Functional characterization of the A 2b adenosine receptor in NIH 3T3 fibroblasts

Abstract

The adenosine (ADO) receptor in NIH 3T3 fibroblasts was characterized using a series of adenosine agonists and selected xanthine and non-xanthine antagonists. The ADO receptor elicited accumulations of cyclic AMP in intact NIH 3T3 fibroblasts and caused activation of adenylate cyclase in membrane preparations. The receptor had characteristics of the A 2b subtype of adenosine receptor. ADO analogs had relatively high EC 50 values at the receptor and were antagonized competitively by xanthines. The rank order of potency for adenosine analogs in NIH 3T3 fibroblasts for cyclic AMP accumulation was: NECA > 2-CIADO > R-PIA ⪢ CV1808, CGS 21680. The EC 50 for 2-CIADO was 4.3 μM in intact cells and 15 μM in membrane preparations. All ADO analogs were more potent at the A 2a receptor of pheochromocytoma PC12 membranes than at the A 2b receptor of fibroblast NIH 3T3 membranes. Structure-activity relationships suggested that the regions of interaction with 5′- and N 6-substituents of ADO were similar for both the PC12 A 2a and NIH 3t3 A 2b receptor. However, ADO analogs with large substituents in the 2′-position, such as 2-cyclohexylethoxyADO and CGS 21680, were highly selective for the A 2a receptor. All ADO analogs tested were stimulatory to adenylate cyclase at the NIH 3T3 A 2b receptor, including 5′-methylthioADO, which was a weak partial agonist. A series of xanthine antagonists were not selective for the NIH 3T3 A 2b versus the PC12 A 2a receptor. In all cases, xanthines were more potent as antagonists in the intact NIH 3T3 membranes. In a series of non-xanthine antagonists, most compounds were equipotent or slightly more potent at the A 2a receptor except for alloxazine, which was approximately 9-fold selective for the A 2b receptor.