Apparent involvement of the A(2A) 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 down-regulate 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 Ca(2+) in mediating these effects. Neutrophils were treated with varying concentrations (0.01-1 microM) of AR agonists operative at A(1) (N(6)-cyclopentyladenosine, CPA), A(2A) (2(4-[(2-carboxyethyl)phenyl]ethylamino)-5'-N-ethylcarboxamidoadenosi ne, 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 microM). Intracellular cAMP, superoxide, and elastase were assayed using radioimmunoassay, lucigenin-enhanced chemiluminescence (LECL), and colorimetric procedures, respectively, while changes in the concentrations of cytosolic Ca(2+) 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 microM). 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 Ca(2+) 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 microM). 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 Ca(2+) handling.