105

Pharmacological Research, Vol . 25, Supplement 1, 1992 LYMPHOCYTES AND ADENOSINE RECEPTORS C . Dianzani, C . Ferretti, D . Colangelo and I . Viano Istituto di Farmacologia e Terapia Sperimentale, University degli Studi di Torino, via P . Giuria 13, Torino (Italy) . Key words : Adenosine, lymphocytes,

receptors

Adenosine has potent effects in many different cells and tissues [1] . A wide range of studies has shown the existence of two types of adenosine receptors [2] . Adenosine has several

inhibitory effects on the immune system and the

lack of adenosine deaminase is related to severe immunodeficiency [3] . The aim of this study was to assess the correlationship between the activity of

adenosine

on

lymphocyte

proliferation, evaluated

tritiated [ 3 H]-

as

(PHA),

thymidine incorporation in response to policlonal mitogenic stimulus the

adenosine

receptors

resting lymphocytes .

and

the intracellular

As shown in tab .

as A1 agonists had an

levels

of

cyclic

AMP

in

1, the compounds which are classified

inhibitory effect on the replication of lymphocytes,

on the contrary A1 antagonist had no significant effect on cell replication . The

binding

experiments

specific binding site,

on

lymphocyte

membranes suggested evidence

of

a

which has been determined according to Bruns et al .

[4], using labeled and unlabeled NECA (10 -4 M) . A Kd value of 61 ± 14 nM and a maximum receptorial

site

density value

calculated by Scatchard analysis .

of

Furthermore,

183

±

23

fmol/mg

prot .

were

by agonists and

competition

antagonists for [ 3 H]-NECA binding was measured to confirm its

specificity .

We found all displacers inhibiting [ 3 H]-NECA binding (tab .1), even if their potency

was

much

less

than

in

other tissues .

lymphocyte adenosine receptors exist

These

data

suggest that

in a low affinity state ;

this

is also

indicated by the high value of Kd . The

intracellular

showed cAMP,

that

the

while the

levels A1

of cAMP,

after

agonists inhibited

A 2 agonist did

1043-6618/92/2510105-02/$03 .00/0

not

treatment with

A1

or A 2

agonists

the intracellular accumulation

significantly modify

the

activity

of of

© 1992 The Italian Pharmacological Society

Pharmacological Research, Vol . 25, Supplement 1, 1992

106 adenylate

cyclase

Although CGS lymphocyte partial

according

21680 was

able

proliferation

and

to

the results

to displace CAMP

displacement of the A1

lymphocyte

[ 3 H]-NECA,

levels .

receptor

on

This

that

it

proliferation .

had

no effect

on

could be related to the

might occur

at

the

highest

concentrations tested . TABLE 1 3 [ H]-NECA binding

cyclic AMP % of controls

>100

0 .69

- 14

NECA (A1=A2 agonist)

51

0 .07

- 28

CPA (A1 agonist)

20

0 .25

- 50

5

0 .36

- 48

3 .21

nd

IC50 (uM)

[3

H]-thymidine incorporation

CGS 21680 (A2 agonist)

2-cloroadenosine (A1>A2 agonist) 8-(p-sulfophenyl) theophylline (A1>A2 antagonist)

>100

The IC50 values were calculated by log-probits analysis . [1251] kit in human Cyclic AMP levels were determined by RIA-NEN CAMP lymphocytes incubated with 1~1 M drugs for 15 min . The basal cyclic AMP concentration was 0 .8 pmol/10 cells References 1 . Daly J .W . Adenosine receptors : targets for future drugs . J . Med . Chem . 1982 ; 25 :197-207 . 2 . Stiles G .L . Adenosine receptors : structure function and regulation . TIPS 1986 ; 7 :486-490 . 3 . Dissing J ., Knudsen B ., Adenosine deaminase deficiency and combined immunodeficiency syndrome . Lancet 1972 ; 2 :1316 . 4 Bruns R ., Lu G .H ., Puusley T .A . Characterization of the A2 adenosine receptors labeled by [ H]-NECA in rat striatal membrane . Mol . Pharmacol . 1986 ; 29 : 331-346 .