Protein-Dependent Activation of Phospholipase C by Adenosine A3 ...

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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

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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


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


(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


(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


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-


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


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


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-

ment. 26. References 1. Fredhoim, B. B., M. P. Abbracchio, G. Burnstock, J. W. Daly, T. K Harden, K A. Jacobson, P. Leff, and M. Williams. Nomenclature and classification of purinoceptors. Pharmacol. Rev. 46:143-156 (1994). 2. van Galen, P. J. M., G. L. Stiles, G. Michaels, and K A. Jacobson. Adenosine A1 and A receptors: structure-function relationships. Med. Res. Rev. 12:423-471 (1992). 3. Tucker, A. L., and J. Linden. Cloned receptors and cardiovascular responses to adenosine. Cardiovasc. Res. 27:62-67 (1993).

A. S. Robeva,

S. M. Rapport. adenosine 44:524-532

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89:7432-7436 (1992). A. L. Tucker, J. H. Stehle, S. A. USA

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pharmacological Trends Pharmacol.

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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


agingand ischemia-associated neuronal cell death 38). It has been known for a long time that adenosine

and characterization

receptor. Proc. J., H. E. Taylor, Rivkees, J. S. Fink, and expression of a sheep A3 tribution. Mol. Pharmacol.

Johnson. speculate

cloning

adenosine 6. Linden,

shown). We

R. Muller-Brechlin, and D. Richter. Molecular cloning of a G protein coupled receptor expressed during rat spermioLett. 284:155-160 (1991). Li, M. E. Olah, R. A. Johnson, G. L. Stiles, and 0. Civelli.

of PLC by Adenosine

Activation 29.

30.

31.

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