Apparent involvement of the A2A subtype adenosine receptor in the anti-inflammatory interactions of CGS 21680, cyclopentyladenosine, and IB-MECA with human neutrophils

This study was undertaken to identify the adenosine receptor (AR) subtypes which downregulate the proinflammatory activities of human neutrophils, as well as the involvement of adenosine 3',5'-cyclic monophosphate (cAMP) and its relationship to cellular handling of Ca2+ in mediating these effects. Neutrophils were treated with varying concentrations (0.01-1 mu M) of AR agonists operative at A(1) (N-6-cyclopencyladenosine, CPA), A(2A) (2(4-[(2-carboxyethyl)phenyl]ethylamino)-5'-N-ethylcarboxamidoadenosine, CGS 21680), and A(3) (N-6-(3-iodobenzyl-5)-N-methylcarbamoyladenosine, IB-MECA) receptors, after which they were activated with the chemoattractant, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP, 1 mu M). Intracellular cAMP, superoxide, and elastase were assayed using radioimmunoassay, lucigenin-enhanced chemiluminescence (LECL), and colorimetric procedures, respectively, while changes in the concentrations of cytosolic Ca2+ were monitored by fura-2-based spectrofluorimetry. CGS 21680, at all concentrations tested, inhibited superoxide production in a dose-related manner, while CPA and IB-MECA were effective only at the highest concentrations tested (0.5-1 mu M). The release of elastase from activated neutrophils was also inhibited by all three AR agonists, but was more sensitive to CGS 21680 and IB-MECA than was superoxide production. The inhibitory effects of all 3 agonists on superoxide production and elastase release were associated with accelerated clearance of Ca2+ from the cytosol of activated neutrophils, and were effectively neutralized by pretreatment of the cells with the highly selective A(2A)R antagonist, ZM 241385 (4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5yl amino]ethyl)phenol). Increased cAMP was detected in neutrophils treated with CGS 21680 and IB-MECA (1 mu M) These data support the involvement of the A(2A)R subtype in the suppression of superoxide production and degranulation by activated human neutrophils, probably by cAMP-mediated alterations in Ca2+ handling. BIOCHEM PHARMACOL 60;7:993-999, 2000. (C) 2000 Elsevier Science Inc.