FUNCTIONAL-CHARACTERIZATION OF THE A(2B) ADENOSINE RECEPTOR IN NIH 3T3 FIBROBLASTS

The adenosine (ADO) receptor in NIH 3T3 fibroblasts was characterized using a series of adenosine agonists and selected xanthine and non-xan thine antagonists. The ADO receptor elicited accumulations of cyclic A MP in intact NIH 3T3 fibroblasts and caused activation of adenylate cy clase in membrane preparations. The receptor had characteristics of th e A(2b) subtype of adenosine receptor. ADO analogs had relatively high EC(50) values at the receptor and were antagonized competitively by x anthines. The rank order of potency for adenosine analogs in NIH 3T3 f ibroblasts for cyclic AMP accumulation was: NECA > 2-ClADO > R-PIA muc h greater than CV1808, CGS 21680. The EC(50) for 2-ClADO was 4.3 mu M in intact cells and 15 mu M in membrane preparations. All ADO analogs were more potent at the A(2a) receptor of pheochromocytoma PC12 membra nes than at the A(2b) receptor of fibroblast NIH 3T3 membranes. Struct ure-activity relationships suggested that the regions of interaction w ith 5'- and N-6-substituents of ADO were similar for both the PC12 A(2 a) and NIH 3T3 A(2b) receptor. However, ADO analogs with large substit uents 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, i ncluding 5'-methylthioADO, which was a weak partial agonist. A series of xanthine antagonists were not selective for the NIH 3T3 A(2b) versu s the PC12 A(2a) receptor. In all cases, xanthines were more potent as antagonists in the intact NIH 3T3 cells than in 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, w hich was approximately 9-fold selective for the A(2b) receptor.