identical receptor con- firmed the sequence as an adenosine receptor based on ago- ... receptors have been cloned. Rat. A3 receptors are relatively insensitive.
0026-895X/95/061038-08$3.OO/O Copyright AH rights
tO by The American Society of reproduction in any form
MOLECULAR
for
48:1038-1045
PHARMACOLOGY,
Pharmacology
and
Experimental
Therapeutics
reserved. (1995).
Protein-Dependent Activation of Phospholipase Adenosine A3 Receptors in Rat Brain
C by
G
MARIA KENNETH
P. ABBRACCHIO, A. JACOBSON,
ROBERTA BRAMBILLA, STEFANIA and FLAMINIO CATTABENI
CERUTI,
HEA
0.
KIM,
DAG
Institute of Pharmacological Sciences, University of Milan, School of Pharmacy, via Balzaretti 9, 20133, and Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes, National Institutes of Health, Bethesda, Mar,4and 20892 (H.O.K., D.K.J.E.v.L., K.A.J.) Received
June
30, 1 995; Accepted
September
K. J.
E. VON
LUBITZ,
Milan, Italy (M.P.A., R.B., S.C., Digestive and Kidney Diseases,
F.C.),
1 2, 1995
The recently
been proposed to play a role in the pathophysiology of cerebral ischemia. Because phospholipase C activation occurs as a very early response to brain ischemia, we evaluated the ability of Aselective and nonselective adenosine analogues to elicit phos-
cloned
guanosine-5’-O-(2-thiodiphosphate), confirming ment of a G protein-coupled receptor. Activation lipase C was higher in the striatum than in the consistent with A3 receptor densities. Stimulation lipase C activity by adenosine analogues was
phoinositide
antagonized
hydrolysis.
hippocampal
nositol
G protein-coupled
slices,
phosphates
adenosine
In myo-[H]inositol-labeled
A3 agonists
stimulated
in a concentration-dependent
A receptor
rat striatal
formation
has
and
of rH]i-
manner.
In
striatum, the potency order was 2-chloro-N-(3-iodobenzyI)adenosine-5’-N-methyluronamide N-(3-iodobenzyl)-adenosine-5’-N-methyluronamide >> N-methyl-i ,3-di-n-butylxanthine7-J3-D-ribofuronamide 5’-N-ethylcarboxamidoadenosine N-2-(4-aminophenyl)-ethyladenosine > N-(p-sulfophenyl)adenosine = 1 ,3-dibutylxanthine-7-riboside, which is identical to the potency order in binding studies at cloned rat A3 receptors. Stimulation of phospholipase C activity was abolished by
Four G protein-coupled adenosine receptors have been cloned and designated A1, AA, A2B, and A3 subtypes (1). These receptors share features common to other members of the G protein-coupled receptor superfamily, including seven hydrophobic putative transmembrane domains linked by regions of greater hydrophilicity (1-3). The first three of these receptors were identified initially on the basis of physiological and pharmacological criteria. However, the A3 receptor
[125l]APNEA,
N-2-(4-amino-3-iodophenyl)-ethyladenosine;
and at much
higher
con-
was defined A3 sequence, from a rat
only after its recent molecular cloning. The rat tgpcrl, was first cloned by Meyerhof et al. (4) testis cDNA library by a polymerase chain reac-
tion-based
strategy.
of a rat firmed
striatal the
Subsequently, cDNA
sequence
thines, (5,
9).
sheep
(6)
as
an
The
and
the
adenosine
(7,
expression receptor
receptor
of adenylyl radioligand human
functional identical
based
cyclase activity [1251]APNEA. 8) A3
receptors
conon
ago-
(5) and More rehave
been
A3 receptors are relatively insensitive to xanknown to be potent A1 and A receptor antagonists However, there are major species differences in the Rat
pharmacological thines
the
encoding
nist-induced inhibition specific binding of the cloned.
ABBREVIATIONS:
derivatives
centrations than needed for blocking adenosine A1 , AA, and A2B receptors. In the presence of an A1/A antagonist, a selective A agonist only weakly inhibited forskolin-stimulated adenylyl cyclase activity in rat striatum. Thus, stimulation of phospholipase C activity represents a principal transduction mechanism for A3 receptors in mammalian brain, and perhaps A3 receptor-mediated increases of inositol phosphates in the ischemic brain contribute to neurodegeneration by raising intraceIlular calcium levels.
cently, M.P.A. and F.C. are involved in the concerted action ADEURO (EU, Biomed 1) (ADEURO is the logo for the Concerted Action “Physiology and pharmacology of brain adenosine receptors-implications for the rational design of neuroactive drugs” supported by the European Union within the Biomedical and Health Research Programme). Part of this research was supported by the Italian Ministero deli’ Universita e della Ricerca Scientifica e Tecnologica; financial support provided by Gilead Sciences of Foster City, California.
by xanthine
the involveof phosphohippocampus, of phosphoonly modestly
(10,
profile
of A3 receptors
with
respect
to xan-
11).
physiological IB-MECA,
role
of the
A3 receptor
N-(3-iodobenzyl)-5’-N-methyuronamide
is mostly
unex-
adenosine;
CHO, Chinese hamster ovary; PLC, phospholipase C; lnsP, inositol phosphate; PIP2, phosphatidylinositol-4,5-bisphosphate; DBXRM, N-methyl1 ,3-di-n-butylxanthine-7-3-D-ribofuronamide; NECA, 5’-N-ethylcarboxamidoadenosine; APNEA, N-2-(4-aminophenyl)-ethyladenosine; SPA, N6(p-sulfophenyl)-adenosine; DBXR, 1 ,3-dibutylxanthine-7-riboside; CPX, 8-cyclopentyl-1 ,3-dipropylxanthine; XAC, 8-[4-[[[[(2-aminoethyl)amino]carbonyl]methyl]-oxy]phenyl]-1 ,3-dipropylxanthine; BWA1433, 1 ,3-dipropyl-8-(4-acrylate)phenylxanthine; KHB, Krebs-Henseleit buffer; ADA, adenosine deaminase; GDPf3S, guanosine-5’-O-(2-thiodiphosphate); [125IJAB-MECA, N-(4-amino-3-iodobenzyl)-adenosine-5’-N-methyluronamide. 1038
Downloaded from molpharm.aspetjournals.org at ASPET Journals on June 9, 2016
SUMMARY
Activation plored.
A3 receptor heart (5, 8),
(4-7), various
brain
responses thus
areas
have
ischemic
variety
from
of
ofthe
heart
mast
cells
of the hampered
the
testes and
chemically
related
stimulate
and
hydrolysis
Receptors
unrelated
of PIP2
A3
in the
I 039
in Rat Brain receptor
agonists
to
brain.
adenosine-elicited
Materials
insensitive,
and
Methods
in hypotension
(14), (12,
and 15).
physiological by
in (12),
xanthine
of A3 receptors
characterization been
found system
as
preconditioning
has
been
immune
A
mediators
receptor
have
characterized
involvement
inflammatory precise
(5-8).
been
suggesting
(13),
transcripts lung (5-8),
of PLC by Adenosine
release
role
lack
a
of the
of selective
Compounds.
of
However,
A3
A3 re-
used: Fig.
chemical
1. These
bition 17,
adenosine
derivatives
in cell
lines
stably
activity
by both
transfected
SPA,
are
characterized
A3 radioligands
and
shown
in
their
A3
for
radioreceptor-binding
and with
were
APNEA,
nucleosides
previously
intrinsic
agonists
NECA,
of these
were
and
receptor
DBXRM,
structures
affinity and with A1, A,
studies
following
Cl-IB-MECA,
The
DBXR.
receptor
ceptor ligands. The first truly selective A3 receptor agonists to be synthesized were the N6’ -uronamides (16-18). IB-MECA was shown to display a 50-fold selectivity for rat brain A3 versus either A1 or AA receptors in binding studies using CHO cells transfected with the cloned A3 receptor (16). More recently, 2-substitution of N-benzyl-adenosine-5 uronamides was shown to further enhance A3 receptor selectivity, leading to Ci-IB-MECA. The latter compound is selective for A3 versus A1 and A2A receptors by 2500and 1400-fold, respectively (17). Insights into the role(s) ofA3 receptors in brain have come from recent in vivo studies using IB-MECA. In mice, administration of this compound produced a locomotor depressant
The
IB-MECA,
adenylyl
rat
cyclase
A3 receptor
inhi-
cDNA
(9,
29).
IB-MECA, Cl-IB-MECA, sized as previously reported xanthine derivatives CPX
DBXRM, SPA, and DBXR were synthe(16, 17, 29, 30). NECA, APNEA, and the and XAC were obtained from Research
Biochemicals
International
(Natick,
of Dr. Susan Park, North Animals.
Daluge
MA);
BWA1433
was
a kind
gift
‘ -
that
was
insensitive
xanthine
antagonists
(18).
was
MECA
found
D-aspartate-induced emia-associated effects, struction
i.e.,
to be
to A1 or AA
The
chronic
receptor-selective
administration
cerebroprotective
of IB-
against
N-methyl-
enhanced mortality in the hippocampus
and extensive neuronal of ischemic animals, were
dein-
Calco,
cycle
2#{176}) and
at
in rooms
of [3H]-InsPs.
Assay
to characterize ways by a variety
Rats
rapidly
dissected
CO2)
on
in the
KHB
ice
and
NaCl
mM),
mM),
MgSO4
cyclase
the
affinities
(9, 17).
In RBL-2H3
a xanthine-insensitive lation of PLC and in this
potency
in this
cells
(15,
21),
binding
activation
of
studies
cell response
line
confirmed
(12); does
the
however, not
match
presence
the
agonist
that
predicted
ofthe
order
Y. Shin,
W. Daly,
and
K A. Jacobson,
unpublished
observations.
(1.2
(2 IU/ml),
slices
chopper
and
15-mm
washes
1 hr
A3
of
for A3
were
ries,
(w/v)
used
a
12-hr (22
±
available
ad
gands
O/5%
purified
for
permit
NaCl
(final
and
the
various
volume,
300
shaken
and
310
d ofdoubly
for
on 0.75
(Bio-Rad form.
To
remove
inositol-1-monophos(KHLiJADA).
were
then
adenosine
pd).
ml of
Laborato-
washed twice with and LiC1 (7.5 mM)
products
slices Vials
added
to
receptor
were
at 37#{176} for 1 hr.
li-
gassed
Previous
(95%
experi-
of [3HJInsPs in rat brain slices selected experiments, the effect of
formation was tested. l of a chloroform/methanol
separation
Incubation was stopped mixture (1:2 w/v);
distilled
water
were
then
by the 310 d
added
to
of phases.
centrifugation
at
1000
aqueous phase, representing tent, was diluted to 3 ml with 0.75 ml of a 50% (w/v) slurry nine
the
ofreaction
containing
two
incubated
1 ml of gravity-sediunder sustained shakBefore each experiresin
to inhibit
detection
shown that accumulation up to 90 mm (34). In of940
were
After
by chromatography
mesh)
(1.2 ADA (95%/5%). and
tissue-
KHB/ADA.
slices
exchange
con-
a Mcllwain
were ADA
in KHLiJADA CO2), capped,
to
shaking,
of gravity-sedimented
on InsP
seven
in fresh
mM),
OdCO2
using
Ger-
KH2PO4
(25
formate
Bio-Vials
After
cut
O/5%
buffer
mM),
NaHCO3
in the
microliters
ofchloroform
mM),
(1.7
myo-{3Hjinositol, slices once with KHB containing
thus
addition
KHB/ADA
CaC12
were
anion
100-200
(95% Mannheim,
to pH 7.4 with m)
slow
ofAg-1 CA;
GDPI3S
cause
extensively
carboxygenated The
suspended
substituted
ments had was linear
28,
(Boehringer
mM),
(11.7
x 350
was
Richmond,
unincorporated KHB/ADA and
Beckman
used
transduction path31, 32). Accumulation
myo-[3H]inositol (3 CiJni; slices in 10 ml of KHB/ADA) myo-[3Hlinositol incorporation.
slurry
and
in
equilibrated
myo-[3Hlinositol
Fifty
KC1 (4.7
a1 37#{176} under
cerebral
a 50%
been
have
adenosine.
immediately
to facilitate
ment,
slices
of ADA
glucose
(350
at 37#{176} with
mented
mg
mM),
previously
Cerebral
phatase
receptors.’ Moreover, RBL-2H3 mast cells and other mast cells also express other adenosine receptor subtypes (21, 22), which makes interpretation of functional responses complex. In the brain, PLC activation and subsequent formation of InsPs and diacylglycerol might be relevant to ischemic damage because this transduction pathway has been proposed to represent a very early pathophysiological response to cerebra! ischemia (23-26). All previous studies (27, 28) have failed to demonstrate either direct activation or inhibition of basal InsP levels in the brain by adenosine agonists. Because A3 receptors appear to trigger PLC activation (12), we investigated the ability of IB-MECA, Cl-IB-MECA, and other 1
(120
isotonically
adenosine receptor resulted in stimuformation of inositol-1,4,5-triphosphate.
cDNA-hybridization
subtype
mast
relative
in
temperature
water
phosphoinositide (27,
placed
presence
tamed
their
and
were
housed
InsPs in rat cerebral slices was measured as described et al. (33), with modifications according to Balduini et al. were killed by decapitation. Hippocampi and striata were
by Berridge
(34).
Rat brain
modulation of ofneurotransmitters
duced by acute IB-MECA administration (20). These data have led to the hypothesis that activation of brain A3 receptors may play an important role in the development of ischemic damage (20) and that desensitization of this receptor after chronic agonist administration may prove to be useful in counteracting ischemia-associated brain damage. Very little is known about transduction mechanisms involved in A3 receptor effects in vivo. In cell culture, two second messenger systems have been found to be coupled to A3 receptors: inhibition of adenylyl cyclase and activation of PLC. Potencies of adenosine agonists in inhibiting adenylyl with
old)
were
at controlled Food
Triangle
libitum.
endogenous
correlated
Research
(2 months
animals
maintained
to remove
A3 receptors
All
humidity.
many)
via
rats
Italy).
standard
of[3H]-labeled
seizures in mice (19) and against ischneurodegeneration in gerbils (20). Opposite
Carolina). Male Sprague-Dawley
River,
light/dark
Co.,
washes
of
the
x g for
10
mm,
the accumulated doubly distilled of Ag-1X8 anion resin
columns
750
il
of the
myo-[3H]inositol water and passed exchange resin. with
3
ml
of
upper
conover After 5
mM
myo-inositol, InsPs were eluted with 2 ml of 1 M ammonium/formate/ 0.1 M formic acid. The eluate was then added to 8 ml of Luma Gel scintillation liquid (Packard Instruments, Gromngen, The Netherlands) and counted in a Packard scintillation spectrometer with an
Downloaded from molpharm.aspetjournals.org at ASPET Journals on June 9, 2016
response
(Charles
(Burroughs-Wellcome
I 040
et aL
Abbracchio
NH2
CH3CH2NHCO/
CH3NI
CI-IB-MECA
HOOH
HOOH
IB-MECA
NECA
I
CH3(CH2)3 0 CH3NHC#{176} 0
OH
HOOH
HO
SPA
DBXRM
APNEA Fig. 1 Structures
of adenosine
.
agonists
used in this study. See Materials
efficiency of 60%. Two hundred microliters ofthe lower lipidic phase were added to 8 ml of the same scintillation liquid to determine the amount ofmyo-[3H]inositol incorporated into membrane phospholipids (mostly as phosphatidylinositol; Ref. 35). Data were calculated as
as
dpmJlO4
This
dpm
incorporated
(to
inositol into phospholipids) basal (unstimulated) InsP stimulated
InsP
correct
and
for
expressed
formation
incorporation
either
of myo-[3H1-
as a percentage
or as a percentage
of
in
formation.
10 mM
EDTA.
TrisHCl,
Homogenates
pH
7.4,
were
containing
centrifuged
0.32 (10
mm
M sucrose at
800
and X
g)
(-40
medium
containing
g
of
protein)
were
0.5 mM ATP,
incubated
0.2 mM EGTA,
with
Ci-IB-MECA
dal
by
protein
and
computer-assisted
allows
expressed
by
ifexperimental
relationship
as
using
data and,
percent
to
curves
analysis
to verify
concentration-effect
ther
two-way
the
Bonferroni
analysis
of variance
or Fisher
correction.
2 to
in
2 mM MgCl2, 1 mr and 0.2 mi papav-
80 mM Tris-maleate, pH 7.4, 10 M GTP, erine to inhibit cAMP phosphodiesterases; a regenerating system consisting of 6.3 mii creatine phosphate and 60 pg/sample creatine phosphokinase; 10” cpm/sample [3H]cAMP (NEN-DuPont, Italy) to measure chromatographic recovery; 1 CiJsample {32P]ATP; and 2 IU/ml ofADA. To better evaluate possible inhibitory effects of Cl-IBMECA on cAMP formation, adenylyl cyclase activity was increased by the addition of the direct activator forskolin (1 M final concentration). After a 15-mm incubation at 33#{176}, samples were stopped by addition of a “stopping solution” containing 2% sodium dodecyl sulfate, 1.3 mM cAMP, and 40 mM ATP and boiled for 4 mm to denature proteins. [32PIcAMP was then purified by double-column chromatography on AG-50X4 (Bio-Rad Laboratories, Richmond, CA) and alumina columns as previously described (36). Results were calculated cAMP,
analyzed analysis
of
names.
adenylyl cyclase activity. data. Concentration-response
of
Analysis were
Parametric
pellet nuclei, and supernatants were collected and centrifuged again (25 mm at 11,500 x g) to pellet membranes. Pellets were resuspended in 80 mM Tris-maleate (2 mM dithiothreitol, pH 7.4), and aliquots
cAMP/mm/mg
forskolin-stimulated
for full chemical
describe
if so,
to
was
of
agonists
Allfit
(37).
a sigmoi-
obtain
EC50
carried
out
and
deterof the
Em
of adenylyl cyclase activity. Adenylyl cyclase activity was assayed as previously described (36) in rat striatal preparations by the conversion of [32PIATP (NEN-DuPont, Italy) to [32P]cAMP. Briefly, tissues were homogenized (10 strokes with a Teflon-glass mM
of
and Methods
values. Nonparametric one-way analysis ofvariance was mined using the Kruskall-Wallis test. Statistical significance single concentration data was determined with the Student’s
of maximally
Assay
potter)
pmol
DBXR
t test.
using
ei-
Results Seven
nucleoside
IB-MECA, tive agonists. agonists and
Fig. NECA
IB-MECA PIP2 on
and
at very InsP
by in
compared
with
ability
A3 receptors. demonstrated their ceptor,
the effects formation
both (Fig.
concentrations.
Table
other
1. The
effects
to inhibit
The by
at A,, adenylyl
rank the
relative affinities as determined
order selective in by
the
stimulation
Em,
and
adenosine A,
cyclase
of potency agonists
radioligand displacement
effects and
was values
satuin
analogues
PIP2 and
of
their
dependent
The
on
A3-selec-
hydrolysis
and
M),
concentration 2A).
1), of which
novel
of these A3-selective in rat striatal slices.
(-10
EC50
and
affinity
(Fig.
were
stimulated
were
these
studied
DBXRM
concentrations
significant
at high
ported the
2 shows on InsP
formation
formation
were
and
Ci-IB-MECA
low
statistically rable
analogues
Cl-IB-MECA,
hydrolysis via
and
be with
cloned
on InsP was
re-
may
A3 receptors activity
InsP
are
rat
formation
consistent
binding at rat A3 of specific binding
with reof
Downloaded from molpharm.aspetjournals.org at ASPET Journals on June 9, 2016
(CH2)3CH3
Activation
A
.
Based
iB-MECA
0 Cl-IB-MECA
on all these
order
profile
rat
250
the 200
ow
elicited 150
;
100
lated
InsP
effects
demonstrate
0
by
activity inN
lOOnM
lOnM
Agonist
1tM
Unlike
10KM
has
been
duced
200 0 0
S
0
potent thine
150
tion 100
.0
to lOnM
lOOnM
Agonist Fig. 2. Effects of (A) IB-MECA,
1pM
1OIM
CI-IB-MECA,
reported
on the x-axis.
lnsPs formation
Materials hydrolysis tion.
and Methods. set to 100%.
Results
(B) DBXRM and NECA on
was measured
as descnbed
in
formation
stimulation
dent
fashion
[‘251]APNEA
transfected using these
with two
as
inhibitors
to be
activation
[‘25I]AB-MECA
rat A3 receptor radioligands
binding
found
adenylyl
or
nearly
were
cyclase
very
activity
enosine
analogues
stimulate
PLC, the
at
cloned
identical
(16).
similar
to IC50
cells
stably
respect
cells
rat
The values
(17,
A3
EC50
receptor values
in
in inhibiting
29).
less
1); than
N-benzyladenosine
both
compounds
full
efficacy
derivatives
appeared in
to
comparison
IB-MECA
and
Cl-
IB-MECA. Modest, PIP2
although
hydrolysis
statistically were
obtained
significant, with
other
stimulations adenosine
agonists (Table 1). Stimulation oflnsP accumulation by 10 pM APNEA, SPA, or DBXR ranged between 48% were
over control, concentration
i.e., less than dependent
full (data
efficacy. Also, not shown).
of receptor induced
23% the
and
effects
4A; over
by IB-MECA
was
of
should
be blocked
not
receptors in
of IB-MECA in hippocampal
tively
lower,
by 1
,tM
InsP
formation)
IB-MECA
42.3).
< 100
esti-
(18). As in accumulation
A1,
A2A,
because
the
K, values
Only
at 100
(2).
with to
tested
by XAC
maxi-
lower
according
which nM
The
was
striatum,
antagonized
presence
of
1
presence
of IB-MECA respect lower
XAC,
tM
stimulation slices.
of InsP A similar,
of xanthine
antagonists
(178
tion with
ability
rat
%CV,
receptor in hippocampus stimulation of InsP
are
InsP a signif-
in a concentration-depen-
basal
4B)
there-
modulate
4) showing
ni,
In the striatum, the of 0. 1 jM potentiated
of both
The
using
over a wide
A2B
receptors of XAC
M
XAC
at was
observed.
presence with the
of ob-
expression
We have
25.6
(Fig.
effect
striatal slices. concentration
the
of 10 ni
striatum
to
(Fig.
formation
at
the
crease tected
mouse
EC50,
antagonism
Curiously,
at
range
IB-MECA slices
concentrations
significant
tested
demonstrated.
of
in
levels ofA3 hippocampal
three
also
obtained
range
studies
experiments
[‘25IIAB-MECA ( 18). Saturable other brain regions, such as hip-
was
of InsP
that
to
stimula-
significant in
hippocampal
(143%
In
were
results,
ability
mated lower the striatum,
all
DBXRM and NECA also stimuin rat striatal slices (Fig. 2B). The than those for the N-benzylad-
(Table with
the
CHO
(9, 17, 29). K1 values same adenosine ana-
cDNA for the
in CHO
The weaker A3 agomsts lated InsP accumulation EC50 values were higher
in
present
cortex,
(Fig.
effect
11).
the
no significant antagonism of PIP2 hydrolysis was
demonstrated
the
icant
with
acidic xanlow antag-
previous
in a concentration
the
in rat
striatal
IB-MECA
derivatives
affinity radioligand to A3 receptors in
and
rat
the The
with (9,
of in-
shown).
was
evaluated
on
receptor
compound
not with
pocampus fore
A3
however, stimulation
any
(data
the high binding
mal
were
rat
ability
obtained
with (11).
BWA1433
2, xanthine
of A3 receptors
are expressed as percent of basal PIP2 p < 0.04, with respect to basal lnsP forma-
*,
with
10 jM Consistent
concentrations
PIP2 hydrolysis in rat striatal slices. myo-[3H]inositol-Iabeled slices were incubated with the various adenosine analogues at the concentrations
logues
the
in Table
served
0
and
the
effect at all. Very reductions of IB-
were and
A3
xanthine
in this
is in agreement
100 jtM concentration; IB-MECA-elicited
Cs
with
at
reported 50
either
of XAC hydrolysis
affinity
formation
rat
to accumulation
concentrations
antagonist XAC at high concentrations
activity
the
we evaluated InsP
PLC
a G protein.
insensitive
antagonize significant,
of InsP
of PIP2
of
(no (n. C(n
IB-MECA
Al/A2A/A2B BWA1433
onistic
jCl)
PLC
maximal
MECA-stimulation
I-
c:: .2w
by
results of
subtypes,
be
basis,
3). These
through
to antagonize
slices (Table 2). CPX did not modest, although statistically
DBXRM NECA
D
this
of xanthines
of slices abolished
stimulation
receptor to
GDPI3S. stimu-
± 8%
IB-MECA alone).
over and
basal
InsP
lower
to hippocampal slices density of A1 receptors of A3
receptors
to
was
versus
obtained
in
CPX
a
modulate
at induced
formation 157
in-
was dequantita-
A, antagonist PLC activation
CPX Such
a significant
formation although
±
degree
in the
6% in the
of potentia-
(Fig. 4B) is consistent in the striatum. adenylyl
cyclase
Downloaded from molpharm.aspetjournals.org at ASPET Journals on June 9, 2016
(1, 5, 9). On
a number
(Fig. slices,
A2B
were
inhibitor potently
completely
occurs
reported
formation
exposure
almost
analogues
with
(9, 17, 29).
in InsP
concomitant
brain
in
DBXRM
experiments
turnover
rat
adenosine
antagonists
B
PIP2
in
the A1, A2A, and
receptor
concentrations
the
on
that
subtype
G protein IB-MECA
GDPI3S
potency
hydrolysis
IB-MECA >> which is consistent
agonists,
of the slices,
1 ), the
PIP2
of G proteins
receptor
or 1 mM
LM
Table on
A3 receptor
accumulation, 100
2 and
DBXR,
=
involvement
adenosine
IB-MECA
5#{176}
SPA
adenosine
in the presence in control brain
to either
-2
(Fig.
I 041
in Rat Brain
analogues
CI-IB-MECA >
the
by
performed Although
0.-
was:
at the
To assess
C-
results
APNEA
affinities
Receptors
adenosine
slices
NECA
0
U)
for
striatal
0 0
of PLC by Adenosine
I 042
Abbracchio
TABLE
1
Affinity
at rat brain
agonists
et aL
A1, A,
and A3 receptors;
inhibition
of adenylate
cyclase;
and effects
in radioligand
Affinities
binding
assaysa
NECA
APNEA DBXMR
IC50
K,A3d
470 56
0.33 ± 0.08 1.1 ± 0.3
66.8 90.0
46.4 184
6.3 14 37,300
10.3 172
113±34 116 ± 229 ± 526 ± 1400 ±
5500
1241
DBXR Values
b
Displacement
from
Displacement Displacement
8900
4250
ND.
27 142 160
18,000 ND.
986
240 ± 5 192 ± 11 170±
34
ND.
148 ±
12g
± 138 ND.
140 123
± 11 ± 9 129 ± 6g
ND.
at
maximum 100,000
44%
± 9.6 ± 65 ±25
[3H]Nphenylisopropyladenosine
binding
from
rat
brain
membranes.
rat striatal
as described
slices,
in Materials
and
of CHO cells stably transfected with the rat A3-cDNA.
Methods.
values
Erna.
are
expressed
with the
as percent
rat A3-cDNA
of control
(5).
lnsPs
formation
set
at 100%.
not determined. Concentration
for APNEA
=
and
DBXR
10
Although < 0.03
LM.
and
p
it was for
SPA
not possible (Student’s
to determine
EC50 values,
there
was
a statistically
activation 0
IB.MECA
+
0
IB-MECA
+ 1 mM GDPfS
100 pM GDPs
of
crease
the
of cAMP
perhaps
300
rise (six to nine
experiments),
with
abolished
200
and
A2A
by CPX
observed
with p
0.001
=
in-
(Fig.
M
5),
A2 receptors
(17).
a concentration
An
iO
K1 value
reported
receptors
at
receptor. at
of stimulatory
previously
at
A3
was
activation
the
Ci-IB-MECA
0
xanthine-insensitive production
reflecting
sistent
0
significant
t test).
con-
of 0.47
This
stimulation
known
to block
for
j.M
was both
A1
A2 receptors.
0.
C
100
Discussion
0
Activation inhibit
0 lOnM
5pM
1iM
of lB-MECA
stimulation
of lnsPs formation
by GDPf3S
in rat striatal slices. myo-[H]inositol-labeled slices were incubated with IB-MECA at the concentrations indicated on the x-axis in the absence or presence of either 1 00 M or 1 mM GDPI3S. PIP2 hydrolysis was assayed as described in Materials and Methods (both curves, p < 0.01 by either Kruskall-Wallis test or Bonferroni).
duction brain
has
enylyl
cyclase
PLC
Xanthine
IB-MECA
was
formation
81.4
BWA1 433 CPx
set to 1OO/
shown
tion
in rat
that
these
effects the
rank
variety
of
tency
in both
adenylyl
ity
tested
by measuring on
the
concentration
forskolin-stimulated
striatal
membranes
cyclase
activity
either
(Fig.
toward
a subtle
inhibition
M. This
inhibitory
effect
5).
alone
cAMP
The
effect
or with
CPX
at concentrations therefore
appears
response formation
of Cl-IB-MECA indicated to be related
of
inability
on
(18).
of 10’#{176}to 10 to the
sus levels results
by
First, of
potency
their (9,
the
involvement
the
and 17, 29).
breakdown
rat
A3
receptor
of CPX to reverse Fourth, PLC stimulation
the
hippocampus, ofA3 show
which
receptors for
the
the
in these first
time
generasuggests
recently
cloned correlation
(5,
that
brain
a po-
stimulation
9,
11) effects in rat with
areas
stimulation
of is comwhich
receptor. modestly
which xanthine
is consistent two
for
IB-MECA GDPS,
is only
in vivo is higher
G
A3 receptor-mediated Second,
of a G protein-coupled
of PIP2
cloned
InsP
is a good
by the selective A3 agonist by the G protein inactivator
the
A3 re-
agonists
on PLC stimulation previously demonstrated
binding
inhibition
by
strongly
there
weakly whereas
and
by a number ofxanthine derivatives, with the previously demonstrated
in
a trend
hydrolysis
intact ad-
very
fashion
mediated
and
modulation
fected ment
only
A3 receptor
evidence
A3 receptor
activity
confirmed
order
agonists
cyclase
Third,
are
was
Extensive
A3 receptor.
with
the
Cl-IB-MECA,
of selective PIP2
slices.
function in Forskolin-stimulated
a dose-dependent
to stimulate
to
transfected
6, 9, 17, 29). However, of this and other trans-
agonist
A variety
brain
PIP2 hydrolysis pletely abolished
was
in
demonstrated
lines
striatum
receptor
stimulated
Striatal slices were incubated with 10 MM IB-MECA in the absence (maximal lnsPs formation) or presence of the indicated xanthine derivatives (all tested at 1 00 M concentration). Levels of lnsPs were determined as described in Materials and Methods. Results are expressed as percent of IB-MECA stimulated lnsPs formation set to 1 00%. IB-MECA stimulation was 1 71 .2 12.8% with respect to basal lnsPs formation. The number of determinations is given in parentheses. * P < 0.025; ‘ P = 0.001, with respect to 10 M IB-MECA, Student’s t test.
to Ci-IB-MECA
previously in cell
in rat
A3
activation.
between
± 6.5* (3) ± 1.8**(3) ± 7.5 (3)
84.3 109.7
XAC
the
protein-coupled
Percent of maximal IB-MECA-stimulated lnsPs
derivative
activity
by
were
2
Effects of several xanthine derivatives on maximal stimulation of PIP2 hydrolysis in rat striatal slices
was activity
mechanisms to A3 receptor been totally unknown.
inhibited ceptor
TABLE
cyclase
the rat brain A3 receptor cDNA (5, until the present study the relevance
10pM
Concentrations
Fig. 3. Abolition
of A3 receptors
adenylyl
af-
is in agreeinsensitivand
with
the
of IB-MECA striatum verthe (18).
estimated Thus,
of PLC
our activ-
Downloaded from molpharm.aspetjournals.org at ASPET Journals on June 9, 2016
g
74
2250
18
of specific [H]CGS 21680 binding from rat striatal membranes. of specific binding of [3HJAPNEA or [125l]AB-MECA from membranes of adenylyl cyclase activity in membranes of CHO cells stably transfected
In myo-[H]inositol-labeled
ND.,
M
%
flM
flM
>io
Emax
9, 1 7, 30.
of specific
e Inhibition t.
Refs.
EC50
820 54
SPA
a
PIP2 hydrolysis’
K,A2AC flM
d
for the adenosine
Cyclase lnhibitionae
KIA,b
Cl-IBMECA IB-MECA
C
on PIP2 hydrolysis
used in the study
Activation
of PLC by Adenosine
Receptors
in Rat Brain
I 043
130
A 200
0
120
at
U
S
:g
#{182}E
=5
.s
150
110
S
a.SS
.
.
St
100
0
-.--
ci-IB-MEcA
---
ci-IB-MEcA
cx
tM
1
+
100
2
90 10.10
St
iO-9
mM
lOnM
lOOnM
1tM
expressed tM iB-MECA
*5
10 tM IB-MECA
+
XAC
C
0
g
on forskolin-stimulated
cAMP formation
as percent
of control
forskolin-stimulated
ason are
activity.
Each
value is the mean ± standard error of eight experiments run in triplicate. Cl-lB-MECA alone: #, p < 0.003 with respect to corresponding control, Bonferroni test; CI-IB-MECA + CPX: § and *, p < 0.03 with respect to corresponding control; **, not statistically different from control, Fisher’s test; and p < 0.004 with respect to #, posthoc test of a two-way analysis of variance.
150
S
adenine
g
moiety,
the
xanthine
riboside
DBXRM
was
shown
to
5*
C
be a full inhibition
2100 0
intermediate cyclase (29).
tion (Fig. 2A; Table partial agonists in
St
MECA
and
0
lOOnM
1pM
xAc
tal
10iM
model
cyclase concentrations
Fig. 4. IB-MECA stimulated lnsPs formation in rat hippocampal slices and effect of XAC. A, myo-[H]inositol-labeled slices were incubated with lB-MECA at the concentrations indicated on the x-axis. PIP2 hydrolysis was assayed as described in Materials and Methods. *, p < 0.02 with respect to basal lnsPs formation. B, myo-[3H]inositol-Iabeled slices were incubated with 10 M IB-MECA in the absence or presence of graded XAC concentrations. InsP formation was assayed as described in Materials and Methods. *, p < 0.01 with respect to basal lnsP formation; **, p < 0.02 with respect to 10 M IB-MECA.
receptors
a principal in mammalian
A discrepancy the
binding
cyclase
assay
discrepancy benzyladenosine-5 differences curves
due
‘
In
in the and
that
study,
DBXRM
and
the
accumulation values agreement pounds
in the
micromolar the rat
17
IC50
for
been
at
human at
classified lower
(Table
thines
29).
functional
A similar
receptors and
moderate
the
functional
xanthine
analogue
slices (Table
riboside NECA
(Fig.
the
2B),
1). These
are
for evalcomplex
9).
very
high and
intermediate (10) and
Thus,
these
findings
in the
PLC
effects.
with results
InsP
the
Adenosine
EC50 are
in
relative binding affinities of these comA3 receptors (9, 29). Although lacking an
inhibit could XAC
A,
brain C8
receptors
be hypothesized or CPX stimulated
position,
very support
histamine-activated
of A1 receptor-mediated
much
as
that
was
was
the
slices). agonists
2-fold.
This
previously
and
previously
reported
A3-mediated
also
did not behave
on InsP
were PLC
antagonizing in radioligand is 29 M (11).
an acidic has
been
phe-
reported
sheep and human A3 receptors (11). ofA3
previously
Such
xan-
in
receptors
demonstrated in rat
that the “paradoxical” PLC, could be related inhibition.
of
in brain
of certain
tM)
involvement
activity
to
as a full
formation
containing which
rela-
inhibition
affinity at cloned weakly at rat the
is consistent A3 receptor
rat
nonselective
(10-100
derivative
in the
derivative
phenomenon
were effective value of XAC A3 receptors
K
The
rat
effects.
stimulated
effects
BWA1433)
is a xanthine
to bind with A3 receptors
study
concentrations
cloned
substitution
in the
to its
activation.
at
this
Cl-IB-MECA the cloned the
which
(9), in this
(XAC
as
for
brain
at other adenosine receptors. SPA, or DBXR with respect
agonist
respect
receptor
rat
ofvarious
agonist
Interestingly,
with
apparent
of APNEA,
as a partial
PLC
nyl
agonist
DBXR,
BWA1433
and
used
Only
partial
agonist
binding
dose-response
of other
agonist
by
IB-MECA and affinity toward
cyclase
A3
study,
A3 receptor,
differs
This
as IB-
in the experimen-
(7); the efficacy
receptors
1; Ref.
1).
cloned
adenosine
one
Table
present
reported
potency
weak
behave
and
systems
range
in
the
to the A3-selective with their lower
2B;
transfected
least
in A3-mediated in PLC activa-
to differences
in the
as a full
The
slices.
formation
striatal
values
of InsP formation by N(Table 1). Such consistent and
the
A3
includes
K1 values in by a factor of 40)
messenger
adenosine
in rat
with at cloned
A3
the
experimental
second
present
the and
lower
1; Refs.
binding
interactions
the
being
to a number
to differences
protein
latter
to stimulation -uronamides
common
binding
between assay
(Table
between are
uating
the
noted applies
for
the
Specifically
already
(Fig.
be related
and,
has
adenylyl
functional
(i.e.,
previously
mechanism
brain.
for A3 agonists
adenylyl
was
transduction
(i.e.,
assay
tively represents
might
potency However,
1), both DBXRM and NECA behaved comparison with the efficacies of
Cl-IB-MECA
discrepancy
50
ity
agonist of of adenylyl
brain
to (28).
effect, in which to antagonism
antagonism
would
It
Downloaded from molpharm.aspetjournals.org at ASPET Journals on June 9, 2016
B 200
i-
(M)
in rat striatum. Forskolin-stimulated adenylyl cyclase activity was sayed in the presence of the CI-IB-MECA concentrations indicated the x-axis in the absence or presence of 1 MM CPX. Results
10iM
concentrations
IB-MECA
i-
concentration
Fig. 5. Effect of CI-IB-MECA
50
io-
10
Agonist
1044
et aL
Abbracchio
lead to a net increase of stimulation of PLC activity in the presence of 10 tM IB-MECA. The disappearance of the stimulation at higher XAC concentrations might be due to antagonism of the A3 receptor-mediated effect, which is in agreement with the K values of this antagonist at cloned rat A3 receptors (11). To further support this hypothesis, the highly selective A, receptor antagonist CPX also potentiated stimulation of PIP2 hydrolysis by IB-MECA in slices (data not
4. Meyerhof, W., novel putative genesis. FEBS 5. Zhou, Q.-Y., C.
Molecular
that
A3 receptor-mediated
increases
of inos-
ito! phosphates
in the ischemic brain may contribute to neurodegeneration by raising intracellular calcium levels. In general, PLC activation leads to a rise in intracellular calcium. “Calcium-mobilizing” receptors in the central nervous system have been involved in pathological changes associated with excessive intracellular calcium concentrations, e.g.,
7.
the
brain
are
hypoxia
(39,
40).
in tive
A3 agonist
ciated bils
massively
that,
antagonist
and might
ministration A
in the
prove
to be
and
to
adaptive
receptor
uating animals In
14.
ad-
demonstrated (20).
of brain
A3
(agonist-induced hypothesized
(41-46).
Future
in ger-
Such regieffects might
receptors
studies
aimed
PLC stimulation with A3 agonists
the
pathophysiology
for
present
at eval-
in the brains of will clarify this
results
provide
a
18.
molecular
be the basis of the previously desystem effects of adenosine A3 recepthe present data support the hypothA3
receptors
of cerebral disorders) drug
17.
19.
adenosine
neurological
16.
after
desensitizafor other adeno-
point.
that
13.
an A3
acute
and
may
play
a
role
in
ischemia
(and,
possibly,
of other
may
represent
a novel
they
20.
21.
the
that
22.
development. 23.
Acknowledgments
We thank statistical
and
Dr. Enrico analysis
and
Dr. A. De Matteis
discussion.
We
thank
Rovati
(University
Dr.
Balduini
W.
(Consorzio Dr.
Norbert
Mario
ofMilan)
for help
(University
Negri
Bischofberger
ofUrbino,
Sud, for
Italy) his
with
the
24.
Italy)
for useful
25.
encourage-
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Jacobson.
ischemia-associated
was
changes
subtypes
conclusion,
target
Thus, on
against
to agonists way to that
mechanism that might scribed central nervous tor agonists. Moreover, esis
mobilization.
Pharmacol.
J.
298-306 (1994). 11. Ji, X.-D., D. A. J.
of
may contribof intracellular
with IB-MECA of IB-MECA-induced
A3 receptor-mediated chronically treated
important
in ger-
activation
cerebroprotective
effect
chronic exposure tion), in a similar sine
brain,
adenosine increases
destruction
treatment inversion
men-dependent related
ischemic
Sci.
C. A., M. A. Jacobson, Molecular cloning and characterization
species-differences
15.
neuronal
chronic
tor. Mol.
10. Linden,
ischemia-asso-
mortality
of an adenosine
Acad.
sine receptor. Proc. Nati. Acad. Sci. USA 90:10365-10369 (1993). 8. Sajjadi, F. G., and G. S. Firestein. cDNA cloning and sequence analysis of the human A3 adenosine receptor. Biochem. Biophys. Acta 1179i105-107 (1993). 9. Van Galen, P. J. M., A. H. Van Bergen, C. Gallo-Rodriguez, N. Melman, M. E. Olah, A. P. IJzerman, G. L. Stiles, and K A. Jacobson. Binding-site model and structure-activity-relationships for the rat A3-adenosine recep-
12.
selec-
in stroke.
cerebroprotective
mortality bils after
levels and
of the
aggravated
calcium
(24-26,
ischemia
enhanced
by endogenous through
consequent
during
administration
in vivo and
degeneration
it is likely
levels
acute
IB-MECA
cerebral A3 receptors ute to neurodegeneration InsP
increased
Because
neuronal (20),
be
Salvatore,
Nati.
E.
von
Species-differences
Lubitz,
M.
in ligand
Downloaded from molpharm.aspetjournals.org at ASPET Journals on June 9, 2016
agingand ischemia-associated neuronal cell death 38). It has been known for a long time that adenosine
and characterization
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