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Adenosine receptors and behavioral actions of methylxanthines. Proc. Natl. Acad. Sci. USA 78, 3260-3264

Authors:

Abstract

Central stimulant actions of 10 methylxanthines in mice correlate with affinities for adenosine receptors labeled with N6-[3H]cyclohexyladenosine. Affinities of methylxanthines for adenosine receptors are consonant with central levels attained at behaviorally effective doses. The much higher concentrations of methylxanthines required to influence benzodiazepine receptor binding do not correlate with behavioral potency. N6-(L-Phenylisopropyl)adenosine (L-PIA), a metabolically stable analog of adenosine with high affinity for adenosine receptors, is an extremely potent behavioral depressant, reducing locomotor activity of mice at doses as little as 0.05 mumol/kg. The D isomer, which has much less affinity for adenosine receptors, is much less active as a central depressant. Theophylline stimulates locomotor activity and reverses depressant effects of L-PIA. Caffeine or 1,7-dimethylxanthine, when administered alone, elicits biphasic effects, with locomotor depression at lower doses and stimulation at higher doses. When administered with L-PIA, even low doses of caffeine produce marked stimulation. 3-Isobutyl-1-methylxanthine given alone elicits only behavioral depression. However, like theophylline and caffeine, isobutylmethylxanthine reverses the L-PIA-evoked depression, converting it into pronounced locomotor stimulation. The data strongly suggest that the behavioral stimulant effects of methylxanthines involve a blockade of central adenosine receptors.
Proc.
Nati.
Acad.
Sci.
USA
Vol.
78,
No.
5,
pp.
3260-3264,
May
1981
Neurobiology
Adenosine
receptors
and
behavioral
actions
of
methylxanthines
[caffeine/theophylline/N6-cyclohexyladenosine/N6-(phenylisopropyl)adenosine]
SOLOMON
H.
SNYDER*,
JEFFERSON
J.
KATIMS*t,
ZOLTAN
ANNAUt,
ROBERT
F.
BRUNS*t,
AND
JOHN
W.
DALYt
*Departments
of
Neuroscience,
Pharmacology
and
Experimental
Therapeutics,
Psychiatry
and
Behavioral
Sciences,
Johns
Hopkins
School
of
Medicine;
tDepartment
of
Environmental
Health
Sciences,
Johns
Hopkins
School
of
Hygiene,
Baltimore,
Maryland
21205;
and
tLaboratory
of
Bioorganic
Chemistry,
National
Institute
of
Arthritis,
Metabolism
and
Digestive.
Diseases,
National
Institutes
of
Health,
Bethesda,
Maryland
20205
Contributed
by
Solomon
H.
Snyder,
February
6,
1981
ABSTRACT
Central
stimulant
actions
of
10
methylxanthines
in
mice
correlate
with
affinities
for
adenosine
receptors
labeled
with
N6-[3H]cyclohexyladenosine.
Affinities
of
methylxanthines
for
adenosine
receptors
are
consonant
with
central
levels
attained
at
behaviorally
effective
doses.
The
much
higher
concentrations
of
methylxanthines
required
to
influence
benzodiazepine
receptor
binding
do
not
correlate
with
behavioral
potency.
N6-(L-Phenyl-
isopropyl)adenosine
(L-PIA),
a
metabolically
stable
analog
of
aden-
osine
with
high
affinity
for
adenosine
receptors,
is
an
extremely
potent
behavioral
depressant,
reducing
locomotor
activity
of
mice
at
doses
as
little
as
0.05
,.mol/kg.
The
D
isomer,
which
has
much
less
affinity
for
adenosine
receptors,
is
much
less
active
as
a
central
depressant.
Theophylline
stimulates
locomotor
activity
and
re-
verses
depressant
effects
of
L-PIA.
Caffeine
or
1,7-dimethylxan-
thine,
when
administered
alone,
elicits
biphasic
effects,
with
lo-
comotor
depression
at
lower
doses
and
stimulation
at
higher
doses.
When
administered
with
L-PIA,
even
low
doses
of
caffeine
pro-
duce
marked
stimulation.
3-Isobutyl-1-methylxanthine
given
alone
elicits
only
behavioral
depression.
However,
like
theophylline
and
caffeine,
isobutylmethylxanthine
reverses
the
L-PIA-evoked
depression,
converting
it
into
pronounced
locomotor
stimulation.
The
data
strongly
suggest
that
the
behavioral
stimulant
effects
of
methylxanthines
involve
a
blockade
of
central
adenosine
receptors.
Although
methylxanthines
such
as
caffeine
and
theophylline
are
among
the
most
widely
used
behavioral
stimulant
substances,
molecular
mechanisms
for
their
stimulant
effects
are
unclear.
Methylxanthines
can
inhibit
phosphodiesterase,
and
thus
pre-
vent
inactivation
of
cyclic
AMP
(1),
but
the
concentrations
of
caffeine
and
theophylline
required
to
inhibit
phosphodiester-
ases
are
substantially
greater
than
those
which
occur
in
brain
at
behaviorally
effective
doses
(2,
3).
Moreover,
several
potent
phosphodiesterase
inhibitors
lack
behavioral
stimulant
actions
and
indeed
are
central
depressants
(4).
Adenosine
receptor
ac-
tivity
is
blocked
by
methylxanthines
in
concentrations
similar
to
those
that
occur
after
stimulant
doses
(5,
6).
Because
the
gen-
eral
neurophysiologic
actions
of
adenosine
are
inhibitory
(7),
it
is
conceivable
that
methylxanthines
exert
stimulant
actions
by
blocking
adenosine
effects.
In
several
attempts
to
measure
binding
of
adenosine-related
ligands
to
membranes,
binding
sites
largely
lacked
the
speci-
ficity
of
physiologic
adenosine
receptors
(8-12).
Recently,
we
(13,
14)
and
others
(15,
16)
have
demonstrated
binding
of
3H-
labeled
ligands
to
adenosine
receptors
in
brain
and
testes
(15,
17).
In
the
present
study
we
show
a
correlation
between
potencies
of
a
series
of
methylxanthines
in
stimulating
locomotor
activity
of
mice
and
in
competing
at
adenosine
receptors
labeled
with
N6-[3H]cyclohexyladenosine
([3H]CHA).
Both
CHA
and
N6-(L-
phenylisopropyl)adenosine
(L-PIA),
stable
and
potent
adeno-
sine
analogs,
are
shown
to
be
extremely
potent
behavioral
de-
pressants.
Low
doses
of
xanthines
dramatically
reverse
the
L-
PIA-evoked
depression.
MATERIALS
AND
METHODS
Biochemical.
[3H]CHA
binding
to
whole
mouse
brain
mem-
branes
was
assayed
as
reported
(13).
Properties
of
[3H]CHA
binding
to
mouse
brain
membrane
were
essentially
the
same
as
for
guinea
pig
brain
(13).
[3H]Flunitrazepam
binding
to
mouse
brain
membranes
was
assayed
as
before
(18).
[3H]CHA
(14
Ci/mmol;
1
Ci
=
3.7
x
101
becquerels)
and
[3H]flunitrazepam
(79
Ci/mmol)
were
obtained
from
New
En-
gland
Nuclear.
The
sources
of
xanthines
were
as
described
(13).
Behavioral.
Naive
adult
male
ICR
mice
(2540
g)
from
Blue
Spruce
Farms
(Altamont,
NY)
were
given
food
and
water
ad
lib.
The
mice
were
housed
20
per
cage
in
26
X
46
X
17
cm
poly-
propylene
cages
and
were
exposed
to
a
12-hr/12-hr
light/dark
cycle
(lights
on,
0700).
Mice
were
permitted
to
adapt
to
their
housing
for
a
minimum
of
48
hr
before
testing.
Behavioral
tests
were
performed
between
0800
and
1800.
For
each
shipment
of
mice,
received
every
4
weeks,
new
control
groups
were
es-
tablished.
Unless
stated
otherwise,
all
drugs
were
administered
as
a
saline
solution
given
intraperitoneally
at
10
t.d/g
of
body
weight.
Mice
received
drugs
10
min
prior
to
the
1-hr
locomotor
ac-
tivity
testing
period
and
were
placed
individually
in
holding
cages
containing
a
sawdust
bedding
similar
to that
of
their
group
cages.
Locomotor
activity
data
were
subjected
to
parametric
statis-
tical
analysis
by
using
repeated
measures
three-way
analysis
of
variance
and
covariance
with
the
least-squares
computation
for
unequal
numbers.
Two
independent
subjects
grouping
factors
consisted
of
the
drugs
and
the
various
doses
in
which
they
were
administered.
The
repeated
measures
of
spontaneous
locomo-
tor
activity
during
a
testing
session
were
considered
the
within-
subject
dependent
repeated
measure.
This
analysis
of
variance
was
repeated
with
a
logarithmic
transformation
of
the
data.
Stu-
dent's
t
test
was
used
for
making
individual
comparisons.
Locomotor
activity
was
measured
in
four
identical
automated
38
X
38
X
38
cm
open-field
devices
built,
in
our
laboratories,
of
black
Plexiglas.
The
ceiling
of
white
Plexiglis
concealed
a
6-
W
fluorescent
light
fixture
to
provide
background
illumination
and
an
exhaust
fan
for
ventilation.
Sixty-four
cadmium
sulfide
photosensitive
devices
were
placed
under
the
transparent
Plex-
iglas
floor
3.8
cm
apart
in
an
8
x
8
array
and
connected
to
an
Intel
microcomputer
that
monitored
the
state
and
location
of
the
photosensitive
elements
10
times
per
sec.
The
printout
(Tele-
type
43)
showed
the
accumulated
time
on
each
of the
64
pho-
tocells
for
the
predetermined
time
period,
the
number
of
pho-
Abbreviations:
IBMX,
3-isobutyl-1-methylxanthine;
PIA,
N6-(pheny-
lisopropyl)adenosine;
CHA,
N6-cyclohexyladenosine.
The
publication
costs
of
this
article
were
defrayed
in
part
by
page
charge
payment.
This
article
must
therefore
be
hereby
marked
"advertise-
ment"
in
accordance
with
18
U.
S.
C.
§1734
solely
to
indicate
this
fact.
)rr
ct
8
m,
Pr
t0Y~o+|
At
Sct
Ofi0
41.
'tt5
r113260
Proc.
Natl.
Acad.
Sci.
USA
78
(1981)
3261
tocells
covered
during
the
interval,
and
cell
changes
from
the
active
to
the
inactive
state.
RESULTS
Effects
of
Methylxanthines
on
Locomotor
Stimulation
and
on
Adenosine
and
Benzodiazepine
Receptor
Binding.
Meth-
ylxanthines
increase
locomotor
activity
of
rodents
(19)
but
much
less
consistently
than
do
amphetamines.
Because
preliminary
open-field
studies
failed
to
show
consistent
locomotor
stimu-
lation
with
caffeine,
we
utilized
a
photoelectric
activity
meter
with
64
sensors.
Drugs
such
as
caffeine
and
theophylline
en-
hanced
locomotor
activity
up
to
4-fold
compared
to
saline
con-
trols
(Fig.
1;
Table
1).
Caffeine
and
1,7-dimethylxanthine
re-
duced
locomotor
activity at
lower
doses
(5
and
10
,umol/kg)
but
stimulated.
activity
at
30
and
100
,umol/kg.
By
contrast,
no
lo-
comotor
depression
occurred
with
any
dose
of
theophylline,
7-
(f3-chloroethyl)theophylline,
or
7-(,f3hydroxyethyl)theophylline.
Isocaffeine
and
3-isobutyl-1-methylxanthine
(IBMX)
moder-
ately
depressed
activity
at
all
doses.
Theobromine,
8-chloro-
theophylline,
and
1,9-dimethylxanthine
had
negligible
influ-
ence
on
locomotor
activity
at
all
doses.
Brain
levels
of
the
alkylxanthines
were
assessed
30
min
after
a
100-,Amol/kg
dose
(Table
1).
Caffeine
and
theophylline
levels
were
about
60
AtM.
Theobromine,
1,7-dimethylxanthine,
7-(p-
hydroxyethyl)theophylline,
and
7-(f3-chloroethyl)theophylline
had
levels
of
about
20-30
AM;
IBMX,
8-chlorotheophylline
and
isocaffeine
levels
were
10-15
A.M.
In
general,
stimulant
potencies
of
the
methylxanthines
cor-
related
with
potencies
in
competing
for
the
adenosine
receptor
labeled
with
[3H]CHA.
The
three
methylxanthines
that
were
most
potent
at
[3H]CHA
sites
also
were
the
most
potent
loco-
motor
stimulants.
The
four
xanthines
that
were
weakest
in
com-
peting
for
[3H]CHA
binding
also
were
weakest
in
eliciting
lo-
comotor
stimulation.
Three
of
these-8-chlorotheophylline,
1,9-dimethylxanthine,
and
isocaffeine-did
not
penetrate
well
into
brain.
However,
bioavailability
cannot
account
for
the
dif-
ferences
in
behavioral
effects.
Thus,
7-(,8-chloro-
ethyl)theophylline,
which
was
most
potent
behaviorally,
had
one
of
the
lowest
brain
levels.
Theobromine,
which
was
be-
._
Q
.
s.
o
o
u
0
o
cQ
~
haviorally
inactive,
had
brain
levels
as
high
or
higher
than
1,7-
dimethylxanthine
and
7-(,8-chloroethyl)theophylline,
which
were
behaviorally
active.
It
has
been
suggested
that
stimulant
effects
of
methylxan-
thines
might
be
attributable
to
blockade
of
benzodiazepine
re-
ceptors
(21).
However,
the
behaviorally
potent
methylxan-
thines
are
about
100
times
more
potent
at
adenosine
than
benzodiazepine
receptors
and
no
correlation
exists
between
behavioral
potencies
and
effects
at
benzodiazepine
receptors.
Influences,
of
L-PIA
on
Mouse
Locomotor
Activity
and
In-
teractions
with
Methylxanthines.
Although
potencies
of
xan-
thines
as
stimulants
largely
correlate
with
their
potencies
in
competing
for
adenosine
receptors,
there
is
one
notable
excep-
tion.
IBMX
was
as
potent
as
caffeine
at
adenosine
receptors
yet
failed
to
stimulate
activity
and,
in
fact,
elicited
locomotor
depression.
Unlike
the
other
xanthines,
IBMX
is
a
potent
phos-
phodiesterase
inhibitor
(22),
and
phosphodiesterase
inhibitors
are
usually
central
depressants
(4).
Another
difficulty
in
ana-
lyzing
behavioral
effects
of
xanthines
is
the
biphasic
action
of
agents
such
as
caffeine
and
1,7-dimethylxanthine,
which
re-
duced
and
stimulated
behavioral
activity
at
low
and
high
doses,
respectively.
Theophylline
failed
to
display
behavioral
depres-
sion
at
any
dose
examined.
Differential
stimulant
and
depres-
sant
potencies
of
various
methylxanthines
might
obscure
their
intrinsic
stimulant
potencies.
To
evaluate
behavioral
actions
of
the
methylxanthines
on
sys-
tems
specifically
regulated
by
adenosine,
we
explored
effects
of
PIA.
In
an
earlier
study,
PIA
elicited
behavioral
depression
in
rats
(23).
We
examined
in
detail
influences
of
both
L
and
D-
PIA
(Fig.
2).
CHA
and
L-PIA
both
were
very
potent
in
eliciting
locomotor
depression.
The
fact
that
L-PIA
is
more
potent
than
D-PIA
suggests
that
these
effects
involve
adenosine
Al-recep-
tors,
which
display
marked
stereospecificity
for
isomers
of
PIA,
rather
than
A2-receptors,
at
which
L-
and
D-PIA
have
nearly
equal
potencies
(13,
24).
At
doses
as
little
as
0.1
,umol/kg,
CHA
or
L-PIA
markedly
reduced
locomotor
activity
of
mice,
and
sig-
nificant
depression
was
detected
at
0.05
,umol/kg.
These
doses,
around
20
/ig/kg,
indicate
that
N6-substituted
adenosines
rank
o0
o0
00
o
-O
O
50F
O
A
B
C
D
E
F
G
H
J
Drug
and
dose,
pAmol/kg
FIG.
1.
Effect
of
alkylxanthines
on
locomotor
activity
of
mice.
Locomotor
activity
values
for
groups
of
10-20
mice
at
each
dose
are
for
the
second
30
min
after
intraperitoneal
injections
of
the
indicated
doses
except
for
7-(3-chloroethyl)theophylline
for
which
the
activity
values
represent
the
first
30-min
period.
Values
represent
the
locomotor
activity
as
percentage
of
the
activity
of
saline-injected
control
mice
and
are
presented
as
the
antilogarithm
of
a
logarithmic
transformation
of
this
data.
An
overall
analysis
of
variance
revealed
a
significant
drug
group
effect
(F
=
9.33;
df
=
6251;
P
<
0.001),
a
significant
drug
group
x
dose
interaction
(F
=
2.78,
df
=
18,251;
P
<
0.001),
and
a
significant
drug
group
X
time
interaction
(F
=
5.71,
df
=
90,125;
P
<
0.001).
Similar
levels
of
significance
were
obtained
for
a
logarithmic
transformation
of
this
data.
*,
Significantly
different
from
saline,
P
<
0.005
by
Student's
t
test.
A,
7-(,3chloroethyl)theophylline;
B,
theophylline;
C,
caffeine;
D,
1,7-dimethylxanthine;
E,
7-(,-hydroxy-
ethyl)
theophylline;
F,
theobromine;
G,
8-chlorotheophylline,
H,1,9-dimethylxanthine;
I,
isocaffeine;
J,
3-isobutyl-1-methylxanthine.
Neurobiology:
Snyder
et
al.
3262
Neurobiology:
Snyder
et
al.
Table
1.
Xanthines:
Behavioral
stimulant
potencies
and
effects
on
adenosine
and
benzodiazepine
receptor
binding
and
brain
levels
Receptor
binding
IC&D,
IiM
Locomotor
stimulation
Brain
concentration,
Xanthine
[3H]Flunitrazepam
[3H]CHA
threshold,
pimol/kg
IuM
1.
7(3-Chloroethyl)theophylline
900
10
5
19
±
2
(3)
2.
Theophylline
2000
23
10
58
±
12
(5)
3.
1,7-Dimethylxanthine
2000
30 30 26
±
8
(5)
4.
3-Isobutyl-l-methylxanthine
(IBMX)
"1000
50
>100
15
±
3
(3)
5.
Caffeine
800
50
30
63
±
13
(5)
6.
7(3-Hydroxyethyl)theophylline
2000
100
100
32
±
2
(2)
7.
Theobromine
>2000
150
>100
29
±
3
(3)
8.
8-Chlorotheophylline
>2000
500
>250
13
±
6
(3)
9.
1,9-Dimethylxanthine
>2000
>1000
>250
10.
Isocaffeine
1000
>1000
>250
11
±
5
(3)
Binding
of
[3H]CHA
(1.0
nM)
or
[3H]flunitrazepam
(0.2
nM)
was
assayed
in
triplicate
with
six
concentrations
of
xanthines.
Data
are
means
of
three
determinations
of
IC50
values
(concentration
to
inhibit
specific
binding
by
50%)
which
varied
less
than
20%.
Locomotor
stimulation
threshold
represents
the
minimal
dose
to
augment
monitored
locomotor
activity
significantly
(tested
by
statistical
analyses).
For
each
methylxanthine,
five
or
six
doses
from
2.5
to
250
,umol/kg
were
evaluated
with
10-20
mice
at
each
dose.
At
250
tumol
of
IBMX
per
kg,
most
mice
died.
To
measure
meth-
ylxanthine
brain
levels,
mice
were
given
100-tumol/kg
doses
and
were
killed
30
min
later.
Brains
were
homogenized
with
5
vol
of
0.01
M
HCl
and
extracted
three
times
with
10
vol
of
chloroform.
The
combined
chloroform
extracts
were
dried
over
anhydrous
sodium
sulfate
and
evaporated
to
dryness.
The
residue
was
dissolved
in
1
vol
of
solvent
for
high-pressure
liquid
chromatography
adapted
from
the
method
of
Blanchard
et
al.
(20).
A
LiChrosorb
18
(4.6
x
250
mm)
reversed-phase
column
(Altex
Scientific,
Berkeley,
CA)
was
used
with
0.01
M
acetate
buffer,
pH
4/acetonitrile,
90:10
(vol/vol),
as
solvent
for
xanthines
3,
7,
and
10,
an
85:15
mixture
for
xanthines
2,
5, 6,
8,
and
9,
and
a
70:30
mixture
for
xanthines
1
and
4.
A
flow
rate
of
1
ml/min
gave
the
following
retention
times
(min)
for
the
10
xanthines:
1,
6.4;
2,
5.5;
3,
11.2;
4,
7.6;
5,
9.0;
6,
13.0;
7,
7.1;
8,
10.2;
9,
4.5;
10,
7.6.
The
injection
volume
was
20
,ul.
The
ultraviolet
detector
was
set
at
273
nm,
and
integrated
peak
heights
were
compared
to
those
of
standard
solutions
of
methylxanthines.
All
values
are
means
±
SEM;
the
number
of
mouse
brains
is
shown
in
parentheses.
All
data
are
corrected
for
recoveries
of
standards
from
control
brains.
Recovery
of
1,9-dimethylxanthine
was
less
than
5%
and
levels
of
this
xanthine
could
not
be
determined
but
appeared
to
be
less
than
10
,uM.
Recoveries
for
the
other
xanthines
were:
1,
90%;
2,
55%;
3,
56%;
4,
100%;
5,
90%;
6,
47%;
7,
70%;
8,
33%;
10,
32%.
among
the
most
potent
psychoactive
drugs,
comparable
to
LSD
and
the
very
potent
butyrophenone
neuroleptic
spiperone
(25).
At
0.2
AmoI
of
L-PIA
per
kg,
the
mice
displayed
virtually
no
spontaneous
motor
activity
at
30
min
and
were
flaccid
with
their
fore
and
hind
limbs
splayed.
However,
at
this
dose
the
animals
were
alert,
responded
to
nociceptive
stimuli
such
as
tail
pinch-
ing,
and
had
intact
righting
and
corneal
reflexes.
At
this
dose
both
tail
and
ears
displayed
a
reddish
coloration
indicative
of
vasodilation.
The
respiratory
rate
was
slowed,
and
respirations
seemed
to
be
deeper.
At
5
tumol/kg
or
higher,
the
righting
re-
flex
was
abolished,
although
the
animals
were
still
awake.
At
progressively
increasing
doses
up
to
600
tumol/kg
the
mice
still
were
alert
but
flaccid.
The
absence
of
lethality
at
doses
thou-
sands
of
times
greater
than
behaviorally
active
doses
suggests
that
behavioral
effects
are
not
related
to
systemic
effects
such
as
hypotension.
After
peripheral
administration
of
[3H]CHA
to
mice,
its
brain
concentrations
are
such
that
a
0.2-kumol/kg
dose
would
give
brain
levels
of
20
nM,
several
times
greater
than
the
Kd,
6nM
for
adenosine
receptors
(unpublished
data).
To
further
15O
r
U43100
*d
0
s.
0
00
,o
50
0
0
CHA
L-PIA
2
aIfl
ooi
.~~¶1-m'iTff
D-PIA
Dose,
pumol/kg
FIG.
2.
Effects
of
CHA
and
L-
or
D-PIA
on
locomotor
activity
of
mice.
Locomotor
activity
for
groups
of
nine
mice
at
each
intraperito-
neal
dose
for
the
20-
to
30-min
period
after
drug
administration
are
expressed
as
percentage
of
activity
of
saline-injected
control
mice.
Val-
ues
presented
are
the
antilogarithm
of
a
logarithmic
transformation
of
this
data
as
in
Fig.
1.
*Significantly
different
from
saline,
P
<0.005
by
Student's
t
test.
ensure
that
L-PIA
depression
is
centrally
mediated,
we
showed
that
8-(p-sulfophenyl)theophylline
(60
mg/kg),
which
is
as
po-
tent
as
theophylline
at
adenosine
receptors
(13)
but
is
polar
and
not
likely
to
enter
the
brain,
failed
to
reverse
L-PIA
behavioral
depression.
To
explore
possible
interactions
between
L-PIA
and
meth-
ylxanthines,
we
administered
these
substances
alone
or
in
com-
bination
at
various
doses
(Fig.
3).
At
5
and
10
Amol/kg,
caffeine
depressed
motor
activity;
at
30
and
100
,umol/kg
it
was
a
stim-
ulant.
Combining
a
"depressant"
dose
of
caffeine
(10
tumoVkg)
with
L-PIA
markedly
enhanced
locomotor
activity.
Theophyl-
line
did not
depress
activity
at
any
dose
examined.
The
com-
bination
of
theophylline
(10
,u
mol/kg)
and
L-PIA
produced
con-
siderably
greater
enhancement
of
locomotor
activity
than
occurred
with
the
same
dose
of
theophylline
alone.
At
5-100
jimol/kg,
IBMX
alone
failed
to
enhance
locomotor
activity
and,
in
fact,
depressed
activity
at
most
time
points.
L-PIA
(0.2
,umol/
kg)
also
depressed
activity.
Strikingly,
the
combination
of
IBMX
(5
,umol/kg)
and
L-PIA,
like
combinations
of
L-PIA
with
either
caffeine
or
theophylline,
greatly
augmented
locomotor
activity,
to
300%
of
control
activity
at
60
min.
A
similar
although
less-
pronounced
reversal
of
L-PIA
depression
occurred
at
2.5
timol
of
IBMX
per
kg.
These
findings
suggest
that
IBMX
indeed
pos-
sesses
intrinsic
stimulant
activity
that
normally
is
masked
by
its
separate
depressant
effects
and
unmasked
by
the
interaction
with
L-PIA.
These
dramatic
interactions
of
methylxanthines
with
L-PIA
are
quite
selective.
No
synergistic
stimulation
of
locomotor
ac-
tivity
occurred
when
amphetamine
was
combined
with
L-PIA
(Fig.
3B).
At
2.5
Aumol/kg,
amphetamine
markedly
augmented
locomotor
activity.
A
combination
of
this
dose
with
L-PIA
(0.2
,umol/kg)
resulted
in
absence
of
stimulation
or
depression
of
activity.
Influences
of
L-PLIA
on
Nociception
and
Drug-Induced
Con-,
vulsions.
The
very
potent
and
stereospecific
behavioral
effects
of
PIA
and
CHA
suggest
that
these
substances
act
upon
aden-
osine
Al-receptors
in
the
brain
and
may
reflect
the
role
of
en-
dogenous
adenosine
in
the
brain.
Accordingly,
we
evaluated
a
Proc.
Natl.
Acad.
Sci.
USA
78
(1981)
Proc.
Natl.
Acad.
Sci.
USA
78
(1981)
3263
300
250
200
150
A
/.
,/
/K
//
P0
10
20
Time,
min
-
'I-
--;
Ie
Is
I/
I
I
10
20
30
40
50
60
Time,
min
10
20
30
40
50
60
FIG.
3.
Interactive
effects
of
alkylxanthines
or
d-amphetamine
and
L-PIA
on
mouse
locomotor
activity.
Mean
values
for
groups
of
10-15
mice
at
each
intraperitoneal
dose
are
expressed
as
percentage
of
saline
injected
controls.
L-PIA
and
methylxanthines
were
given
intraperitoneally
at
the
same
time;
20
min
later,
the
mice
were
placed
in
activity-monitoring
cages.
(A)
IBMX.
o,
5
1ttmol/kg;
10
,umol/kg;
30
,umol/kg;
A,
100
,umol/kg;
*
L-PIA
at
0.2
jumol/kg;
*,
L-PIA
at
0.2
/lmol/kg
plus
IBMX
at
5
pmol/kg.
(B)
d-Amphetamine.
o,
2.5
Amol/kg;
0,
L-PIA
at
0.2
,umol/
kg;
*,
L-PIA
at
0.2
Amol/kg
plus
amphetamine
at
2.5
,tmol/kg.
(C)
Theophylline.
Open
symbols,
doses
as
in
A;
*,
L-PIA,
0.15
,umol/kg;
*,
L-PIA
at
0.15
Amol/kg
plus
theophylline
at10
,umol/kg.
(D)
Caffeine.
Open
symbols,
doses
as
in
A;
*,
L-PIA,
0.15
,tmol/kg;
*,
L-PIA
at
0.2
Amol/kg
plus
caffeine
at
10
,tmol/kg.
600
r
c
0
0
0
C.)
0
e-
cu
cd
$.4
To
-4
To
550
F
500
F
450
F
400
F
350
h
300
F
250
1
200
[
150
F
100
50
Neurobiology:
Snyder
et
al.
3264
Neurobiology:
Snyder
et
al.
possible
role
of
adenosine
in
nociceptive
and
convulsant
systems.
Convulsions
were
elicited
in
groups
of
15
mice
by
adminis-
tration
of
strychnine
(5
,umoVkg)
or
pentylenetetrazole
(0.4
mmol/kg).
Diazepam
at
5
mg/kg
completely
prevented
both
strychnine
and
pentylenetetrazole
convulsions.
L-PIA(10
,u
moV
kg)
reduced
the
two
types
of
convulsions
by
50%.
However,
at
5
,umoVkg
or
lower,
L-PIA
lacked
anticonvulsant
effects
despite
its
profound
behavioral
actions.
In
the
tail
flick
test
(26)
in
groups
of
15
mice,
L-PIA
(5-10
umoVkg)
displayed
some
an-
tinociceptive
effects
that
were
blocked
by
caffeine
(0.2
mmoV
kg)
but
not
by
naloxone
(3
gmoVkg).
Thus,
whereas
L-PIA
dis-
plays
some
anticonvulsant
and
antinociceptive
activity,
these
actions
require
doses
at
least
10
times
the
effective
dose
for
behavioral
depression.
DISCUSSION
The
present
study
strongly
suggests
that
the
behavioral
stim-
ulant
effects
of
methylxanthines
involve
blockade
of
adenosine
receptors.
Potencies
of
methylxanthines
in
competing
at
aden-
osine
receptor
binding
sites
correlate
with
locomotor
stimula-
tion.
The
failure
of
the
potent
adenosine
receptor
blocker
IBMX
to
stimulate
locomotor
activity
directly
may
reflect
a
"contam-
inating"
behavioral
depressant
effect,
perhaps
related
to
phos-
phodiesterase
inhibition.
The
conversion,
by
low
IBMX
doses,
of
L-PIA-induced
depression
into
a
pronounced
behavioral
ac-
tivation
suggests
an
intrinsic
stimulant
activity
of
IBMX.
Al-
though
certain
of
the
behaviorally
inactive
methylxanthines
display
reduced
brain
penetration,
variations
of
bioavailability
do
not
account
for
differences
in
behavioral
potency,
and
brain
levels
of
most
methylxanthines
are
sufficient
to
occupy
adeno-
sine
receptors.
There
appear
to
exist
at
least
two
distinct
adenosine
recep-
tors.
Al-sites
are
associated
with
decreases
in
cyclic
AMP
levels,
are
influenced
by
nanomolar
concentrations
of
adenosine
and
related
agents,
and
respond
stereoselectively
to
PIA
(13,
27-
29).
A2-receptors,
on
the
other
hand,
are
associated
with
aug-
mentation
of
cyclic
AMP
levels,
respond
to
micromolar
con-
centrations
of
adenosine,
and
do
not
markedly
differentiate
be-
tween
the
isomers
of
PIA.
In
the
present
study,
we
evaluated
effects
of
xanthines
only
on
Al-receptors
labeled
by
[3H]CHA.
Previously,
we
labeled
apparent
A2-receptors
in
brain
mem-
branes
with
1,3-[3H]diethyl-8-phenylxanthine
(13).
Most
of
the
methylxanthines
used
in
the
present
study
have
similar
poten-
cies
on
adenosine
receptors
labeled
with
[3H]diethyl-
phenylxanthine
and
[3H]CHA.
Because
PIA-induced
depres-
sion,
which
is
strikingly
reversed
by
methylxanthines,
appears
to
be
stereoselective,
it
seems
probable
that
stimulant
effects
of
methylxanthines
involve
blockade
of
Al-receptors.
However,
a
role
for
A2-receptors
cannot
be
excluded.
The
extremely
po-
tent
locomotor
depressant
but
nonhypnotic
actions
of
PIA
sug-
gest
that
adenosine
analogs
may
exert
useful,
possibly
thera-
peutic,
behavioral
effects.
The
authors
thank
Pamela
Butts
for
skilled
analysis
of
brain
levels
of
methylxanthines
by
high-pressure
liquid
chromatography.
We
thank
Claire
McHugh,
Theodore
Hoehn-Saric,
Richard
Lebovitz,
and
Ste-
phen
Peroutka
for
preliminary
behavioral
studies
and
Lynda
Hester
for
technical
assistance.
This
work
was
supported
by
Public
Health
Service
Grants
MH-18501
and
DA-00266,
grants
from
the
McKnight
Foun-
dation
and
International
Life
Sciences
Institute,
and
Research
Scientist
Award
DA-00074
to
S.
H.S.
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... (a) Approximately 30 min after oral intake, caffeine reaches the central nervous system and blocks adenosine A 1 and A 2A receptors. There is evidence in the animal domain that caffeine's main metabolite paraxanthine has similar affinity as caffeine (Snyder et al., 1981) to both receptors (Chou & Vickroy, 2003) and disturbs NREM sleep (Okuro et al., 2010). Beside caffeine itself, paraxanthine may therefore contribute to the wake-promoting potential. ...
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For hundreds of years, mankind has been influencing its sleep and waking state through the adenosinergic system. For ~100 years now, systematic research has been performed, first started by testing the effects of different dosages of caffeine on sleep and waking behaviour. About 70 years ago, adenosine itself entered the picture as a possible ligand of the receptors where caffeine hooks on as an antagonist to reduce sleepiness. Since the scientific demonstration that this is indeed the case, progress has been fast. Today, adenosine is widely accepted as an endogenous sleep‐regulatory substance. In this review, we discuss the current state of the science in model organisms and humans on the working mechanisms of adenosine and caffeine on sleep. We critically investigate the evidence for a direct involvement in sleep homeostatic mechanisms and whether the effects of caffeine on sleep differ between acute intake and chronic consumption. In addition, we review the more recent evidence that adenosine levels may also influence the functioning of the circadian clock and address the question of whether sleep homeostasis and the circadian clock may interact through adenosinergic signalling. In the final section, we discuss the perspectives of possible clinical applications of the accumulated knowledge over the last century that may improve sleep‐related disorders. We conclude our review by highlighting some open questions that need to be answered, to better understand how adenosine and caffeine exactly regulate and influence sleep.
... Additionally, the effect of caffeine on adenosine receptors A 1 and A 2a has been widely established in animal models. A 2a receptors are ubiquitously distributed in brain areas known as primary memory regions, including ventral and dorsal striatum, selected areas of cortex, and hippocampus (Borea et al., 2018;Snyder et al., 1981). Habitual caffeine can reverse memory impairments in the animal model of Alzheimer's disease by mimicking the effects of selective inhibitors of A2a receptors (Da Silva et al., 2016), while acute coffee treatment increased plasma level of anti-inflammatory cytokines and granulocyte-colony stimulating factors associated with WM improvements (Cao et al., 2011). ...
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Caffeine is a widely used nootropic drug, but its effects on memory in healthy participants have not been sufficiently evaluated. Here we review evidence of the effects of caffeine on different types of memory, and the associated drug, experimental, and demographical factors. There is limited evidence that caffeine affects performance in memory tasks beyond improved reaction times. For drug factors, a dose-response relationship may exist but findings are inconsistent. Moreover, there is evidence that the source of caffeine can modulate its effects on memory. For experimental factors, past studies often lacked a baseline control for diet and sleep and none discussed the possible reversal of withdrawal effect due to pre-experimental fasting. For demographic factors, caffeine may interact with sex and age, and the direction of the effect may depend on the dose, individual tolerance, and metabolism at baseline. Future studies should incorporate these considerations, as well as providing continued evidence on the effect of caffeine in visuospatial, prospective, and implicit memory measures.
... The influence of caffeine on vasomotor control (i.e., skin blood flow) has two components: as a locally mediated adenosine receptor antagonist, and as a central nervous system stimulant through catecholamine release (38,39). Adenosine assists vasodilation through nitric oxide release (3) and acts as a synergistic vasodilator during exercise and environmental heat stress (40). ...
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