Me. Olah et al., I-125 4-AMINOBENZYL-5'-N-METHYLCARBOXAMIDOADENOSINE, A HIGH-AFFINITY RADIOLIGAND FOR THE RAT A(3) ADENOSINE RECEPTOR, Molecular pharmacology, 45(5), 1994, pp. 978-982
The rat A, adenosine receptor (AR) is a recently characterized AR subt
ype cloned from testis and brain cDNA libraries. N-6-2-(4-Amino-3-[I-1
25]iodophenyl)ethyl adenosine, a high affinity A(1)AR agonist, has ser
ved as the only radioligand available for study of the A(3)AR. The rel
atively low affinity of N-6-2-(4-amino-3-[I-125] iodophenyl)ethyladeno
sine for the A(3)AR and its greater A(1)AR selectivity necessitate the
development of more appropriate radioligands for A(3)AR analysis. Thi
s report characterizes I-125-4-aminobenzyl-5'-N-methylcarboxamidoadeno
sin e (I-125-AB- MECA), a high affinity radioligand for the A(3)AR, in
two cell lines that express this AR subtype. Membranes from Chinese h
amster ovary (CHO) cells expressing the rat A(3)AR and from the rat ma
st cell line RBL-2H3 bound I-125-AB-MECA with K-d values of 1.48 +/- 0
.33 nM and 3.61 c 0.30 nM, respectively. As determined by I-125- AB-ME
CA binding, levels of A(3)AR expresssion in the A(3)AR-CHO cell line a
nd RBL-2H3 cells were 3.06 +/- 0.21 pmol/mg and 1.02 +/- 0.13 pmol/mg,
respectively. Binding of I-125-AB-MECA was characterized in competiti
on assays. In the A,AR-CHO cell line a potency ord er of cyclohexyl-5'
-N-ethylcarboxamidoadenosine (cyclohexyl-NECA) = benzyl-NECA > (-)-N-6
-[(R)-phenylisopropyl]adenosine = NECA was observed, and in RBL-2H3 ce
lls (-)-N-6-[(R)-phenylisopropyl]adenosine and NECA were equipotent. X
anthine amine congener (XAC) and 3-cyclopentyl-1,3-dipropylxanthine di
d not significantly inhibit I-125-AB-MECA binding. The parent compound
, AB-MECA, dose-dependently inhibited forskolin-stimulated adenylyl cy
clase activity in A(3)AR-CHO cell membranes. I-125-AB-MECA bound to th
e rat A(1)AR and canine A(2a)AR expressed in COS-7 cells with K-d valu
es of 3.42 +/- 0.43 nM and 25.1 +/- 12.6 nM, respectively. This bindin
g was significantly reduced in the presence of 1 mu M XAC. In RBL-2H3
cells, XAC had no effect on I-125-AB-MECA affinity and reduced the lev
el of radioligand binding by similar to 5%.