Depart Pharmacology,
University of Michigan Medical School, Ann
Arbor, MI.USA
Abstract Rationale Identification of
behaviors specifically mediated by the dopamine
D2 and D3 receptors would allow for the
determination of in vivo receptor selectivity
and aide the development of novel therapeutics
for dopamine-related diseases.
Objectives These studies were
aimed at evaluating the specific receptors
involved in the mediation of D2/D3
agonist-induced yawning and hypothermia.
Materials and methods The
relative potencies of a series of D2-like
agonists to produce yawning and hypothermia were
determined. The ability of D3-selective and
D2-selective antagonists to inhibit the
induction of yawning and hypothermia were
assessed and a series of D2/D3 antagonists were
characterized with respect to their ability to
alter yawning induced by a low and high dose of
PD-128,907 and sumanirole-induced
hypothermia.
Results D3-preferring agonists
induced yawning at lower doses than those
required to induce hypothermia and the
D2-preferring agonist, sumanirole, induced
hypothermia at lower doses than were necessary
to induce yawning. The rank order of D3
selectivity was pramipexole > PD-128,907 =
7-OH-DPAT = quinpirole = quinelorane >
apomorphine = U91356A. Sumanirole had only D2
agonist effects. PG01037, SB-277011A, and U99194
were all D3-selective antagonists, whereas
haloperidol and L-741,626 were D2- selective
antagonists and nafadotride's profile of action
was more similar to the D2 antagonists than to
the D3 antagonists.
Conclusions D3 and D2
receptors have specific roles in the mediation
of yawning and hypothermia, respectively, and
the analysis of these effects allow inferences
to be made regarding the selectivity of D2/D3
agonists and antagonists with respect to their
actions at D2 and D3 receptors.
Introduction Dopamine D2 and D3
receptors are both members of the D2-like family
of dopamine receptors, which are known to
possess a high degree of sequence homology (52%
overall and 75% in the transmembrane domains;
Sokoloff et al. 1990) and a partially
overlapping pattern of distribution in the
brain. For example, D2 receptors are expressed
at while the D3 receptor has been shown to
possess a much more restricted limbic pattern of
distribution in both the rat (Levesque et al.
1992) and human brains (Gurevich
and Joyce 1999).
These high levels of expression within the
limbic brain regions have led many to
hypothesize that the D2 and D3 receptors are of
particular interest as pharmacologic targets for
the treatment of a variety of movement and
psychiatric disorders including Parkinson's
disease, restless leg syndrome, depression, and
schizophrenia (e. g., Joyce 2001; Happe and
Trenkwalder 2004) and a variety of aspects of
drug abuse (e. g., Heidbreder et al. 2005;
Newman et al. 2005). Due in part to the lack of
highly selective agonists and antagonists, the
receptor(s) mediating either the therapeutic or
mechanistic effects are yet to be fully
elucidated.
Although several agonists and antagonists
have been reported to be over 100-fold selective
for either the D3 (e. g., Stemp et al. 2000;
Grundt et al. 2005) or D2 (e. g., Vangveravong
et al. 2006) receptors based on in vitro binding
studies, a large degree of variability exists
with respect to the reported in vitro binding
affinities and D2/D3 selectivity ratios.
A variety of factors may account for these
differences in affinity and selectivity
including differences in receptor species,
expression systems, radioligands, and/ or assay
conditions. For example, reported binding
affinities for pramipexole at the D2 receptor
range from 3.9 to 955 nM depending upon whether
agonist or antagonist radioligands were used
(Mierau et al. 1995; Millan et al. 2002), while
reported D3 selectivity ratios range from 2-
fold to 488-fold selective for the D3 receptor
over the D2 receptor depending upon whether
binding affinities from cloned human receptor
cell systems or human brain tissue are used to
make the determinations (Seeman et al. 2005;
Gerlach et al. 2003).
Furthermore, in vitro binding studies often
provide greater affinity and selectivity values
than those obtained through functional studies
suggesting that differences in D2 and D3
efficacy may also greatly influence a ligand's
receptor selectivity. For example, in three
separate studies, which characterized D2/D3
agonists based on their binding affinities for
the D2 and D3 receptors and ability to stimulate
mitogenic activity, quinpirole was found to be
either 9-fold, 15-fold or 36- fold selective for
the D3 receptor over the D2 receptor as
determined by radioligand binding, but the D3
selectivity ratios for quinpirole dropped to
2.5-fold, 1.3-fold, and 3.3- fold when the ED50
values for the induction of mitogenic activity
were compared (Pugsley et al. 1995; Chio et al.
1994; Sautel et al. 1995).
The identification of agonists and
antagonists highly selective for the D2 and/or
D3 receptors has been complicated by a lack of
well-characterized behavioral effects
specifically mediated by either the D2 or D3
receptor. While D2/D3 agonists have been
shown to modulate body temperature, locomotor
activity, and certain neuroendocrine responses
in addition to other behavioral measures (Faunt
and Crocker 1987; Millan et al. 1995; Depoortere
et al. 1996; Smith et al. 1997; Boulay et al.
1999a), few of these effects have been fully
characterized and well-validated.
There is strong pharmacological and genetic
evidence in support of subtype selective in vivo
effects for the induction of hypothermia
resulting from D2 receptor activation, and
significant pharmacological evidence for the
induction of yawning resulting from agonist
activation of the D3 receptor. The first
indication that D2/D3 agonist-induced
hypothermia was mediated by the D2 receptor, not
D3, was the finding that D3 receptor-deficient
mice displayed a normal hypothermic response to
D2/D3 agonists, while the effect was completely
absent in D2 receptor-deficient mice (Boulay et
al. 1999a, b).
This was later supported by pharmacologic
studies in rats that demonstrated that the
D2-preferring antagonist, L-741,626, produced a
dosedependent inhibition of D2/D3
agonist-induced hypothermia, whereas the
D3-preferring antagonist A-437203 failed to
alter the hypothermic response at any dose
tested (Chaperon et al. 2003). Yawning behavior
in rats has been a long studied phenomenon and
is known to be regulated by a variety of
neurotransmitter systems including cholinergic
(Urba- Holmgren et
al. 1977; Yamada
and Furukawa 1980), serotonergic (Stancampiano
et al. 1994), and dopaminergic (Mogilnicka
and Klimek 1977;
Holmgren and Urba- Holmgren 1980) systems
associated with the paraventricular nucleus of
the hypothalamus (Argiolas
and Melis 1998).
Recently, a specific role for the D3
receptor in the induction of yawning behavior
has also been demonstrated. A series of
D3-preferring agonists induced dose-dependent
increases in yawning behavior over low doses
with inhibition of yawning occurring at higher
doses resulting in a characteristic inverted
U-shaped dose-response curve. Several
D3-preferring antagonists were also shown to
selectively inhibit the induction of yawning
behavior, while the D2-preferring antagonist,
L-741,626, produced a selective rightward and
upward shift in the descending limb of the
dose-response curve for D2/D3 agonist induced
yawning (Collins et
al. 2005).
Thus, although it has been suggested that
the induction of yawning is mediated by
activation of the D2 receptor (Millan et al.
2000), our data indicated that the induction of
yawning by D2/D3 agonists is mediated by a
selective activation of the D3 receptor, while
inhibition of yawning behavior at higher doses
is a result of a concomitant D2 receptor
activation. The present studies were aimed at
further characterizing the roles of the D2 and
D3 receptors in the regulation of body
temperature and yawning behavior. Thus, a series
of D2-like agonists with a range of reported in
vitro selectivities for the D3 receptor over the
D2 receptor (pramipexole ³ PD-128,907 =
7-OH-DPAT > quinpirole = quinelorane >
apomorphine > U91356A > sumanirole) and
two D4-preferring agonists (ABT-724 and
PD-168,077) were assessed for their ability to
induce yawning and hypothermia, while a series
of D2/D3 antagonists with a similar range of
reported in vitro selectivities (PG01037 =
SB-277011A >> U99194 > nafadotride >
haloperidol > L- 741,626) were characterized
for their ability to modulate the induction of
yawning and hypothermia in the rat.
Convergent evidence
support the hypotheses that the induction of
hypothermia and yawning behavior are mediated by
the selective activation of the D2 and D3
receptors.
Furthermore, these studies suggest that the
minimal effective dose (MED) for the induction
and inhibition of yawning behavior and
hypothermia may provide a means for the
determination of in vivo D3 and D2 receptor
potency measures for agonists and antagonists,
respectively.
Discussion The current studies
replicate and extend the findings of a previous
study that suggested that the induction of
yawning by low doses of D2/D3 agonists is
mediated by the selective activation of the D3
receptor, whereas the inhibition of yawning
occurring at higher doses is mediated by a
concomitant activation of the D2 receptor
(Collins et al. 2005). As was demonstrated in
the earlier paper, yawning induced by a low dose
of the D3-preferring agonist PD- 128,907 was
selectively and dose-dependently inhibited by
the D3 antagonists, PG01037, SB-277011A, and
U99194, whereas the inhibition of yawning
observed at high doses of PD-128,907 was
reversed by the selective D2 antagonist L-
741,626, but not PG01037, SB-277011A nor
U99194.
The current studies extend the previous
findings in several ways. In addition to the
evaluation of agonist and antagonist
interactions on yawning, the effects of the D2/
D3 agonists alone and in combination with
selective antagonists were evaluated on core
body temperatures to test the notion that the
hypothermic effects of these agonists are
mediated by the activation of the D2 receptor,
but not the D3 or D4 receptor (Boulay et al.
1999a, b; Chaperon et al. 2003). Several lines
of evidence presented herein support this
notion. The selective D2 agonist, sumanirole,
produced decreases in body temperature at
relatively low doses that did not induce
yawning. The hypothermic effects of sumanirole
were prevented by prior administration of the
D2-preferring antagonists, haloperidol and
L-741,626. L- 741,626 also inhibited the
hypothermic effects of high doses of all of the
D3-preferring agonists in addition to producing
dramatic increases in yawning when combined with
the same high doses of D3-preferring
agonists.
The latter is likely to reflect the reversal
of the D2-mediated inhibition of yawning
produced at high doses of the agonists and is
consistent with the notion that these
antagonists are D2-selective and that the
suppression of yawning and hypothermic effects
observed at relatively high doses of D2/D3
agonists are D2 agonist-mediated effects. It is
important to note that these differential
effects of D3 and D2 antagonists on yawning
induced by low and high doses of D2/D3 agonists
were observed with all of the D3-preferring
agonists tested in the current study (Table 2)
and occurred at doses of PG01037 that do not
alter the induction of yawning by physostigmine
or TFMPP (Collins et al. 2005) and a dose of
L-741,626 that does not alter the induction of
hypothermia by the serotonin 1A agonist,
8-OH-DPAT (Table 2), suggesting that these
effects are a result of a selective antagonist
activity at D3 and D2 receptors, respectively.
These in vivo measures of selective D3 (yawning)
and D2 (hypothermia) activation were used to
characterize ten D2-like agonists and six D2/D3
antagonists.
This extensive evaluation, comparing the
potency of each agonist to produce increases in
yawning with its potency to produce hypothermia
(Table 1), indicated that pramipexole was the
most selective D3 agonist, followed by
PD-128,907, quinelorane, quinpirole, and
7-OH-DPAT with nearly equal D3 selectivity. Both
apomorphine and U91356A were relatively
nonselective D2/D3 agonists, inducing yawning at
doses that were only slightly lower than those
required to decrease body temperature.
Sumanirole was a selective D2 agonist. Although
sumanirole slightly increased yawning at doses
that were higher than those necessary to
decrease body temperature, this yawning was not
sensitive to the D3- selective antagonist,
PG01037, but was inhibited by the cholinergic
antagonist scopolamine and may therefore
represent cholinergic rather than D3 activation.
McCall et al. (2005) reported a 200% increase in
striatal acetylcholine release in rats at doses
of sumanirole roughly equivalent to those which
induced yawning.
The two D4-preferring agonists, given at
behaviorally active doses (Brioni et al. 2004;
Enguehard-Gueiffier et al. 2006), did not
produce either yawning or hypothermia suggesting
that at these doses, they are devoid of
significant D2 and D3 receptor agonist activity.
As was seen with the agonists, distinct
behavioral profiles emerged for D3-preferring
and D2-preferring antagonists. Three of the four
D3-preferring antagonists, PG01037, SB-277011A,
and U99194, inhibited yawning at doses that did
not alter hypothermia suggesting that they
function as selective D3 antagonists in vivo.
The doses of these antagonists that were able to
be tested was limited by solubility (PG01037 and
SB-277011A) and anticholinergic activity
(U99194), and thus in vivo D2/D3 selectivity
ratios were indeterminate other than being
slightly greater than 1.
It is interesting to note that nafadotride,
which is mildly D3- preferring in vitro and
generally considered to be a D3- preferring
antagonist in vivo (e. g., Richtand et al. 2000;
Leriche et al. 2003), displayed a profile of
activity that was more like those of the D2
antagonists, haloperidol and L- 741,626, than of
the other D3-preferring antagonists. L- 741,626,
haloperidol, and nafadotride were all more
potent at inhibiting the induction of
hypothermia and increasing high dose yawning;
however, suppression of low dose yawning was
also observed with each of these antagonists,
and thus were all determined to be
°„3-fold selective for the D2 receptor
over the D3 receptor in vivo. Evidence provided
in the current and past (Collins et al. 2005)
studies support distinct roles for the D2 and D3
receptors mediating the hypothermic and yawning
effects of D2/D3 agonists although these
generalizations are contrary to earlier
characterizations (see, Millan et al. 2000).
These investigators determined that the
hypothermic effects of 7- OH-DPAT were mediated
by agonist activity at both the D2 and D3
receptors as it was attenuated by the D3
antagonists, S33084 and GR218231, and the D2
antagonist, L-741,626.
Furthermore, they concluded that
7-OH-DPATinduced yawning was mediated by the D2
receptor, not D3, as they observed inhibition of
yawning with L-741,626, but not S33084 or
GR218321. Although our data do not support this
interpretation, we recognize that relatively
large doses of D3-preferring agonists induce
hypothermia and likewise that relatively large
doses of L-741,626 suppress yawning induced by
D3-preferring agonists. However, these effects
likely represent a loss of receptor selectivity
rather than a primary effect of the agonists and
antagonists. A notion that is supported by the
biphasic nature of the D2/D3 agonists and
antagonists with respect to their effects on
yawning and hypothermia. In the current study,
all D3-preferring agonists, including 7-OH-DPAT,
induced yawning at low doses with inhibition of
yawning and induction of hypothermia occurring
at higher, presumably less selective,
doses.
Similarly, at relatively low doses,
L-741,626, haloperidol, and nafadotride
equipotently increased high dose yawning and
inhibited hypothermia, while inhibition of
yawning induced by a low, presumably
D3-selective, dose of PD-128,907 was not
observed until higher doses. Moreover, in the
current study, the D3 antagonists PG01037,
SB-277011A, and U99194 all selectively inhibited
PD-128,907-induced yawning while failing to
alter the induction of hypothermia by
sumanirole, suggestive of a selective D3
antagonist activity. While the MEDs for the
inhibition of yawning by PG01037 and SB-277011A
(32.0 mg/kg for both) are slightly higher than
those reported for SB-277011A on a variety of
operant behaviors (3.0-24 mg/kg; Andreoli et al.
2003; Di Ciano et al. 2003; Xi et al. 2004,
2005; Gilbert et al. 2005; Cervo et al. 2007)
and are likewise higher than might be expected
based on in vitro D3 affinities of 0.7 and 10.7
nM, respectively (Stemp et al. 2000; Grundt et
al. 2005), there is no evidence to suggest that
the inhibition of yawning by these antagonists
results from anything other than an antagonist
activity at the D3 receptor.
Not only did PG01037 and SB-277011A not
inhibit sumanirole-induced hypothermia or
increase yawning induced by high doses of
PD-128,907 in the current studies at doses up to
56.0 mg/kg, but SB-277011A also failed to induce
catalepsy and increases plasma prolactin levels
at doses up to 78.8 and 93 mg/kg; p. o.,
respectively (Reavill et al. 2000).
However, this is not to say that these
antagonists are completely devoid of D2
antagonist activity as U99194 has been reported
to inhibit the induction of hypothermia with an
ED50 of 12.9 mg/kg (Audinot et al. 1998),
suggesting that inhibition of sumanirole-induced
hypothermia by PG01037, SB- 277011A, and U99194
would have been observed if higher, less
selective doses would have been assessed.
Unequivocal resolution of these issues will
depend on greater selectivity of ligands for
these receptors. The rank order of the in vivo
D3 selectivity ratios obtained for these
agonists and antagonists (Table 1) is in general
agreement with similar determinations reported
for in vitro binding studies. The magnitudes of
the in vivo selectivities reported herein are
much lower than those obtained by in vitro
binding studies.
However, similar differences have been
reported when in vitro binding and functional
assays are compared (Pugsley et al. 1995; Chio
et al. 1994; Sautel et al. 1995) and are
therefore not surprising. These data suggest
that while comparisons of in vitro binding
affinities provide an estimation of receptor
selectivity, the utilization of in vitro
functional assays and behavioral measures may
provide a more accurate measure of an agonist or
antagonist's selectivity as they allow for both
potency and efficacy measures to be made and may
therefore be more informative in interpreting
the in vivo pharmacology of D2-like agonists and
antagonists. To summarize, the results of these
studies provide further support for specific
roles for the D3 and D2 receptors in the
mediation of D2/D3 agonist-induced yawning
behavior and hypothermia, respectively, and
demonstrate the usefulness of yawning and
hypothermia in the characterization of in vivo
D3 and D2 receptor activity.
They are the first to provide in vivo
determinations and comparisons of D3 receptor
selectivities for a series of D2/D3 agonists
with a range of in vitro selectivities for the
D3 or D2 receptor. Thus, these data suggest that
yawning and hypothermia may provide useful
endpoints for the evaluation of in vivo
antagonist activity and selectivity of future
antagonists with improved solubility and
selectivities for the D3 or D2 receptor.
-Collins
C, Truccone A et al. Pro-erectile Effects of
Dopamine D2-like Agonists are Mediated by the D3
Receptor in Rats and Mice. J J Pharmacol Exp
Ther 2009
-Canales JJ,
Iversen SD Psychomotor activating effects
mediated by dopamine D2 and D3 receptors in the
nucleus accumbens Pharmacol Biochem Behav 2000;
67;161-168
-Canales
JJ, Iversen SD Dynamic dopamine receptor
interactions in the core and shell of nucleus
accumbens differentially coordinate the
expression of unconditioned motor behaviors
Synapse 2000; 36; 297-306
-Collins GT, JM
Witkin et al Dopamine agonist-induced
yawning in rats: a dopamine d3 receptor mediated
behavior. J Pharmacol Exp Ther
2005;314(1):310-319
-Collins GT, Newman
AH,Woods JH et al.Yawning and hypothermia in
rats: effects of dopamine D3 and D2 agonists and
antagonists. Psychopharmacology
(Berl).
2007;193(2):159-170
-Collins GT. et
al. Food restriction alters
pramipexole-induced yawning, hypothermia, and
locomotor activity in rats: Evidence for
sensitization of dopamine D2 receptor-mediated
effects. JEPT 2008;325:691-697
-Collins
GT et al. Narrowing in on compulsions:
dopamine receptor functions Exp Clin
Psychopharmacol 2008,16(4):498,502
-Chen J, Gregory T.
Collins, et al. Design, Synthesis, and
Evaluation of Potent and Selective Ligands for
the Dopamine 3 (D3) Receptor with a Novel in
Vivo Behavioral Profile. J Med. Chem.,
2008;51(19):5905&endash;5908
-Collins GT,
Truong YN, et al. Behavioral sensitization
to cocaine in rats: evidence for temporal
differences in dopamine D(3) and D (2) receptor
sensitivity. Psychopharmacology (Berl).
2011;215(4):609-20.
Li SM,
Collins GT et al. Yawning and locomotor
behavior induced by dopamine receptor agonists
in mice and rat. Behav Pharmacol. 2010; 21(3):
171&endash;181.
Distribution of
dopamine D3 receptor expressing neurons in the
human forebrain: comparison with D2 receptor
expressing neurons. Gurevich EV, Joyce
JN.
Based on studies in the rat, Sokoloff and
Schwartz (1990-1995) have made the valuable
suggestion that the D3 receptor is a
particularly important target for antipsychotics
in the mesolimbic DA system. The present study
in the human demonstrates that the distribution
of D3 receptors and D3 mRNA-bearing neurons is
consistent with relative segregation of the D3
subtype to the limbic striatum as well as it
primary and secondary targets and many sources
of its afferents.
The nucleus accumbens and adjacent ventral
putamen, referred to as the ventral or limbic
striatum, exhibit the highest expression of D3
receptor binding sites and corresponding levels
of D3 mRNA. These regions are the targets of the
mesolimbic DA system receiving input from the
medial and dorsal components of the SN.
Additional sources of input to the limbic
striatum include regions enriched in D3
receptors such as the anterior nuclei of the
thalamus and NBM. The ventral striatum projects
primarily to the VP, the most medial part of the
GPi and SNr, which are also enriched with D3
receptors and D3 mRNA positive neurons.
These regions provide input to the anterior
nuclei of the thalamus. The AV of the thalamus,
which exhibits the highest expression of D3
receptors within the thalamus, receives a
nigrothalamic GABAergic innervation from the
SNr, a region of relatively higher expression of
D3 and D2 receptors. This provides multiple
sites at which drugs such as antipsychotics
might act to regulate activity in the "limbic"
loop via interaction with the D3 receptor.
However, in contrast with the rat, in the human
brain the D3 receptor is also expressed at high
levels in the motor striatum. Other brain
regions thought to be involved in sensory
(sensory thalamic nuclei), hormonal (mmt) and
association (amygdala) functions also express
the D3 receptor, often co-expressed with the D2
receptor.
The extensive co-localization of D2 and D3
receptors in human forebrain raises a question
about the functional significance of the
simultaneous presence of pharmacologically
similar DA receptors in the same cell. As has
been proposed, the significantly higher affinity
of the D3 receptor for DA may enable it to serve
in vivo as an extrasynaptic receptor responding
to low concentrations of DA at distances remote
from the point of DA release. Conversely, the D2
receptor with its low affinity to DA is likely
to respond to high synaptic concentrations of
DA. Thus, brain areas with relatively high
densities of D3 receptors may be under tonic
regulatory influence of the brain dopaminergic
system, even if the dopaminergic innervation is
sparse. D2 and D3 receptors may elicit opposing
responses in neurons and may respond differently
to loss of DA or antipsychotic treatment.
These data provide initial insight into
details of distribution and co-expression of DA
D2 and D3 receptors in the human brain.
Intricate interplay of signaling by related but
not identical DA receptors in different brain
areas and individual neurons remains to be
elucidated. Perhaps, circuits with high D3
receptor expression and other functionally
connected systems with predominance of the D2
receptor provide different target systems for DA
antagonists in regulation of psychotic symptoms.
Determination
of in vivo dopamine D3 receptor selectivity for
agonists and antagonists utilizing induction of
yawning and hypothermia in rats.
Gregory Thomas Collins and James Henry
Woods
Dept of Pharmacology,
University of Michigan Medical School, Ann
Arbor, MI, USA
Accurate assessment of in vivo selectivity
for preferential D3 agonists and antagonists has
been slowed by the absence of behaviors
specifically mediated by the D3 receptor as well
as the relative lack of agonists and antagonists
highly selective for the D3 receptor. We have
previously demonstrated that the induction of
yawning behavior by D2/D3 agonists is mediated
by an agonist activity at the D3 receptor while
the inhibition of yawning at higher doses
results from a concomitant D2 agonist activity
(Collins et al., 2005). In addition to the
induction of yawning behavior, all D2/D3
agonists produce significant decreases in core
body temperature, an effect that has been shown
to be mediated by their D2 agonist activity
(Chaperon et al., 2003). We have compared
several dopaminergic agonists with a wide range
of in vitro D3 selectivity, including;
PD-128907, quinelorane, pramipexole, 7-OH-DPAT,
quinpirole, apomorphine and sumanirole with
respect to their ability to induce yawning
behavior and hypothermia. In addition, the D2
antagonist, L-741626, and the D3 antagonists,
U99194 and PG01037 have been used to
pharmacologically validate the specific
involvement of the D3 and D2 receptor in yawning
and hypothermia respectively. Therefore, the
induction and, at higher doses, suppression of
yawning in conjunction with hypothermia allows
the inference of an in vivo selectivity ratio
for D3 compared to D2 receptor activity. Using
this analysis, pramipexole is the most selective
of the agonists tested, while apomorphine is the
least.
