Fredholm BB, Chen JF, Masino SA, Vaugeois JMActions of adenosine at its receptors in the CNS: Insights from knockouts and drugs. Annu Rev Pharmacol Toxicol 45:385-412

Abstract

Adenosine and its receptors have been the topic of many recent reviews. These reviews provide a good summary of much of the relevant literature--including the older literature. We have, therefore, chosen to focus the present review on the insights gained from recent studies on genetically modified mice, particularly with respect to the function of adenosine receptors and their potential as therapeutic targets. The information gained from studies of drug effects is discussed in this context, and discrepancies between genetic and pharmacological results are highlighted.

Full text

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10.1146/annurev.pharmtox.45.120403.095731
Annu. Rev. Pharmacol. Toxicol. 2005. 45:385–412
doi: 10.1146/annurev.pharmtox.45.120403.095731
Copyright
c
 2005 by Annual Reviews. All rights reserved
First published online as a Review in Advance on September 27, 2004
ACTIONS OF ADENOSINE AT ITS RECEPTORS IN THE
CNS: Insights from Knockouts and Drugs
Bertil B. Fredholm,
1
Jiang-Fan Chen,
2
Susan A. Masino,
3
and Jean-Marie Vaugeois
4
1
Department of Physiology and Pharmacology, Karolinska Institutet,
S-17177 Stockholm, Sweden
2
Department of Neurology, Boston University School of Medicine,
Boston, Massachusetts 02118
3
Department of Psychology and Neuroscience Program, Trinity College,
Hartford, Connecticut 06106
4
CNRS FRE2735, IFRMP 23, Faculty of Medicine and Pharmacy, 76183 Rouen, France
KeyWords A1 receptor, A2A receptor, caffeine, ischemia, Parkinson’s disease,
pain
■ Abstract
Adenosine and its receptors have been the topic of many recent reviews
(1–26). These reviews provide a good summary of much of the relevant literature—
including the older literature. We have, therefore, chosen to focus the present review on
the insights gained from recent studies on genetically modified mice, particularly with
respect to the function of adenosine receptors and their potential as therapeutic targets.
The information gained from studies of drug effects is discussed in this context, and
discrepancies between genetic and pharmacological results are highlighted.
GENETICALLY MODIFIED MICE
Adenosine Receptor Knockouts
Mouse strains lacking the genes for three of the four adenosine receptors have been
generated (Table 1). Two groups have generated adenosine A
1
receptor knockouts
(A
1
RKO) (27, 28). A
1
RKOmice develop normally. They are fertile, but appear to
have a smaller number of offspring per litter, perhaps because sperm capacitation
is compromised in these animals (29). Their body temperature is normal, but, as
expected, the hypothermia elicited by A
1
R agonists is absent in A
1
RKOmice.
Interestingly, A
1
RKOmice had reduced survival rates as compared to A
1
R wild-
type (WT) mice (30), although the maximal life span was unaffected. The increased
mortality in midlife may be linked to disturbances in cardiovascular, hepatic, and
renal systems, where A
1
Rs are likely to play an important role in the normal
physiology.
0362-1642/05/0210-0385$14.00 385
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386 FREDHOLM ET AL.
TABLE 1 Target disruption of adenosine receptors and adenosine kinase and deaminase in mice
Target Disrupted portion of the gene Parent strains Congenic Lethality References
A
1
receptor Major portion of coding exon 2 129/OlaHsd × C57BL/6 No No (27)
+∼5kbadjacent 3

genomic seq
3

portion of coding exon 1 + intron 129/SvJ × C57BL/6 No No (28)
5

portion of coding exon 2
A
2A
receptor Entire coding exon 2 129/Sv × CD1 CD1, N12 No (31)
3

portion of coding exon 2 129/Sv × C57BL/6 129/Sv, N = 1No (32, 104)
∼1.0 kb immediate intron seq C57BL/6 N = 6No
A
3
receptor Entire coding exon 1 129 × C57BL/6 No No (34)
+ 7.5 kb immediate intron seq 129 × B6D2 No No
Adenosine kinase In frame insertion at exon 129/JEms × C57BL/6 No Die at P4 (39)
amino acid Gly169-Thr225
Adenosine In frame insertion at exon 5 129/Sv × C57BL/6 No Perinatal death, (42)
deaminase die at 3 weeks
after trophoblast
rescue
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NERVE PHENOTYPES OF ADENOSINE KNOCKOUTS
387
Two groups of scientists have generated adenosine A
2A
receptor knockout
(A
2A
RKO) mice. In the line generated by Ledent et al. (31), the mice were on a
CD1 outbred background, whereas in the two lines generated by Chen et al. (32)
the mice were bred onto C57BL/6 and Sv-129 backgrounds. All three lines of
A
2A
RKOmice were viable and bred normally. Blood pressure and heart rate were
increased, as well as platelet aggregation, in mice on a CD1 background (31), but
blood pressure and heart rate were not affected in mice with a C57BL/6 or Sv-129
background (32, 33).
Adenosine A
3
receptors have been implicated in a variety of peripheral organ
system functions, including the regulation of cellular components of the immune
system (34) and cardiovascular function (35). The A
3
RKOmouse also had signifi-
cantly lower intraocular pressure, suggesting that these receptors might be a target
for the development of drugs against glaucoma (36). However, an understanding of
the functions of A
3
receptors in the central nervous system (CNS) has been impeded
both by a lack of specific ligands and the low density of these receptors (37).
All the KO mice mentioned so far lack one adenosine receptor subtype from a
very early developmental stage. Recently, a mouse with LoxP elements flanking the
second coding exon of the A
1
gene was reported, as well as the combination with
an adenovirus expressing Cre recombinase. This opens the interesting possibility
of time- and tissue-specific inactivation of the adenosine A
1
R (38).
Metabolic Pathways
In addition to mice lacking specific adenosine receptor subtypes, there are mouse
strains in which the metabolic pathways controlling the levels of adenosine have
been genetically modified (Table 1). It is well known that adenosine levels are
regulated by adenosine kinase (phosphorylates adenosine to AMP) and adenosine
deaminase (converts adenosine to inosine).
A mouse strain with a targeted disruption of the adenosine kinase gene was
recently reported (39). These mice developed normally until birth, but they died
soon after birth. Low levels of adenine nucleotides and high levels of S-adenosyl
homocysteine are signature features of this genetic manipulation (39). It has been
shown in studies on yeast KOs that adenosine kinase plays an important role in
methyl transfer reactions (40). The fatal outcome in adenosine kinase KO mice
may be due, in particular, to the high levels of S-adenosyl homocysteine and
the consequent depression of several transmethylation reactions. For this reason,
we have to await the generation of region- and time-dependent KOs for adenosine
kinase before we can get clear information about its roles in the CNS. It is also worth
noting that adenosine may play a role in the association between cardiovascular
morbidity and hyperhomocysteinemia (41).
An adenosine deaminase KO mouse has been generated and provides a model
for increased adenosine levels (23, 42). Lack of adenosine deaminase is classically
associated with immune deficiency, but this is probably due to an accumulation of
2-deoxy-adenosine and subsequent accumulation of dATP, and not to adenosine
accumulation (43). Indeed, blockade of adenosine kinase in adenosine deaminase
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388 FREDHOLM ET AL.
KO mice, which is expected to massively increase adenosine accumulation, de-
creased thymocyte death in parallel with decreased dATP accumulation (44).
Deamination of adenosine to inosine largely, but not completely eliminates the
actions of adenosine on adenosine receptors: The A
1
and particularly the A
3
R
can also respond to inosine, although inosine is not a full agonist (45–47). Indeed,
studies on KO mice have shown that the A
3
receptor can mediate some of the effects
of inosine in the immune system, but for other effects of administered inosine only
mice lacking both the A
2A
and the A
3
receptor were unresponsive (48), suggesting
that A
2A
receptors are also involved in mediating the effects of inosine. This does
not necessarily mean that inosine acts on A
2A
receptors, however. It could mean
that inosine increases levels of adenosine, which in turn acts on A
2A
receptors. In
addition, inosine may influence energy levels and polyADP-ribosylation (49).
Use ofHeterozygotes
Attention is most often paid to the phenotype of the homozygous KO. However,
detailed examination of heterozygotes (HZ) can also be very revealing.
a) How well adjusted is the receptor level? If receptor number is directly pro-
portional to gene dosage—as is the case in A
1
R and A
2A
R HZ—this argues
against strong autoregulation of transcription. Therefore, it seems likely that
neither A
1
nor A
2A
Rlevels are regulated to a major extent by the ongoing
signaling via these receptors.
b) Heterozygotes often provide a better model for the effects likely to be seen
with antagonists because it is only rarely the case that antagonists can be
given at a dose that will inhibit all the receptors all the time. An especially
relevant aspect is that caffeine in doses commonly consumed by humans
gives plasma concentrations very close to the K
D
for caffeine at human A
1
and A
2A
Rs (3). Because responses to adenosine are shifted to the right, and
because there are only half the normal number of receptors in heterozygous
mice, it seems possible that heterozygous mice can be used as a genetic
model for caffeine use.
c) Heterozygous mice have also been used to circumvent the problems asso-
ciated with the developmental effects that can potentially confound studies
on homozygous KO mice (50). In this approach, pharmacological agents
are given to heterozygous mice at doses that are subthreshold in WT mice.
There is no biological effect in WT mice treated with a subthreshold dose of
the drug or in HZ mice treated with vehicle, but a subthreshold dose elicits
a biological effect when combined with heterozygous genetic inactivation
of the target molecule (51). This approach may be particularly useful in
examining some adenosine receptor functions where discrepancies between
the pharmacological and genetic approaches have been reported (such as the
psychostimulant effect of A
2A
RKOand A
2A
R antagonists; see below under
the section on striatum and dopamine receptors).
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NERVE PHENOTYPES OF ADENOSINE KNOCKOUTS
389
d) Heterozygotes can also be used to examine aspects of coupling, e.g., the
so-called receptor reserve in different tissues (52).
Limitations and Alternatives to Genetic Knockout Approaches
As powerful as it may be, the genetic KO approach also has its intrinsic limita-
tions that may confound the correct interpretation of the phenotypic analysis of
KO mice. The two major limitations are the confounding developmental effect
and the lack of tissue specificity. The genes for adenosine receptors or enzymes
affecting adenosine level are depleted from early development throughout life in
KO mice, resulting in a phenotype that represents a developmental effect, rather
than an immediate consequence of receptor inactivation. Reproduction of KO phe-
notypes by adenosine receptor antagonists—given acutely or long-term—would
rule out developmental confounding effects. If developmental confounding ef-
fects are strongly suspected, the development of inducible knockouts (53–55) may
be worth the effort. However, differences between acute effects of a purportedly
selective drug and phenotype in a KO could have many other causes than devel-
opmental effects. One potentially useful method to test specificity of drugs, and
the consequences of incomplete blockade, is to administer subthreshold doses of
pharmacological antagonists to heterozygotes (as described above). To address
the issue of lack of tissue specificity, a LoxP strategy has been used to create a
brain-specific depletion of A
1
Rs (38), and a similar approach can be applied to
other adenosine receptor knockouts. Finally, many studies have demonstrated the
effects of genetic backgrounds on differential phenotypic expression (56–58). An
example could be the differences in blood pressure and heart rate of A
2A
RKO
mice in CD1 versus C57BL/6 or Sv-129 backgrounds, but methodological differ-
ences might also explain the different results. Another interesting example is the
finding that the A
1
RKOmouse possesses the Ren-2 renin gene derived from the
129 strain, whereas WTs do not (59). This is related to the fact that the Ren-2 gene
is positioned relatively close to the A
1
R gene (some 850 kb apart) on chromosome
1. Thus it is imperative to employ appropriate breeding strategies to control for
potentially confounding genetic backgrounds and flanking genes (60).
PRE- AND POSTSYNAPTIC EFFECTS
As a neuromodulator, adenosine affects synaptic transmission in a number of brain
regions (see Reference 6). Thus far, studies manipulating adenosine receptors have
focused primarily on characterizing responses in brain regions where that receptor
subtype is known to be important; subtle alterations or compensations in these or
other brain regions may yet be revealed.
The A
1
R subtype tonically inhibits synaptic transmission both pre- and post-
synaptically in brain regions with a high concentration of A
1
Rs, such as the
hippocampus (see Reference 6). Heterozygote mouse brain contains half of the
number of receptors, and the EC
50
for adenosine in the hippocampus is exactly
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390 FREDHOLM ET AL.
Figure 1 Critical role of A
1
Rinmediating inhibition of excitatory neurotransmission
by adenosine and ATP (insert) in hippocampus. Note that neither adenosine nor ATP
had any clear effect in A
1
RKOmice, and that the dose-response curve is shifted to the
left in the heterozygous mice, with no change in the maximal effect. Redrawn from
data in (27, 62).
twice that of the WT, whereas E
MAX
is unaffected (see Figure 1). Despite the
loss of tonic inhibition, no compensatory responses were found in other receptor
subtypes mediating similar G protein–coupled presynaptic inhibition of synaptic
transmission (27), but a wide range of potential compensatory mechanisms remain
to be explored.
Slices from homozygous A
1
RKOshow no evidence of any remaining endoge-
nous inhibitory influence of adenosine in the Schaffer collateral pathway in the
CA1 region of the hippocampus or at the mossy fiber synapses in the CA3 re-
gion (61). Furthermore, there is no inhibition of synaptic transmission when large
concentrations of adenosine (100 µM) are applied exogenously (27). Using the
Cre-loxP system and an adeno-associated viral vector, a targeted deletion of the
A
1
Rwas induced separately in the CA1 and in the CA3 region of the hippocampus
(38). This approach holds promise for dissecting out specific pre- and postsynap-
tic actions of adenosine and its synaptic interactions with other molecules and
neurotransmitters, but so far no major results have been reported. Similar to the
constitutive KO model, there was no response to adenosine in the targeted inducible
knockout. Application of adenine nucleotides such as ATP was also ineffective in
hippocampal slices from KO mice (62) (see Figure 1). Together, these data sug-
gest that the inhibitory effects of both adenosine and adenine nucleotides in the
hippocampus are mediated ultimately through the adenosine A
1
receptor (27, 38),
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NERVE PHENOTYPES OF ADENOSINE KNOCKOUTS
391
or alternatively, that effects of the other receptors require the presence of A
1
Rs, as
suggested for A
2A
Rs (63). It will be important to determine the potency of adeno-
sine in regulating neurotransmission via A
1
Rinmice lacking the other receptors
to determine if there is such an interaction.
Dunwiddie and coworkers, using pharmacological tools (64), reported some
role of A
3
Rs in modulating the responses to A
1
stimulation. This question was
re-examined recently and no significant interactions between A
1
and A
3
Rs were
discovered using a host of different methods, including binding studies and elec-
trophysiological studies (65). Thus, if A
3
Rs do play a role it is likely to be small
and indirect.
ISCHEMIA
It is generally believed that adenosine can protect tissues against the negative conse-
quences of hypoxia or ischemia (1, 66), and that A
1
Rs play a particularly important
role. Hence, survival after a hypoxic challenge may be reduced if A
1
Rs are absent
or blocked (27). One consequence is that use of caffeine or other methylxanthines
in doses that would completely block A
1
Rs may be hazardous in hypoxic human
newborns. In keeping with the proposed role for adenosine acting at A
1
Rs, hip-
pocampal slices taken acutely from adult mice do show greater functional recovery
from both hypoxic and ischemic insults when A
1
Rs are intact (27, 67). Moreover,
acute administration of an A
1
R antagonist did enhance ischemic damage in vivo,
giving further evidence that compensatory mechanisms may be providing protec-
tion in the knockout (68).
However, the severity of ischemic damage either in vivo or in organotypic
hippocampal slice cultures is not increased in the A
1
RKOmodel (68). The lack
of any obvious difference between the WT and the KO after pathophysiological
insult where A
1
Rs are considered neuroprotective is surprising. In immature brain,
blockade of A
1
Rs in fact attenuated ischemic injury. For example, the loss of white
matter that is a typical consequence of hypoxia in the newborn actually appears
to be mediated by adenosine acting on A
1
Rs (69). Thus, blockade of adenosine
receptors—even incomplete blockade like that achieved by caffeine—reduces such
white matter loss (69). In addition, the consequences of prenatal hypoxic ischemia
in rats are reduced if the dams have been given caffeine (70).
Brain damage after focal ischemia has been reported to be attenuated in adult
A
2A
RKOmice compared with WT mice (32). On the other hand, aggravated brain
damage is observed after hypoxic ischemia in immature seven-day-old A
2A
RKO
mice (71). These results suggest that, in contrast to the situation in adult animals,
A
2A
Rs play an important protective role against hypoxic ischemic brain injury in
neonates. Interestingly, a recent study using a novel approach where A
2A
RKOis
combined with bone marrow transplantation demonstrated that selective reconsti-
tution of the A
2A
Rinbone marrow–derived cells of A
2A
RKOmice abolished the
neuroprotection against ischemic brain injury afforded by global depletion of A
2A
R
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392 FREDHOLM ET AL.
(72). Conversely, selective A
2A
R inactivation by transplantation of bone marrow
cells from A
2A
RKOmice into WT mice reduced the volume of MCAO-induced
infarct in brain. This neuroprotection did not relate to the number of infiltrating
neutrophils and macrophages, but was associated with reduced MCAO-induced
expression of IL-6, IL-1, and IL-12 in the ischemic brain after gene inactivation.
These findings reveal a critical role for A
2A
Rs on bone marrow–derived cells fol-
lowing transient focal ischemia and suggest that targeting peripheral A
2A
Rs in
bone marrow–derived cells may be therapeutic against ischemic brain injury.
The role of A
3
Rs is enigmatic. Part of the reason for this is that the drugs
used to test their importance are not very selective, especially on rodent recep-
tors (7). Indeed, some of the purportedly selective antagonists have effects in A
3
R
KO mice (36). A
3
receptors are, however, clearly implicated in ischemia in the
heart, where the knockout shows significantly improved tolerance (21, 35, 73; J.
Yang, H. Sommerschild, G. Valen & B.B. Fredholm, unpublished data). In par-
ticular, recovery after myocardial ischemia was improved (74). By contrast, in
a model of carbon monoxide–induced hypoxia, the hippocampal neuronal dam-
age was increased in A
3
RKOmice (75). The histological changes, along with
possible cognitive consequences, were also observed after administration of the
A
3
R antagonist MRS 1523 [5-propyl-2-ethyl-4-propyl-3-(ethylsulfanylcarbonyl)-
6-phenylpyridine-5-carboxylate 1 mg/kg i.p.]. These results, and the observation
that deletion of the A
3
R had a detrimental effect in a model of mild hypoxia, sug-
gest the possible use of A
3
R agonists in the treatment of ischemic, degenerative
conditions of the CNS (75).
The effects observed in these models and in in vivo models for other diseases
are summarized in Tables 2 and 3.
CAFFEINE
One reason why studies of adenosine and its receptors attract interest is that adeno-
sine receptors (A
1
,A
2A
, and A
2B
) are the targets for the most widely used of all
psychoactive drugs, caffeine. Studies on KO animals have provided compelling
evidence that the psychostimulant effects of caffeine require blockade of A
2A
Rs.
Caffeine has a mild stimulant effect in A
2A
RWTmice, but becomes a depressant of
locomotor activity in A
2A
RKOmice (31). Thus, A
2A
Rs appear to be required for the
stimulant effect of caffeine (see Figure 2). In fact, caffeine dependently decreases
locomotion in A
2A
RKOmice over a wide range of doses (76). This effect probably
results from the other biological effects of caffeine, the blockade of A
1
Rs being a
candidate. Examining immediate early gene expression in WT and A
2A
RKOmice,
the Schiffmann group also concluded that A
1
R blockade was important for some
of the high-dose effects of caffeine (77). However, the role of A
1
Rinthe effects of
caffeine on motor activity is less clear. Recently, Halldner et al. (78) showed that
the A
1
Risnot crucial for the stimulatory effect of caffeine, although the effect is
facilitated in the A
1
RKOmice. The results also suggest that the inhibitory effects
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NERVE PHENOTYPES OF ADENOSINE KNOCKOUTS
393
TABLE 2 Adenosine receptor knockouts show a variety of behavioral changes that
differ according to receptor subtype. The different phenotypes may help to define brain
functions of adenosine and to discover novel targets for drugs against neurologic and
psychiatric disorders
Receptor
Function knockout Modifications
Aggressiveness A
1
Increased
A
2A
Increased
A
3
Not determined
Anxiety A
1
Increased/no change
A
2A
Increased
A
3
No change
Despair-like A
1
Not determined
A
2A
Decreased
A
3
Increased
Memory A
1
No change
A
2A
Not determined
A
3
Not determined
Motor activity A
1
No change/decreased
A
2A
Decreased/slightly increased
A
3
Slightly increased
Neuroprotection A
1
No effect in adults, beneficial in newborns
A
2A
Beneficial in adults, detrimental in newborns
A
3
Detrimental effect
Sensorimotor gating A
1
Not determined
A
2A
Reduced startle inhibition and prepulse inhibition
A
3
Not determined
Thermal nociception A
1
Hyperalgesia
A
2A
Hypoalgesia
A
3
Hyperalgesia/no change
of higher doses of caffeine are not due to blockade of the A
1
R. Rather, this effect
is likely to be independent of adenosine receptors. Clearly, many more studies of
the actions of caffeine in single and double KOs are necessary to delineate which
effects are entirely due to adenosine receptor blockade and which are not.
SLEEP
One of the best-known effects of caffeine is its effect on sleep (3). There is also
considerable evidence that adenosine is an endogenous promoter of sleep (for
references see 79). Adenosine levels, particularly in the basal forebrain, increase
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394 FREDHOLM ET AL.
Figure 2 Biphasic effects of caffeine on locomotor behavior in mice. Data redrawn
from (78) and (76). The studies using A
1
R mice (squares) used mice on a mixed
C57BL/6 × 129OlaHsd background; those on A
2A
mice (circles) used CD1 mice.
Furthermore, the exact experimental setup differs. Hence, the two sets of data are not
strictly comparable. Filled symbol, WT mice; open symbols, KO mice.
during wakefulness and become particularly high during prolonged wakefulness
(80). Part of the reason for these changes in adenosine levels could be changes in
adenosine kinase and 5

-nucleotidase activities in the basal forebrain (81), but it
remains unclear if the basal forebrain differs from other brain regions and if there
are any changes upon sleep deprivation (82).
Most of the pharmacological data implicate A
1
Rinthe regulation of sleep
(79). Thus, A
1
R agonists induce sleep and sleep-like EEG (83, 84), whereas
antagonists reduce sleep (85). There are several mechanisms by which A
1
R stim-
ulation may induce sleep. First, there is evidence that A
1
Rs are present on the cell
bodies of long cholinergic neurons and reduce their firing rate tonically (86), pre-
sumably by increasing potassium conductances. In hypothalamic slices, adenosine
disinhibits the GABAergic input to ventrolateral preoptic neurons (87). One possi-
ble additional substrate are the orexin-containing neurons, which express A
1
R (88).
Further support for an important role of A
1
Rwas given by studies showing
that an A
1
R antisense construct infused into the basal forebrain could decrease the
amount of REM sleep and increase wakefulness (89). However, the A
1
RKOmouse
did not show any difference from controls in the amount of sleep or in rebound
after sleep deprivation, even though an A
1
R antagonist produced the expected
effect in the control animals (90). Thus, there is evidence that the A
1
Risimportant
in sleep regulation in normal animals, but also that it is not absolutely necessary
for sleep regulation. Thus, when A
1
Rs are eliminated (and presumably when they
are profoundly antagonized for long periods of time) other regulatory mechanisms
take over. The nature of these adaptive changes is not known, and it remains to be
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NERVE PHENOTYPES OF ADENOSINE KNOCKOUTS
395
shown if some level of adaptation also occurs after long-term, high-dose exposure
to caffeine.
Despite the fact that most attention has been focused on the role of A
1
receptors,
there is increasing evidence that A
2A
receptors also play a role. For example, in
fetal sheep there is evidence for a tonic role of A
2A
Rs in regulating REM sleep
state (91), and administration of A
2A
R agonists into the subarachnoid space close
to the preoptic area increases sleep (92). The possibility exists that the abundant
A
2A
Rs present in the nucleus accumbens (92) or tuberculum olfactorium (93)
play a role. Thus, there are changes in A
2A
Rs and the corresponding mRNA in
tuberculum olfactorium following sleep deprivation (93). Furthermore, the sleep-
inducing effect of the A
2A
R agonist CGS 21680 was eliminated in A
2A
RKOmice
(94). It will be of considerable interest to examine sleep in mice that lack both A
1
and A
2A
Rs and to determine whether caffeine has any effect in such mice.
STRIATUM AND INTERACTIONS WITH DOPAMINE
Although A
2A
Rs are commonly believed to couple to G
s
proteins, it is now es-
tablished that in striatum, A
2A
Rs (as well as dopamine D
1
receptors) signal via
G
olf
proteins instead (95, 96). Indeed, full activity of A
2A
and D
1
ligands requires
the presence of the normal number of G
olf
molecules, as shown by the reduced
response in mice heterozygous for G
olf
deletion (96). In agreement with this, the
same authors also found that the disruption of A
2A
or D
1
receptors led to an altered
expression of G
olf
.
There is excellent evidence that A
2A
Rs and dopamine D
2
receptors are coex-
pressed on striatopallidal neurons and that they are functionally antagonistic (97,
98). Thus, blockade of D
2
receptors would be expected to increase activity mediated
by A
2A
Rs, and vice versa. One might therefore expect adaptive changes in KOs.
Indeed, in dopamine D
2
receptor KO mice there is a functional decrease in A
2A
R
signaling (99), and A
2A
RKOmice are somewhat hypodopaminergic (100). This
might explain why A
2A
R deficiency selectively attenuates amphetamine-induced
and cocaine-induced locomotor responses (101), even though A
2A
receptor ago-
nists can also attenuate psychostimulant responses (101, 102).
Even though A
2A
and D
2
receptors are colocalized, interact at the membrane
level, and may form functional heterodimers, studies using A
2A
and D
2
KO mice
show that endogenous adenosine can act independently of D
2
receptors on A
2A
Rs
and exerts a tonic influence. This tonic A
2A
stimulation is opposed by dopamine
acting at D
2
receptors (98, 103–105).
Locomotion
Basal locomotion is marginally affected in A
1
RKOmice (30, 78). Thus, no differ-
ences in overall spontaneous motor activity were detected over a 23-h monitoring
period, but activity was reduced in some parts of the light-dark cycle.
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396 FREDHOLM ET AL.
A
2A
Rs are highly expressed in the dorsal and ventral striatum, where they
could be involved in the physiological control of motor activity, and a major role
of A
2A
R stimulation is to modulate locomotor activity. In most studies performed
so far (31, 32, 101, 106), the exploratory behavior of A
2A
RKOmice was reduced
as compared to A
2A
RWTmice. As expected, treatment with the A
2A
agonist CGS
21680 strongly reduced locomotor activity in A
2A
RWTmice and had no significant
effect on A
2A
RKOmice (31, 32). However, this reduction of locomotor activity
is not an invariable characteristic of A
2A
RKOmice because as
˚
Ad´en et al. (71)
observed there is a small increase in basal locomotor activity at four weeks of
age in A
2A
RKOmice compared with A
2A
RWTmice. Locomotor behavior was
reported to be increased in A
3
RKOmice (75).
Parkinson’s Disease
Among new therapeutic approaches for Parkinson’s disease, one possibility being
investigated is modulation of dopamine-mediated striatal functions through the
blockade of A
2A
Rs. The past ten years have witnessed significant progress in the
development and characterization of a new generation of A
2A
R antagonists for
use in Parkinson’s disease. The motor enhancement afforded by A
2A
antagonists
was well documented in early pharmacological studies (19), and activity at A
2A
Rs
reduces motor responses in both normal and dopamine-depleted animals. The
multiple benefits of A
2A
R inactivation (seen either after genetic deletion, as in
A
2A
RKOmice, or after treatment with A
2A
antagonists) advance the prospects of
A
2A
R antagonists as a novel treatment strategy for Parkinson’s disease (18, 107)
(see Table 3).
Proof of principle comes from large epidemiological studies that firmly establish
an inverse relationship between caffeine consumption and the risk of developing
Parkinson’s disease (108–110). Studies carried out in mice support these epidemi-
ological findings, providing evidence for neuroprotective effects of caffeine and
specific A
2A
R antagonists, as well as genetic deletion of the A
2A
R (111). Several
other pharmacological studies employing various A
2A
R antagonists also support
this neuroprotective effect (112, 113).
A
2A
R antagonism also provides symptomatic relief of surgical lesion or drug-
induced motor dysfunction. Catalepsy, a state where the animals remain immobile
for long periods, even if they are placed in awkward postures, can be induced by
dopamine D
1
or D
2
receptor antagonists or a muscarinic acetylcholine receptor ag-
onist. In A
2A
RKOmice, such catalepsy was reduced as compared with A
2A
RWT
mice (104, 114). These results suggest that A
2A
Rs influence not only dopamine D
1
and D
2
receptor–mediated neurotransmission but also that mediated via muscarinic
acetylcholine receptors. Interestingly, caffeine and muscarinic antagonists act in
synergy to inhibit haloperidol-induced catalepsy (115). The results on catalepsy
show that deletion of the A
2A
receptor alleviates dysfunction of basal ganglia motor
circuitry caused by drugs acting at dopamine and acetylcholine receptors. These
preclinical studies led to the clinical trial of the A
2A
R antagonist KW6002 in pa-
tients with Parkinson’s disease, and the initial results were encouraging (116, 117).
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NERVE PHENOTYPES OF ADENOSINE KNOCKOUTS
397
TABLE 3 Therapeutic implications for neurological disorders as suggested by genetic
knockout and pharmacological analysis
Disorder Animal models Effect References
Parkinson’s MPTP Reduced neurotoxicity in A
2A
RKOmice (111)
disease MPTP Reduced neurotoxicity by A
2A
R antagonists (111, 153)
6-OHDA Reduced SN neuron loss by A
2A
R antagonists (112)
Haloperidol-catalepsy Enhanced locomotor activity by A
2A
RKO (111, 114)
D
2
RKO-induced
hypolocomotion
MPTP-bradykinesia Reversed by A
2A
R antagonists (154, 155)
in monkey
Huntington’s 3-NP Reduced striatal damage in A
2A
RKO (111)
disease 3-NP Reduced striatal damage by A
2A
R antagonists (111)
3-NP Enhanced/reduced striatal damage (120)
in A
2A
RKO
3-NP Reduced striatal damage by A
1
agonists (156)
Multiple Experimental Increased demyelination and axonal (132)
sclerosis autoimmune degeneration in A
1
RKO
encephalomyelitis
(EAE)
Stroke (ischemic Hypoxia No change in organotypic hippocampus (27)
brain injury) slices in A
1
RKOmice
Hypoxic-ischemia Reduced white matter in neonate A
1
RKO (69)
Prenatal hypoxic Aggravated damage in neonate A
2A
RKO (71)
ischemia
MCAO Reduced infarct volume and neurological (32)
deficit score in A
2A
RKOmice
MCAO No effect on damage in A
1
RKO (68)
MCAO Reduced infarct volume in chimeric (72)
mice with selective depletion of
bone marrow-derived cells
Carbon monoxide Increased hippocampus neuronal (75)
damage in A
3
RKOmice
Alzheimer’s Beta-amyloid Reduced neurotoxicity in cerebellum (157)
disease aggregation neurons
Finally, recent studies with A
2A
RKOmice and pharmacological agents sug-
gest the possibility of another potentially beneficial effect of A
2A
R antagonists,
namely prevention of the development of dyskinesia after repeated treatment with
L-DOPA (118, 119). Debilitating motor complications such as dyskinesia are the
major limiting factors of management in the later stages of Parkinson’s disease.
Thus the finding that repeated administration of
L-DOPA did not lead to behavioral
sensitization in A
2A
RKOmice indicates that the A
2A
R may be required for the de-
velopment of maladaptive changes after long-term treatment with
L-DOPA (113).
This notion is further supported by recent studies in MPTP-treated nonhuman
primates, showing that coadministration of KW6002 with the dopamine agonist
apomorphine completely abolished apomorphine-induced dyskinesia (119).
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398 FREDHOLM ET AL.
Huntington’s Disease
The cellular localization of the A
2A
Rinstriatopallidal neurons suggests that the
A
2A
R may contribute to selective vulnerability to neurotoxins in Huntington’s
disease. Indeed, there is also evidence that A
2A
Rs may play a role in Huntington’s
disease (16, 120) (Table 3). In a neurochemical model of Huntington’s disease,
pharmacological and genetic inactivation of the A
2A
Rhave been shown to attenuate
striatal damage induced by the mitochondrial toxin 3-nitropropionic acid (121) or
the excitotoxin quinolinic acid (122). However, the role of the receptors is complex,
and it is difficult at present to envision A
2A
antagonism as a therapy in this disorder.
The complex actions were interpreted as resulting from a balance between negative
effects owing to blockade of presynaptic A
2A
receptors regulating glutamate release
and positive effects owing to blockade of postsynaptic receptors (120). However,
glutamate levels are also regulated by glutamate transporters on glial cells, which
express A
2A
R. Therefore, the interpretation of glutamate changes is not yet clear.
Schizophrenia
Another pathological condition involving both adenosine and dopamine in the
striatum—and where A
2A
agonists might be beneficial—is schizophrenia. Patients
with schizophrenia show impaired sensorimotor gating. Normally, this gating pre-
vents excessive irrelevant sensory stimuli from disturbing integrative mental pro-
cesses in the brain. In schizophrenic patients, the impairment in sensorimotor
gating results in reduced prepulse inhibition (PPI) and reduced startle habituation.
In experimental animals, both parameters are modulated by dopaminergic and
adenosine receptor agonists and antagonists. Wang et al. (123) recently found that
startle amplitude, startle habituation, and PPI were significantly reduced in A
2A
R
KO mice, which provides evidence that this receptor may be involved in the regu-
lation of these phenomena. In addition, responses to an NMDA antagonist and am-
phetamine were altered (123). These data suggest substances with A
2A
receptor ag-
onist properties may be of interest in the development of antipsychotic drugs (124).
ACTIONS IN OTHER PARTS OF THE NERVOUS SYSTEM
Addictive Drugs
A popular belief is that coffee can antagonize the intoxicating effects of alcohol.
However, the molecular mechanisms that might underlie this offsetting action of
coffee remain poorly identified. To investigate the possible involvement of the
A
2A
Rinthe behavioral sensitivity to high doses of ethanol, the hypnotic effect
of ethanol on A
2A
RKOmice and A
2A
RWTmice has been assessed (125). The
righting reflex was lost following acute ethanol administration, but the effect lasted
longer in A
2A
RWTmice than in A
2A
RKOmice. The fall in body temperature was
not different between the two phenotypes. Dipyridamole, an inhibitor of adenosine
uptake, increased the sleep time observed following administration of ethanol in
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399
A
2A
RWTmice but not in A
2A
RKOmice. The selective A
2A
R antagonist SCH
58261, but not the selective A
1
receptor antagonist DPCPX, shortened the duration
of the loss of righting reflex induced by ethanol, thus mimicking the lack of the
receptor in A
2A
R-deficient mice. Caffeine (25 mg/kg) also reduced ethanol-induced
hypnotic effects. These results indicate that the activation of A
2A
receptors plays
a role in the hypnotic effect of ethanol.
The cessation of chronic ethanol intake or “ethanol withdrawal” is an exper-
imental procedure recognized to produce seizures in mice. This convulsant ac-
tivity is associated with an increase in excitatory neurotransmission in the brain.
Whereas A
2A
RKOmice and controls ingested similar amounts of ethanol during
forced ethanol consumption, the severity of handling-induced convulsions during
withdrawal was significantly lower in the A
2A
RKOmice than in A
2A
RWTmice.
Because the selective A
2A
R antagonist ZM 241385 also attenuated the intensity
of withdrawal-induced seizures, it was suggested that selective A
2A
R antagonists
may be useful in the treatment of alcohol withdrawal (126). The role of A
2A
Rs
in ethanol consumption and neurobiological responses to this drug of abuse was
further characterized by Naassila et al. (127). Male and female A
2A
RKOmice con-
sumed more ethanol than WT mice. This slightly higher ethanol consumption was
also related to ethanol preference. Relative to A
2A
RWTmice, A
2A
RKOmice were
found to be less sensitive to the sedative and hypothermic effects of ethanol. No
major difference in the development of tolerance to ethanol-induced hypothermia
was found between the two phenotypes, although female A
2A
RKOmice showed
alower tolerance-acquisition rate. These results suggest that activating the A
2A
Rs
may play a role in suppressing alcohol-drinking behavior and be associated with
sensitivity to the intoxicating effects of acute ethanol administration.
There is also evidence that morphine dependence is modified by A
2A
Rs. Opiate
withdrawal was enhanced in mice lacking A
2A
receptors, and this enhancement
was abolished when both the cannabinoid CB1 receptor and A
2A
R were eliminated
(106).
Because there is considerable evidence for interactions between adenosine re-
ceptors and central stimulants (see above), for a role of adenosine in some actions
of morphine (17), for various interactions between adenosine and ethanol (128),
and because adenosine receptors are very important in regulating dopaminergic
transmission in the reward pathways in nucleus accumbens, it is important to
further examine the effects of addictive drugs in AR KO mice.
Seizures
It has long been known that adenosine can suppress repetitive neuronal firing, and a
role of adenosine as an endogenous modifier of seizures has been suspected. This
notion recently received support (129) when it was found that seizure-inducing
lesions can increase the level of adenosine kinase in astrocytes, and that this,
by reducing adenosine levels, contributes to increased seizure susceptibility. This
raises the possibility that modifying the extracellular adenosine level in brain may
be of therapeutic value against seizures. Indeed, cells that generate adenosine have
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400 FREDHOLM ET AL.
been transplanted into rat brain and this has led to decreased seizure susceptibility
(130, 131). In particular, activation of A
1
Rs appears to be an interesting target
for therapy in drug-resistant epilepsy (131). Unless there are major compensatory
mechanisms in effect, seizure thresholds would be expected to be lower in A
1
R
KO animals, but this awaits further investigation.
Multiple Sclerosis
There is some evidence that adenosine may play a role in multiple sclerosis—at
least there are effects in an experimental model (132). Thus, in A
1
RKOmice the
demyelination and axonal degeneration was much more pronounced than in WT
littermates. There was also a stronger activation of microglia/macrophages. Fur-
thermore, macrophages from A
1
RKOanimals exhibited increased expression of
the proinflammatory genes IL-1β and matrix metalloproteinase-12 on immune ac-
tivation compared to control cells from A
1
RWTanimals (132). This would imply
first that A
1
Rs are very important in regulating macrophages/microglial cells. How-
ever, this is not immediately obvious from other data where these cells have been
examined (e.g., 133). Furthermore, the role of A
1
Rs in regulating oligodendrocyte
function and survival appears to differ between the adult spinal cord (132) and the
immature brain (69). This again emphasizes that the roles of adenosine receptors
may be complex, and that they could differ with age, location, and pathology.
Memory
Despite some hints from experiments with drugs that affect adenosine receptors,
the evidence from KO animals does not reveal any clear effect of the A
1
RKO
genotype on memory (30, 134). Minor effects in the water maze were suggested
(134) to be due to the altered emotional stability reported for these mice (27,
30). Long-term potentiation (LTP), an in vivo model of memory formation, has
generally been observed to be inhibited by A
1
R activation (135) and enhanced by
A
2A
R activation (136, 137). Deletion of adenosine A
2A
Rs did not alter ongoing
synaptic transmission in either striatum (138) or nucleus accumbens (137), but
accumbens neurons showed significantly reduced LTP when the effects of the
A
2A
R were removed (137). LTP was reduced greatly in the mossy fiber pathway in
hippocampal slices from A
1
RKOmice as well as rat hippocampal slices pretreated
with an A
1
R antagonist (61), providing strong evidence that adenosine acting at
the A
1
R augments LTP in this pathway.
Anxiety
The neurobiology of anxiety, including the role of adenosine, was recently compre-
hensively reviewed (139). Interestingly, anxiety-related behavior in the classical
light/dark box test was increased in the A
1
RKOmice, as shown by a reduction
in the number of entries into as well as the total time spent in the lit compartment
compared with A
1
RWTmice (27, 30). The A
1
RKOmice also showed a decrease
in exploratory behavior in the open-field and in the hole-board, results that could
reflect an anxiogenic state in A
1
RKOmice. However, another strain of A
1
RKO
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NERVE PHENOTYPES OF ADENOSINE KNOCKOUTS
401
mice with a similar genetic background displayed a normal overall level of motor
activity, with very modest behavioral changes in the direction of increased anxiety
(134). It is likely that different environmental conditions have contributed substan-
tially to the behavioral discrepancies between the two lines. This might prompt
us to ask whether the increased sensitivity to caffeine reported in patients with
panic disorders (140) is indeed linked to a disorder of adenosine neuromodulation
at A
1
Rs in the brain.
A
2A
RKOmice showed higher rates of spontaneous anxiety-like responses
in two different anxiety-like behavioral tests, the elevated plus-maze and the
light/dark box (31, 106, 141). Thus, A
2A
RKOmice and at least one strain of
A
1
RKOmice exhibit increased anxiety, consistent with the well-known, pro-
nounced, anxiogenic effects of high doses of caffeine. High doses of caffeine will
presumably block most of these adenosine receptor subtypes, but low doses will
not. Despite several studies using pharmacological tools and performed in rodent
models (141–144) there is no clear consensus concerning the role of A
1
and A
2A
Rs
in anxiety. However, on the basis of screening tests, it has been proposed that A
1
R
agonists exert anxiolytic effects, whereas A
1
R antagonists in some cases, but not
consistently, exert anxiogenic effects. On the other hand, it is still unclear whether
the A
2A
R also plays a major role in anxiety states. Selective A
2A
R antagonists
seem to be devoid of effects in tests on rodents (141). However, recent data from
humans shed fresh light on the potential role of A
2A
Rs in the anxiogenic effects
of caffeine. In a study conducted by Alsene et al. (145), the association between
variations in anxiogenic responses to caffeine and polymorphisms in the adeno-
sine A
1
and A
2A
R genes has been examined. They found a significant association
between self-reported anxiety after oral administration of 150 mg of caffeine and
two linked polymorphisms on the A
2A
R gene. Individuals with the 1976T/T and
the 2592Tins/Tins genotypes reported greater increases in anxiety after caffeine
administration than the other genotypic groups. Moreover, in patients with panic
disorder, a psychiatric condition characterized by recurrent panic attacks and an-
ticipatory anxiety, a single-nucleotide polymorphism haplotype in the A
2A
R gene
was found to be associated with the disease (146). Alpha-melanocyte-stimulating
hormone (alpha-MSH) influences anxiety, aggressiveness, and motor activity, all
of which are also influenced by A
2A
R gene disruption. In A
2A
RKOmice, signif-
icantly increased alpha-MSH content was observed in the amygdala and cerebral
cortex. Plasma corticosterone concentration was significantly higher in A
2A
RKO
mice, revealing hyperactivity of their pituitary-adrenocortical axis. Results suggest
that A
2A
Rs are involved in the control of POMC gene expression and biosynthesis
of POMC-derived peptides in pituitary melanotrophs and corticotrophs (147).
Aggression
Several studies have suggested that adenosine receptors are involved in the modu-
lation of aggressive behavior. In agreement with the decrease of offensive behavior
induced by a selective stimulation of A
1
Rs (148), A
1
RKOmice isolated for the
resident-intruder aggression test showed enhanced aggressive behavior (27). A
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402 FREDHOLM ET AL.
similar enhancement was also observed in isolated male A
2A
RKOmice in the
resident-intruder test (31). The increased aggressiveness observed in both A
1
R
KO mice and A
2A
RKOmice is in agreement with the increase of offensive be-
havior induced by selective blockade of either A
1
or A
2A
Rs (M. El Yacoubi &
J.M.Vaugeois, unpublished observations). These results suggest that both adeno-
sine receptor subtypes are involved in the effect of adenosine on aggressiveness.
The link between these effects and the increase in nervousness and irritability re-
ported in humans (3) after chronic administration of high doses of caffeine remains
a matter of speculation.
Depression
In behavioral procedures used to screen potential antidepressants, such as tail
suspension and forced swim tests, A
2A
RKOmice were found to be less sensitive
to “depressant” challenges than their WT littermates, which were less immobile
than A
2A
RWTmice in both tests (149). Consistently, A
2A
R blockers reduced
the immobility times in tail suspension and forced swim tests. Taken together,
the results support the hypothesis that blockade of the A
2A
R might be an interesting
target for the development of effective antidepressant agents. Although their mode
of action in potentially alleviating mood disorders is unknown, modulation of
dopamine transmission might play a role (149). Future clinical trials with selective
A
2A
R antagonists as potential therapeutic agents for major depressive episodes will
help to delineate the role of adenosine in the pathophysiology of mood disorders.
Whereas A
2A
R antagonists have been proposed as antidepressants (149), A
3
RKO
mice showed an increase in the amount of time spent immobile in the two tests of
behavioral depression, the forced swim test and the tail suspension test (75).
Pain
The role of adenosine as an endogenous analgesic substance has also been evaluated
(27). A
1
Rs are abundant in mouse spinal cord, with the highest levels in the outer
lamina of the dorsal horns, where the density of receptors was close to that observed
in the hippocampus. A
1
Rs are responsible for the analgesic effects of intrathecally
administered A
1
agonists. A
1
RKOmice react faster to thermal pain than A
1
R
WT mice. However, this increase is not matched by an increased sensitivity to
mechanical stimulation. The authors suggested that endogenous adenosine acting
at A
1
Rs decreases nociception, mediated via C fibers. These results also suggest
that the A
1
R may be a target for the development of antinociceptive drugs.
The response of A
2A
RKOmice to acute pain stimuli is slower in the hot
plate and tail-flick tests compared to A
2A
RWTmice (31). Similar reduced pain
responses were also found when a tail-immersion test was used (106). This higher
nociceptive threshold suggests that the peripheral lack of A
2A
Rs predominates over
the spinal defect. Thus, depending on the site of action and the receptor activated
(A
1
or A
2A
), adenosine may exert very different effects on pain. This variety of
effects may explain why caffeine has analgesic effects against some, but not all,
types of pain (3).
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403
A
3
RKOmice show decreased sensitivity to some painful stimuli, as evidenced
by the increase in latency in the hot plate but not tail-flick test (75). Another study
(150) found no evidence for a role of A
3
Rinnociception or in the antinociceptive
effect of the adenosine analog R-phenylisopropyl adenosine (R-PIA). Thus, no
difference was seen between A
3
RKOand A
3
RWTmice in nociceptive response
to mechanical or radiant heat stimuli. The antinociceptive response to intrathecal
R-PIA was also unchanged in the A
3
RKOmice. In contrast, heat hyperalgesia,
plasma extravasation, and edema following carrageenan-induced inflammation
in the hind paw were significantly reduced in A
3
RKOmice compared to the
A
3
RWTcontrols. Thus, mice lacking A
3
R had deficits in generating the localized
inflammatory response to carrageenan, supporting a proinflammatory role of A
3
Rs
in peripheral tissues.
CONCLUSIONS
Whereas deletion of genes for enzymes critically involved in adenosine metabolism
leads to lethal phenotypes, deletion of A
1
,A
2A
, and A
3
receptors has rather subtle
effects and the mice are remarkably normal. This agrees well with the conclusion
drawn before, i.e., that adenosine receptors are involved in modulating physiolog-
ical responses and that they are particularly important under pathophysiological
conditions. Thus, to determine the roles of the adenosine receptors, the genetically
modified mice must be subjected to various types of challenges.
The results obtained so far have both confirmed previous data and yielded some
surprises. The important role of the A
1
Rinmodulating excitatory transmission and
its role in pain transmission was expected, as was the critically important role of
A
2A
Rs in striatal function. Among the major surprises were the noncritical role of
A
1
Rs in brain ischemia and in sleep and the finding that A
2A
Rs mediate aggravated
brain damage mainly via peripheral receptors. Our examination of the literature has
also indicated studies that can and should be performed to further define the roles
of adenosine receptors in the nervous system. Because the goal of these studies is to
examine the possibility for novel drug therapies, the use of KO mice to determine
that the drugs are indeed selective is very important. Indeed, data obtained already
suggest that some of the drugs used to delineate adenosine receptor effects are not
as selective as previously hoped (36, 151, 152).
The Annual Review of Pharmacology and Toxicology is online at
http://pharmtox.annualreviews.org
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December 10, 2004 15:28 Annual Reviews AR232-FM
Annual Review of Pharmacology and Toxicology
Volume 45, 2005
CONTENTS
FRONTISPIECE—Minor J. Coon xii
CYTOCHROME P450: NATURE’S MOST VERSATILE BIOLOGICAL
CATALYST, Minor J. Coon 1
CYTOCHROME P450 ACTIVATION OF ARYLAMINES AND
HETEROCYCLIC AMINES, Donghak Kim and F. Peter Guengerich 27
GLUTATHIONE TRANSFERASES, John D. Hayes, Jack U. Flanagan,
and Ian R. Jowsey 51
PLEIOTROPIC EFFECTS OF STATINS, James K. Liao and Ulrich Laufs 89
FAT CELLS:AFFERENT AND EFFERENT MESSAGES DEFINE NEW
APPROACHES TO TREAT OBESITY, Max Lafontan 119
FORMATION AND TOXICITY OF ANESTHETIC DEGRADATION
PRODUCTS, M.W. Anders 147
THE ROLE OF METABOLIC ACTIVATION IN DRUG-INDUCED
HEPATOTOXICITY, B. Kevin Park, Neil R. Kitteringham, James L. Maggs,
Munir Pirmohamed, and Dominic P. Williams 177
NATURAL HEALTH PRODUCTS AND DRUG DISPOSITION, Brian C. Foster,
J. Thor Arnason, and Colin J. Briggs 203
BIOMARKERS IN PSYCHOTROPIC DRUG DEVELOPMENT:INTEGRATION
OF
DATA ACROSS MULTIPLE DOMAINS, Peter R. Bieck
and William Z. Potter 227
NEONICOTINOID INSECTICIDE TOXICOLOGY:MECHANISMS OF
SELECTIVE ACTION, Motohiro Tomizawa and John E. Casida 247
GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE,APOPTOSIS,
AND NEURODEGENERATIVE DISEASES, De-Maw Chuang,
Christopher Hough, and Vladimir V. Senatorov 269
NON-MICHAELIS-MENTEN KINETICS IN CYTOCHROME
P450-CATALYZED REACTIONS, William M. Atkins 291
EPOXIDE HYDROLASES:MECHANISMS,INHIBITOR DESIGNS,
AND BIOLOGICAL ROLES, Christophe Morisseau
and Bruce D. Hammock 311
v
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December 10, 2004 15:28 Annual Reviews AR232-FM
vi CONTENTS
NITROXYL (HNO): CHEMISTRY,BIOCHEMISTRY, AND
PHARMACOLOGY, JonM.Fukuto, Christopher H. Switzer,
Katrina M. Miranda, and David A. Wink 335
TYROSINE KINASE INHIBITORS AND THE DAWN OF MOLECULAR
CANCER THERAPEUTICS, Raoul Tibes, Jonathan Trent,
and Razelle Kurzrock 357
ACTIONS OF ADENOSINE AT ITS RECEPTORS IN THE CNS: INSIGHTS
FROM
KNOCKOUTS AND DRUGS, Bertil B. Fredholm, Jiang-Fan Chen,
Susan A. Masino, and Jean-Marie Vaugeois 385
R
EGULATION AND INHIBITION OF ARACHIDONIC ACID
(OMEGA)-HYDROXYLASES AND 20-HETE FORMATION,
Deanna L. Kroetz and Fengyun Xu 413
CYTOCHROME P450 UBIQUITINATION:BRANDING FOR THE
PROTEOLYTIC SLAUGHTER? Maria Almira Correia, Sheila Sadeghi,
and Eduardo Mundo-Paredes 439
PROTEASOME
INHIBITION IN MULTIPLE MYELOMA:THERAPEUTIC
IMPLICATION, Dharminder Chauhan, Teru Hideshima,
and Kenneth C. Anderson 465
CLINICAL AND TOXICOLOGICAL RELEVANCE OF CYP2C9:
D
RUG-DRUG INTERACTIONS AND PHARMACOGENETICS,
Allan E. Rettie and Jeffrey P. Jones 477
CLINICAL DEVELOPMENT OF HISTONE DEACETYLASE INHIBITORS,
Daryl C. Drummond, Charles O. Noble, Dmitri B. Kirpotin, Zexiong Guo,
Gary K. Scott, and Christopher C. Benz 495
THE MAGIC BULLETS AND TUBERCULOSIS DRUG TARGETS,
Ying Zhang 529
MOLECULAR MECHANISMS OF RESISTANCE IN ANTIMALARIAL
CHEMOTHERAPY:THE UNMET CHALLENGE, Ravit Arav-Boger
and Theresa A. Shapiro 565
SIGNALING NETWORKS IN LIVING CELLS, Michael A. White
and Richard G.W. Anderson 587
HEPATIC FIBROSIS:MOLECULAR MECHANISMS AND DRUG TARGETS,
Sophie Lotersztajn, Boris Julien, Fatima Teixeira-Clerc, Pascale Grenard,
and Ariane Mallat 605
ABERRANT DNA METHYLATION AS A CANCER-INDUCING
MECHANISM, Manel Esteller 629
THE CARDIAC FIBROBLAST:THERAPEUTIC TARGET IN MYOCARDIAL
REMODELING AND FAILURE, R. Dale Brown, S. Kelley Ambler,
M. Darren Mitchell, and Carlin S. Long 657
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December 10, 2004 15:28 Annual Reviews AR232-FM
CONTENTS vii
EVALUATION OF DRUG-DRUG INTERACTION IN THE HEPATOBILIARY
AND
RENAL TRANSPORT OF DRUGS, Yoshihisa Shitara, Hitoshi Sato,
and Yuichi Sugiyama 689
DUAL SPECIFICITY PROTEIN PHOSPHATASES:THERAPEUTIC TARGETS
FOR
CANCER AND ALZHEIMER’S DISEASE, Alexander P. Ducruet,
Andreas Vogt, Peter Wipf, and John S. Lazo 725
INDEXES
Subject Index 751
Cumulative Index of Contributing Authors, Volumes 41–45 773
Cumulative Index of Chapter Titles, Volumes 41–45 776
ERRATA
An online log of corrections to Annual Review of Pharmacology and
Toxicology chapters may be found at
http://pharmtox.annualreviews.org/errata.shtml
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