Characterization by flow cytometry of fluorescent, selective agonist probes of the A3 adenosine receptor

Various fluorescent nucleoside agonists of the A(3) adenosine receptor (AR) were compared as high affinity probes using radioligands and flow cytometry (FCM). They contained a fluorophore linked through the C2 or N-6 position and rigid A(3)AR-enhancing (N)-methanocarba modification. A hydrophobic C2-(1-pyrenyl) derivative MRS5704 bound nonselectively. C2-Tethered cyanine5-dye labeled MRS5218 bound selectively to hA(3)AR expressed in whole CHO cells and membranes. By FCM, binding was A(3)AR-mediated (blocked by A(3)AR antagonist, at least half through internalization), with t(1/2) for association 38 min in mA(3)AR-HEK293 cells; 26.4 mm in sucrose-treated hA(3)AR-CHO cells (K-d 31 nM). Membrane binding indicated moderate mA(3)AR affinity, but not selectivity. Specific accumulation of fluorescence (50 nM MRS5218) occurred in cells expressing mA(3)AR, but not other mouse ARs. Evidence was provided suggesting that MRS5218 detects endogenous expression of the A(3)AR in the human promyelocytic leukemic HL-60 cell line. Therefore, MRS5218 promises to be a useful tool for characterizing the A(3)AR. Published by Elsevier Inc.