I-125 4-AMINOBENZYL-5'-N-METHYLCARBOXAMIDOADENOSINE, A HIGH-AFFINITY RADIOLIGAND FOR THE RAT A(3) ADENOSINE RECEPTOR

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%.