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 subtype cloned from testis and brain cDNA libraries. N-6-2-(4-Amino-3-[I-125]iodophenyl)ethyl adenosine, a high affinity A(1)AR agonist, has served as the only radioligand available for study of the A(3)AR. The relatively low affinity of N-6-2-(4-amino-3-[I-125] iodophenyl)ethyladenosine for the A(3)AR and its greater A(1)AR selectivity necessitate the development of more appropriate radioligands for A(3)AR analysis. This report characterizes I-125-4-aminobenzyl-5'-N-methylcarboxamidoadenosin 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 hamster ovary (CHO) cells expressing the rat A(3)AR and from the rat mast 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-MECA binding, levels of A(3)AR expresssion in the A(3)AR-CHO cell line and 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 competition 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 cells (-)-N-6-[(R)-phenylisopropyl]adenosine and NECA were equipotent. Xanthine amine congener (XAC) and 3-cyclopentyl-1,3-dipropylxanthine did not significantly inhibit I-125-AB-MECA binding. The parent compound, AB-MECA, dose-dependently inhibited forskolin-stimulated adenylyl cyclase activity in A(3)AR-CHO cell membranes. I-125-AB-MECA bound to the rat A(1)AR and canine A(2a)AR expressed in COS-7 cells with K-d values of 3.42 +/- 0.43 nM and 25.1 +/- 12.6 nM, respectively. This binding 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 level of radioligand binding by similar to 5%.