitussive effect of dihydrocodeine
neurotransmitters are involved in the regulation of might have been due to the changes in the sensitivity
the cough reflex. For example, we reported that tran- of 5-HT receptors.
ylcypromine (TCP), an inhibitor of monoamine ox- Serotonergic receptors in the central nervous system
idase, depressed the cough reflex.14 Moreover, 5-HTP have been classified in up to seven different types.16,17
in combination with TCP rapidly caused a profound Several studies have further subdivided the 5-HT1
depression of the cough reflex.14 Thus, it appears that receptors into four subtypes: 5-HT1A, 5-HT1B, 5-HT1C
brain monoamines may be involved in the mechanism and 5-HT1D. 8-Hydroxy-2-(di-n-propylamino)tetralin
of action of antitussive drugs. Therefore, the effect of (8-OH-DPAT) is a potent and highly selective ligand
drugs which modify the concentrations of mono- for 5-HT1A receptors. This drug also causes bio-
amines in the brain on the cough-depressant effect of chemical and behavioral changes indicative of the
antitussive drugs are discussed. stimulation of central 5-HT receptors.18–23 As shown
Table 1 shows the antitussive ED50 (AtD50) of each in Fig. 1, systemic administration of 8-OH-DPAT, at
drug tested in rats. Morphine and dihydrocodeine doses of 0.1 and 0.3mg/kg, ip, markedly reduced
both had potent antitussive activity. The antitussive the number of coughs in rats in a dose-dependent
effects of dextromethorphan was about 25% of the manner.24 The antitussive effect of 8-OH-DPAT was
antitussive effect of dihydrocodeine. attenuated by the antagonist of 5-HT1A receptors,
Reserpine produced a profound depression in levels spiperone, and by the non-selective antagonist of 5-HT
of NA, DA and 5-HT in the whole brain. This treat- receptors, methysergide. Spiperone and methysergide
ment resulted in a substantial reduction of the anti- are also well-known antagonists of 5-HT2 receptors.
tussive effects of the drugs tested, as evidenced by an However, ketanserin, a selective antagonist of 5-HT2
increase in the AtD50 of morphine, dihydrocodeine receptors, had no effect on the antitussive effect of 8-
and dextromethorphan. The AtD50 of these three OH-DPAT. Thus, spiperone and methysergide prob-
antitussive drugs in reserpinized rats were between ably exerted their effect through the 5-HT1A receptors
two- and four-fold higher than the AtD50 for these in this case. The distinction between pre- and post-
drugs in normal rats (Table 1). Parachloro- synaptic sites of antitussive effect of 8-OH-DPAT is
phenylalanine (PCPA) reduced the level of 5-HT in supported by the effect of PCPA. PCPA did not affect

A B
100 100
*
% Inhibition of the
% Inhibition of the
number of coughs
number of coughs
50 * 50
0 0
Saline 0.1 0.3
8-OH-DPAT
(mg/kg, ip)
Fig. 1 (A) Effect of 8-OH-DPAT on the number of capsaicin-induced coughs. (B) Effects of methysergide (Ε), ketanserin (∆) and
spiperone (Ρ) on the antitussive effects of 8-OH-DPAT, dihydrocodeine and dextromethorphan. The data represent the mean with SEM
of the percent inhibition of the number of coughs for at least five rats, 15 min after administration of each antitussive drug.
Methysergide, ketanserin and spiperone were administered ip 15 min before administration of each antitussive drug. Significant
differences from saline control (Φ) are indicated as ∗P<0.05.
the antitussive effect of 8-OH-DPAT. As inhibition of
5-HT synthesis is a major pharmacological action
of PCPA, the antitussive effect of 8-OH-DPAT is
consistent with a predominantly postsynaptic action
of 8-OH-DPAT. Taken together, these findings indicate
that an agonist of 5-HT1A receptors has an antitussive
effect, and it is probable that this effect is due to
activation of postsynaptic 5-HT1A receptors. In the
same manner as 8-OH-DPAT, the antitussive effects
of dihydrocodeine and dextromethorphan were sig-
nificantly reduced by pretreatment with spiperone
and methysergide, but not by ketanserin (Fig. 1).
Therefore, it is possible to speculate that the 5-HT1
receptors, in particular the 5-HT1A receptors, may be
more important than others with respect to the cough-
depressant activities of centrally-acting antitussive
drugs.
It is not yet possible, however, to conclude un-
equivocally that these antitussive drugs interact dir-
ectly with the 5-HT receptors involved in the
regulation of coughing. Moreover, it has been sug-
gested that dextromethorphan does not interact dir-
ectly with receptors for 5-HT.25 Dihydrocodeine and
dextromethorphan significantly increase the K+-
evoked release of [3H]5-HT from slices of rat nucleus
of the solitary tract (NTS).26 Serotonin has been found
in visceral primary sensory neurons that project to
the NTS.27,28 The NTS is the site of the first central
synapse for primary afferent fibers that originate from
airway receptors and the NTS plays an important
role in the regulation of the cough reflex.1 In addition,
among the various direct projections from central
structures, a significant contribution to the NTS arises
from the serotonergic brainstem raphe nucleus.29
Opioids and Serotonin in Cough 351
*
*
*
* *
*
8-OH-DPAT Dihydrocodeine Dextromethorphan
0.3 mg/kg, ip 1 mg/kg, ip 3 mg/kg, ip
Furthermore, we demonstrated that the antitussive
effects of dihydrocodeine and dextromethorphan were
markedly reduced in PCPA-treated rats.2 Therefore,
one possible hypothesis to be considered is that
antitussive drugs, such as dihydrocodeine and dex-
tromethorphan, interact with 5-HT receptors in-
directly by causing the release (and/or blocking the
uptake) of 5-HT perhaps by interaction with site(s)
on 5-HT nerve terminals in the NTS. On the other
hand, several pieces of evidence indicate that 5-HT
cell bodies and/or dendrites in the raphe nucleus
contain 5-HT1A receptors.30–34 The antitussive effects
of dihydrocodeine and dextromethorphan are reduced
by sc administration of 8-OH-DPAT (0.1mg/kg). Since
sc administration of 0.1mg/kg 8-OH-DPAT, by itself,
had no significant effect on the number of coughs, it
is unlikely that a postsynaptic agonistic action of 8-
OH-DPAT would appear at this dose with this route
of administration. Hutson et al.31 demonstrated that
8-OH-DPAT acts on autoreceptors in the dorsal raphe
to cause decreased release of 5-HT from the terminal
region and decreased synthesis of 5-HT therein. Sub-
cutaneous administration of 0.1mg/kg 8-OH-DPAT
significantly decreased 5-HT turnover and decreased
or prevented the increase in 5-HT turnover induced
by dihydrocodeine and dextromethorphan. Thus, it
is reasonable to speculate that the sc administration
of 0.1mg/kg 8-OH-DPAT induced the inhibition of
the antitussive effect of dihydrocodeine and dextro-
methorphan through its action on somatodendritic 5-
HT autoreceptors. These findings, therefore, strongly
support the above mentioned hypothesis that anti-
tussives interact indirectly with postsynaptic receptors
for 5-HT1A by facilitating the release of 5-HT.
352 J. Kamei

100 100

*
*
*
% Inhibition of the
number of coughs

% Inhibition of the
*

number of coughs
50 * *
50

* *
*

0
Saline
0.
1.
3.

0.
0. 3
0.

0. 1

10
1.
3.
3
0
0

3
0
00

03

0
0

.0
Morphine DAMGO U-50,488H DPDPE 0
nmol, ic Morphine DAMGO U-50,488H
3 nmol, ic 0.03 nmol, ic 3 nmol, ic
Fig. 2 Effects of morphine, DAMGO, U-50,488H and DPDPE
on the number of capsaicin-induced coughs in rats. The Fig. 3 Effect of DPDPE on the antitussive effects of morphine,
antitussive effect was determined 5 min after ic administration of DAMGO and U-50,488H, 5 min after ic administration of drugs
drugs. Each column represents the mean with SEM for at least in rats. DPDPE (∆) was injected ic 5 min before administration
five rats. Significant differences from saline control are indicated of each antitussive drug. Naltrindole (Ε), a selective d-opioid
as ∗P<0.05. receptor antagonist, was injected icv 5 min before administration
of DPDPE. Each column represents the mean with SEM for at
least five rats. Significant differences from saline control are
indicated as ∗P<0.05.
OPIOID RECEPTOR TYPES AND
ANTITUSSIVE EFFECTS

Involvement of opioid receptor types in the antitussive
effect
Opioids such as morphine and codeine are well known
antitussive agents. The analgesic effect of morphine
is mainly mediated by sites with characteristics of l-
opioid receptors. However, recent investigations have
demonstrated that an antinociceptive response can be
mediated at both supraspinal and spinal sites by l-,
d- and j-opioid receptors.35,36 Therefore, not only l-
opioid receptors but also d- and j-opioid receptors
may play an important role in the antitussive effects
of opioids. In this paper, we discuss the relative
contribution of opioid l-, d- and j-opioid receptors
to antitussive activity, through the use of the selective
agonists for the l-, d- and j-opioid receptors that are
currently available.
It has been hypothesized that d-opioid receptor
agonists may function both to initiate directly, as well
as to modulate, some forms of supraspinal l- and j-
opioid receptor-mediated antinociception.37,38 It was,
therefore, of interest to determine whether d-opioid
receptor agonists modulate l- and j-opioid receptor
agonist-induced antitussive activity. We also discuss
the interaction of l-, d- and j-opioid receptors in
opiate-mediated antitussive effects.
The selective l-opioid receptor agonist, DAMGO
(0.003–0.03nmol), has a dose-related cough-de-
pressant effect when given intracisternally (ic) in rats
(Fig. 2). U-50,488H, a highly selective agonist of j-
opioid receptors, has potent antitussive effects when
administered ic in rats (Fig. 2). Furthermore, nor-
binaltorphimine, a selective antagonist of j-opioid
receptors, antagonized the antitussive effects U-50,
488H and U-62066E, but it had no effect on the
antitussive effect of morphine. Although morphine at
a dose of 3nmol caused a more than 70% reduction
in the number of coughs, we did not observe a cough-
depressant effect of the selective d-opioid receptor
agonist DPDPE even with a dose of 10nmol (Fig. 2).
It is possible, however, that DPDPE might produce
a cough-depressant effect at even higher doses. None-
theless, it appears that d-opioid receptors play a minor
role in the cough-depressant effect of opiates. These
results indicate that the antitussive effects of opioids
are mediated predominantly by l- and j-opioid re-
ceptors.39,40
A dose of DPDPE that does not produce a sig-
nificant antitussive effect when given alone can inhibit
DAMGO-, morphine-, U-50,488H-mediated anti-
tussive effects (Fig. 3). The inhibition of the antitussive
effect of l- and j-opioid receptor agonists associated
with DPDPE was prevented by blockade of d-opioid
receptors by naltrindole (NTI). This finding suggests
that the modulation of the l- and j-opioid receptor
agonists-mediated antitussive effects results from the
action of DPDPE at d-opioid receptors. Therefore, it
seems likely that d-opioid receptors may play an
inhibitory role in antitussive processes mediated by
the l- and j-opioid receptors.40,41
On the other hand, NTI administered ip caused a
dose-dependent and significant antitussive effect in

both rats and mice.42 Pretreatment with DPDPE par-
tially but significantly reduced the antitussive effect
of NTI. It is possible, therefore, that the antitussive
effect of NTI results in the inhibition of d-opioid
receptor-mediated inhibitory system for antitussive
processes. Furthermore, blockade of j-opioid re-
ceptors by pretreatment with nor-binaltorphimine also
partially antagonized the antitussive effect of NTI.
However, the antitussive effect of NTI was not ant-
agonized by b-funaltrexamine, a selective l-opioid
receptor antagonist. Takemori et al.43 reported that
NTI had a agonist activity that appears to be mediated
mainly by j-opioid receptors. Thus, it seems likely
that the antitussive effect of NTI may be mediated,
in part, by j-opioid receptors but not by l-opioid
receptors.
l-Opioid receptor subtypes and antitussive effect
Various studies have demonstrated the existence of
two l-opioid receptor subtypes which have been re-
ferred to as l1-opioid and l2-opioid receptors.44 In
this regard, we previously reported that naloxonazine,
a selective l1-opioid receptor antagonist, had no effect
on the antitussive effects associated with ip morphine
and icv administred DAMGO in mice.4 Furthermore,
there was no significant difference between the mor-
phine-induced antitussive effect in CXBK, l1-opioid
receptor deficient mice, and in C57BL/6 mice.45 Fur-
thermore, the antitussive effects of morphine in both
the CXBK and C57BL/6 mice were not antagonized
by pretreatment with naltrexonazine. These results
indicate that l2- rather than l1-opioid receptors are
involved in l-opioid receptor-mediated antitussive
effects.
Differential modulation of l-opioid receptor-mediated
antitussive activity by d-opioid receptor agonists
Recent reports have presented evidence that at least
two subtypes of d-opioid receptors can be phar-
macologically identified in mice.46–49. It has been
proposed that DPDPE is selective for d1-opioid re-
ceptors, while [D-Ala2]deltorphin II is selective for d2-
opioid receptors.47 Given the possible existence of d-
opioid receptor subtypes and the observation of the
modulation of l-opioid receptor-mediated antitussive
activity by the d1-opioid receptor agonist, DPDPE,
it would be interesting to know whether d2-opioid
receptors have a modulatory role in l-opioid receptor-
mediated antitussive effects.
When DPDPE was injected icv 5 min before the
administration of DAMGO, the antitussive effect of
DAMGO was significantly reduced. The decrease in
the antitussive effect of DAMGO caused by DPDPE
was prevented by pretreatment with NTI. However,
Opioids and Serotonin in Cough 353
100
% Inhibition of the
number of coughs
50 *
*
* * *
*
* *
0
DTG Dextrometorphan Noscapine Morphine
3 mg/kg, i p 3 mg/kg, i p 10mg/kg, i p 3 mg/kg, i p
Fig. 4 Effects of rimcazole on the antitussive effects of DTG,
dextromethorphan, noscapine and morphine when assessed 30
min after administration of drugs in mice. Rimcazole (1mg/kg,
Ρ; 3mg/kg, ∆; 10mg/kg, Ε) was injected ip 15 min before
administration of antitussive drugs. Each column represents the
mean with SEM for at least five mice. Significant differences
from saline control are indicated as ∗P<0.05.
naltriben (NTB), a selective d2-opioid receptor ant-
agonist, had no effect on the decrease in the antitussive
effect of DAMGO caused by DPDPE. Unlike
DPDPE, [D-Ala2]deltorphin II administered icv 5 min
before the administration of DAMGO significantly
enhanced the antitussive effect of DAMGO. The in-
crease in the antitussive effect of DAMGO caused by
[D-Ala2]deltorphin II was prevented by pretreatment
with both NTI and NTB. There is also evidence that
not only d-opioid receptor agonists but also d-opioid
receptor antagonists themselves had no significant
effect on the number of coughs. Thus, it seems likely
that modulation of the DAMGO-mediated antitussive
effect by d-opioid receptor agonists may not result
from the synergistic or the antagonistic effect of l-
opioid receptor agonists and d-opioid receptor agon-
ists on the number of coughs. These results lead us
to conclude that d1-opioid receptors may play an
inhibitory role, whereas d2-opioid receptors may play
a synergistic role, in antitussive processes mediated
by l-, especially l2-opioid receptors.50
a-BINDING SITES AND ANTITUSSIVE
EFFECT
Haloperidol-sensitive [3H]N-allylnormetazocine
3
([ H]SKF10 047) binding sites, first termed ‘r-opioid
receptors’,51 are now commonly referred to as ‘r-
sites’.52,53 The r-sites are believed to be involved in
various central nervous system (CNS) disorders. These
binding sites have been characterized in the CNS using
radioligand binding assay employing high affinity and
selective ligands such as (+)-SKF-10 047, (+)-3-(3-
hydroxyphenyl)-N-(1-propyl)-piperidine ((+)-3-PPP)
354 J. Kamei
DNY?
Inhibitory
interneuron
Receptors for non-narcotic
antitussives ( -sites?)
5-HT 1A
Calcium channel
NMDA
receptor
NTS
Fig. 5 A proposed mechanism for the action of antitussive drugs. NTS: nucleus of the solitary tract, NRM: nucleus of raphe magnus,
b-END: b-endorphinergic fibers, DYN: dynorphinergic fibers.
and N,N′-di(ortho-tolyl)guanidine (DTG).54–57. Re-
cent studies indicated that dextromethorphan, a com-
monly available non-opioid antitussive drug, has a
high affinity for the r binding site.57,58 Furthermore,
we previously reported that pentazocine, a ligand for
haloperidol-sensitive r binding site, exhibits a potent
antitussive effect.59 The r-site is found in all areas
throughout the brain, including the NTS.60. The NTS
is adjacent to the site that controls the basic central
mechanism of the cough reflex.1 Thus, it is possible
that r-sites might be involved in the control of cough
reflex. The possible role of r-sites in the regulation
of the cough reflex is discussed next.
Two selective r-ligands, (+)-SKF-10 047 and DTG,
have a marked dose-dependent cough depressant effect
in rats.61 The antitussive effects of these drugs were
antagonized by pretreatment with haloperidol. Fur-
thermore, the antitussive potencies of r-ligands were
similar to that of dextromethorphan. Therefore, these
results raise the possibility that haloperidol-sensitive
r-sites are involved in the regulation of coughs. This
possibility is supported by the result that the anti-
tussive effects of (±)-pentazocine and dextro-
methorphan were also significantly reduced by
pretreatment with haloperidol. Furthermore, ip in-
jection of rimcazole, a specific antagonist of r sites,
in doses from 1 to 10mg/kg, significantly antagonized
the cough-depressant effect of DTG, a r ligand, dose-
dependently (Fig. 4). Cough-depressant effects of dex-
tromethorphan and noscapine were also significantly
and dose-dependently reduced by pretreatment with
rimcazole (Fig. 4). However, rimcazole (10mg/kg,
ip) did not have a significant effect on the antitussive
ß - END?
NMR
Serotonergic neuron
Receptors for non-narcotic
antitussives ( -sites?)
Glutamatergic neurones
Afferent input from the sensory
receptors of the airway
effect of morphine (Fig. 4). Furthermore, rimcazole
by itself (10 and 30mg/kg, ip) had no significant
effect on the number of coughs. These results suggest
that r sites may be involved in the antitussive
mechanism of centrally acting non-narcotic anti-
tussive drugs.62
There is considerable evidence which suggests that
there is a functional interaction between the r-site
and the N-methyl-D-aspartate (NMDA) receptors
(see review by Su,63). Furthermore, the release of endo-
genous glutamate from rat hippocampal slices during
ischaemic insult is markedly attenuated by a r ligand,
such as DTG, suggesting the possibility that the r-
sites may be involved in the modulation of the release
of glutamate.64 Moreover, several lines of evidence
suggest a possible association of r-sites with Ca2+
channels.65,66 Previously, we demonstrated that both
NMDA receptor antagonists and voltage-dependent
Ca2+ channel antagonists have a marked cough-de-
pressant effect.3,67 These findings led us to the spec-
ulation that the cough-depressant effect of non-opioid
antitussive drugs, such as dextromethorphan, may
be due to the inhibition of glutamatergic synaptic
transmission of the afferent information of the cough
reflex, an effect mediated not only by the inhibition
Ca2+-dependent release of glutamate but also by the
direct inhibition of NMDA receptor-activated Ca2+
channels.3,67 Thus, it is possible that r ligands exhibit
their antitussive effect through an inhibition of glu-
tamatergic synaptic transmission of the afferent in-
formation of the cough reflex as a result of modulation
of NMDA receptors and/or voltage-dependent Ca2+
channels which are associated with r-sites.

CONCLUSIONS
A proposed mechanism for the antitussive drugs is
summarized in Fig. 5. It is now clear that centrally-
acting antitussive drugs exhibit their effect through
an inhibition of glutamatergic synaptic transmission
of the afferent input from the sensory receptors of
the airway as a result of facilitation of serotonergic
mechanisms. This hypothesis could open new avenues
in developing antitussive drugs with more potency
and safety than drugs used presently.
ACKNOWLEDGMENTS
I wish to thank my colleagues who collaborated in
the research reported here.
REFERENCES
1. Korpas J, Tomori Z. Cough and other respiratory reflexes.
In: Herzog E S ed. Progress in Respiration Research. Basel:
Karger, 1979: 15–179.
2. Kamei J, Ogawa M, Kasuya Y. Monoamines and the
mechanisms of action of antitussive drugs in rats. Arch int
Pharmacodyn Ther 1987; 290: 117–127.
3. Kamei J, Tanihara H, Igarashi H, Kasuya Y. Effect of N-
methyl-D-aspartate antagonists on the cough reflex. Eur J
Pharmacol 1989; 168: 153–158.
4. Kamei J, Iwamoto Y, Kawashima N, Suzuki T, Nagase H,
Misawa M, Kasuya Y. Possible involvement of l2-mediated
mechanisms in l-mediated antitussive activity in the mouse.
Neurosci Lett 1993; 149: 169–172.
5. Bolme P, Fuxe K. Pharmacological studies on a possible role
of central noradrenergic neurons in respiratory control. J
Pharm Pharmacol 1973; 25: 351–352.
6. Bolme P, Corrodi H, Fuxe K, Hokfelt P L, Goldstein M.
Possible involvement of central adrenaline neurons in
vasomotor and respiratory control. Studies with clonidine
and its interaction with piperoxone and yohimbine. Eur J
Pharmacol 1974; 28: 89–94.
7. Florez J, Corrodi H, Fuxe K, Hokfelt P L, Goldstein M.
Adrenergic and serotonergic mechanisms in morphine
induced respiratory depression. Psychopharmacology 1972;
24: 258–274.
8. Hedner J, Hedner T, Jonasan J, Lundberg D. Evidence for a
dopamine interaction with the central respiratory control
system in the rat. Eur J Pharmacol 1982; 81: 603–615.
9. Lundberg D, Breese G R, Mueller R A. Dopaminergic
interaction with the respiratory control system in the rat.
Eur J Pharmacol 1979; 54: 153–159.
10. Armijo J A, Florez J. The influence of increased brain 5-
hydroxytryptamine upon the respiratory activity of cats.
Neuropharmacology 1974; 13: 977–986.
11. Lundberg D, Mueller R A, Breese G R. An evaluation of the
mechanism by which serotonergic activation depresses
respiration. J Pharmacol Exp Ther 1980; 212: 397–404.
12. McCrimmon D R, Lalley P M. Inhibition of respiratory
neuronal discharges by clonidine and 5-hydroxytryptophan. J
Pharmacol Exp Ther 1982; 222: 771–777.
13. Mueller R A, Lundberg D, Breese G R. Evidence that
respiratory depression by serotonin agonists may be exerted
in the central nervous system. Pharmacol Biochem Behav
1980; 13: 247–255.
14. Kamei J, Hosokawa T, Yanaura S, Hukuhara T.
Involvement of central serotonergic mechanisms in the cough
reflex. Jpn J Pharmacol 1986; 42: 531–538.
Opioids and Serotonin in Cough 355
15. Kamei J, Ogawa M, Kasuya Y. Supersensitivity of 5,7-
dihydroxytryptamine-treated rats to the respiratory
depressant and antitussive effects of dihydrocodeine. Eur J
Pharmacol 1988; 153: 305–308.
16. Peroutka S J, Snyder S H. Multiple serotonin receptors:
differential binding of [3H]5-hydroxytryptamine, [3H]lysergic
acid diethylamine and [3H] spiperidol. Mol Pharmacol 1979;
16: 687–699.
17. Hoyer D, Clarke D E, Fozard J R, Hartig P R, Martin G R,
Mylecharane E J, Saxena P R, Humphrey P P A.
International Union of Pharmacology classification of
receptors for 5-hydroxytryptamine (serotoin). Pharmacol
Rev 1994; 46: 157–203.
18. Ahlenius S, Larsson K, Sevensson L, Hjorth S, Carlsson A,
Lindberg P, Wikstrom H, Sanchez D, Arvidsson L-E,
Hacksell U, Nilsson J L G. Effects of a new type of 5-HT
receptor agonist on male sexual behavior. Pharmacol
Biochem Behav 1981; 15: 785–792.
19. Arvidsson L-E, Hacksell U, Nilsson J L G, Hjorth S,
Carlsson A, Lindberg P, Sanchez D, Wikstrom H. 8-
Hydroxy-2-(di-n-propyl-amino)tetralin, a new centrally
acting 5-hydroxytryptamine receptor agonist. J Med Chem
1981; 24: 921–923.
20. Eide P K. Hole K, Subsensitivity of serotonin and substance
P receptors involved in nociception after repeated
administration of a serotonin receptor agonist. J Neural
Transm 1989; 77: 1–10.
21. Hjorth S, Carlsson A, Lindberg P, Sanchez D, Wikstrom H,
Arvidsson L-E, Hacksell U, Nilsson J L G. 8-Hydroxy-2-di-
(n-propylamino)tetralin, 8-OH-DPAT, a potent and selective
simplified ergot congener with central 5-HT receptor
stimulating activity. J Neural Transm 1982; 55: 169–188.
22. Middlemiss D N, Fozard J R. 8-Hydroxy-2-(di-n-
propylamino)tetralin discriminates between subtypes of the
5-HT recognition site. Eur J Pharmacol 1983; 90: 151–153.
23. Tricklebank M D, Forler C, Fozard J R. The involvement of
subtypes of the 5-HT1 receptor and of catecholaminergic
systems in the behavioral response to 8-hydroxy-2-(di-n-
propylamino)tetralin in the rat. Eur J Pharmacol 1985; 106:
271–282.
24. Kamei J, Mori T, Igarashi T, Kasuya Y. Effects of 8-
hydroxy-2-(di-n-propylamino)tetralin, a selective agonist of
5-HT1A receptors, on the cough reflex in rats. Eur J
Pharmacol 1991; 203: 253–258.
25. Craviso G L, Musacchio J M. High-affinity
dextromethorphan binding sites in guinea pig brain. II.
Competition experiments. Mol Pharmacol 1983; 23: 629–640.
26. Kamei J, Mori T, Igarashi H, Kasuya Y. Serotonin release in
nucleus of the solitary tract and its modulation by
antitussive drugs. Res Commun Chem Pathol Pharmacol
1992; 76: 371–374.
27. Gaudin-Chazal G, Segu L, Seyfritz N, Puizillout J J.
Visualization of serotonin neurons in the nodose ganglia of
the cat. An autoradiographic study. Neuroscience 1981; 6:
1027–1037.
28. Gaudhi-Chazal G, Portalier P, Puizillout J, Vigier D.
Simultaneous visualization of aortic and [3H]5-
hydroxytryptamine-accumulating cell bodies in the nodose
ganglia of the cat. J Physiol (London) 1982; 337: 321–330.
29. Pierce T E, Foote W E, Hobson J A. The efferent
connections of the nucleus raphe dorsalis. Brain Res 1976;
107: 137–144.
30. Hutson P H, Dourish C T, Curzon G. Neurochemical and
behavioral evidence for mediation of the hyperphagic action
of 8-OH-DPAT by 5-HT cell body autoreceptors. Eur J
Pharmacol 1986; 129: 347–352.
31. Hutson P H, Sarna G S, O’Connell M T, Curzon G.
Hippocampal 5-HT synthesis and release in vivo is decreased
by infusion of 8-OH-DPAT into the nucleus raphe dorsalis.
Neurosci Lett 1989; 100: 276–280.
32. Sprous J S, Aghajanian G K. Electrophysiological responses
of serotonergic dorsal raphe dorsalis. Synapse 1987; 1: 3–9.
33. Verg D, Daval G, Patey A, Gozlan H, Mestikawy S E,
Hamon M. Presynaptic 5-HT autoreceptors on serotonergic
cell bodies and/or dendrites but not terminals are of the 5-
HT1A subtype. Eur J Pharmacol 1985; 113: 463–464.
356 J. Kamei
34. Weissmann-Nanopoulos D, Mach E, Magre J E, Demassy Y,
Pujol J F. Evidence for the localization of 5-HT1A binding
sites on serotonin containing neurons in the raphe dorsalis
and raphe centralis nuclei of the rat brain. Neurochem Int
1985; 7: 1061–1072.
35. Heyman J S, Mulvaney S A, Mosberg H I, Porreca F.
Opioid d receptor involvement in supraspinal and spinal
antinociception in mice. Brain Res 1987; 420: 100–108.
36. Porreca F, Heyman J S, Mosberg H I, Omnaas J R, Vaught
J L. Role of l and d receptors in the supraspinal and spinal
analgesic effects of [D-Pen2, D-Pen5]enkephalin in the mouse.
J Pharmacol Exp Ther 1987; 241: 393–400.
37. Heyman J S, Vaught J L, Mosberg H I, Haaseth R C,
Porreca F. Modulation of l-mediated antinociception by d
agonists in the mouse: selective potentiation of morphine
and normorphine by [D-Pen2, D-Pen5]enkephalin. Eur J
Pharmacol 1989; 165: 1–10.
38. Heyman J S, Jiang Q, Rothman R B, Mosberg H I, Porreca
F. Modulation of l-mediated antinociception by d agonists:
characterization with antagonists. Eur J Pharmacol 1989;
169: 43–52.
39. Kamei J, Tanihara H, Kasuya Y. Antitussive effects of two
specific j-opioid agonists, U-50,488H and U-62,066E, in
rats. Eur J Pharmacol 1990; 187: 281–286.
40. Kamei J, Tanihara H, Kasuya Y. Modulation of l-mediated
antitussive activity in rats by a d agonist. Eur J Pharmacol
1991; 203: 153–156.
41. Kamei J, Tanihara H, Kasuya Y. Modulation of j-mediated
antitussive activity in rats by a d agonist. Res Commun
Chem Pathol Pharmacol 1992; 76: 375–378.
42. Kamei J, Iwamoto Y, Suzuki T, Misawa M, Nagase H,
Kasuya Y. Antitussive effects of naltrindole, a selective d-
opioid receptor antagonist, in mice and rats. Eur J
Pharmacol 1993; 249: 161–165.
43. Takemori A E, Sultana M, Nagase H, Portoghese P S.
Agonist and antagonist activities of ligands derived from
naltrexone and oxymorphone. Life Sci 1992; 50: 1491–1495.
44. Pasternak G W, Wood P L. Multiple mu opiate receptors.
Life Sci 1986; 38: 1889–1898.
45. Kamei J, Iwamoto Y, Suzuki T, Misawa M, Nagase H,
Kasuya Y. The role of the l2-opioid receptor in the
antitussive effect of morphine in l1-opioid receptor-deficient
CXBK mice. Eur J Pharmacol 1993; 240: 99–101.
46. Jiang Q, Takemori, A E, Sultana M, Portoghese P S, Bowen
W D, Mosberg H I, Porreca F. Differential antagonism of
opioid delta antinociception by [D-Ala2, Leu4,
Cys6]enkephalin and naltrindole 5-isothiocyanate: evidence
for delta receptor subtypes. J Pharmacol Exp Ther 1991;
257: 1069–1075.
47. Mattia A, Farmer S C, Takemori A E, Sultana M,
Portoghese P S, Mosberg H I, Bowen W D, Porreca F.
Spinal opioid delta antinociception in the mouse; mediation
by single subtype of opioid delta receptor. J Pharmacol Exp
Ther 1992; 260: 518–525.
48. Sofuoglu M, Portoghese P S, Takemori A E. Differential
antagonism of delta opioid agonists by naltrindole (NTI)
and its benzofuran analog (NTB) in mice: evidence for delta
opioid receptor subtypes. J Pharmacol Exp Ther 1991; 257:
676–680.
49. Sofuoglu M, Portoghese P S, Takemori A E. Cross-tolerance
studies in the spinal cord of b-FNA-treated mice provides
further evidence for delta opioid receptor subtypes. Life Sci
1991; 49: PL153–156.
50. Kamei J, Iwamoto Y, Suzuki T, Nagase H, Misawa M,
Kasuya Y. Differential modulation of l-opioid receptor-
mediated antitussive activity by d-opioid receptor agonists in
mice. Eur J Pharmacol 1993; 234: 117–120.
51. Su T-P. Evidence for sigma opioid receptor: binding of [33H]-
SKF-10047 to etorphine-inaccessible sites in guinea-pig
brain. J Pharmacol Exp Ther 1982; 223: 284–290.
52. Quirion R, Chicheportice R, Contreras P C, Johnson K M,
Lodge D, Tam S W, Woods J H, Zukin S R. Classification
of nomenclature of phencyclidine and sigma receptor site.
Trends Neurosci 1987; 10: 444–446.
53. Quirion R, Bowen W D, Itzhak Y, Junien J L, Musacchio J
E, Rothaman R B, Su T-P, Tam S W, Taylor D P. A
proposal for the classification of sigma binding sites. Trends
Pharmacol Sci 1992; 13: 85–86.
54. Gundlach A L, Largent B L, Snyder S H. Autoradiographic
localization of r-receptor binding sites in guinea pig and rat
central nervous system with (+)-[3H]-3-(3-hydroxyphenyl)-N-
(1-propyl)-piperidine. J Neurosci 1986; 6: 1757–1770.
55. Largent B L, Gundlach A L, Snyder S H. Sigma receptors
on NBC-20 hybrid neurotumor cells labeled with
(+)[33H]SKF10047 and (+)[3H]3-PPP. Eur J Pharmacol
1986; 124: 183–187.
56. Su T-P. Psychotomimetic opioid binding: specific binding of
[3H]-SKF-10047 to etorphine-inaccessible sites in guinea-pig
brain. Eur J Pharmacol 1981; 75: 81–82.
57. Weber E, Sonders M, Quarum M, McLean S, Pou S, Keana
J F W. 1,3-Di(2-[5-3H]tolyl)guanidine: a selective ligand that
labels r-type receptors for psychotomimetic opiates and
antipsychotic drugs. Proc Natl Acad Sci USA 1986; 83:
8747–8788.
58. Klein M, Santiago L J, Musacchio J M. Effect of calcium
channel blockers on the binding of the novel anticonvulsant
dextromethorphan. Fed Proc 1987; 46: 708.
59. Kamei J, Katsuma K, Kasuya Y. Involvement of l-opioid
receptors in the antitussive effects of pentazocine. Naunyn-
Schmiedeberg’s Arch Pharmacol 1992; 345: 203–208.
60. McLean S, Weber E. Autoradiographic visualization of
haloperidol-sensitive sigma receptors in guinea-pig brain.
Neuroscience 1988; 25: 259–269.
61. Kamei J, Iwamoto Y, Kawashima N, Hitosugi H, Misawa
M, Kasuya Y. Involvement of haloperidol-sensitive r-sites in
antitussive effects. Eur J Pharmacol 1992; 224: 39–43.
62. Kamei J, Iwamoto Y, Misawa M, Kasuya Y. Effects of
rimcazole, a specific antagonist of r sites, on the antitussive
effects of non-narcotic antitussive drugs. Eur J Pharmacol
1993; 242: 209–211.
63. Su T-P. r-Receptors: putative links between nervous,
endocrine and immune systems. Eur J Biochem 1991; 200:
633–642.
64. Lobner D, Lipton, P. r-Ligands and non-competitive
NMDA antagonists inhibit glutamate release during cerebral
ischemia. Neurosci Lett 1990; 117: 169–174.
65. Watkins J C, Evans R H. Excitatory amino acid transmitters.
Annu Rev Pharmacol Toxicol 1981; 21: 165–204.
66. Rothman R B, Reid A, Mahboubi A, Kim C-H, DeCosta R
B, Jacobson A E, Rice K C. Labelling by [3H]1,3-di(2-tolyl)-
guanidine of two high affinity binding sites in guinea pig
brain: evidence for allosteric regulation by calcium channel
antagonists and pseudoallosteric modulation by sigma
ligands. Mol Pharmacol 1991; 39: 222–232.
67. Kamei J, Kasuya Y. Antitussive effects of Ca2+ channel
antagonists. Eur J Pharmacol 1992; 212: 61–66.