Accelerated resequestration of cytosolic calcium and suppression of the pro-inflammatory activities of human neutrophils by CGS 21680 in vitro

1 We have investigated the effects of the adenosine A(2A) receptor agonist CGS 21680 (0.01-1 mu M) on reactive oxidant production by, and elastase release from FMLP-activated human neutrophils, as well as on cytosolic Ca2+ fluxes and intracellular concentrations of cyclic AMP. 2 Oxidant production, elastase release and cyclic AMP were assayed using lucigenin-enhanced chemiluminescence, colourimetric and radioimmunoassay procedures respectively, while cytosolic Ca2+ fluxes were measured by fura-2 spectrofluorimetry in combination with radiometric procedures which distinguish between net efflux and influx of the cation. 3 Treatment of neutrophils with CGS 21680 did not affect the FMLP-activated release of Ca2+ from intracellular stores, but resulted in dose-related acceleration of the rate of decline in fura-2 fluorescence, as well as decreases in both efflux and store-operated influx of Ca2+, compatible with enhancement of resequestration of the cation by the endo-membrane Ca2+-ATPase. These effects on neutrophil Ca2+ handling were associated with increased intracellular cyclic AMP and with inhibition of oxidant production and release of elastase. 4 In contrast, treatment of neutrophils with the selective A(2A) receptor antagonist, ZM 241385 (2.5 mu M), prevented the transient increase in cyclic AMP in FMLP-activated neutrophils which was associated with delayed sequestration of incoming Ca2+ during store-operated influx. 5 The CGS 21680-mediated reduction of Ca2+ efflux from FMLP-activated neutrophils was also antagonized by pretreatment of the cells with ZM 241385 (2.5 mu M), as well as by thapsigargin (1 mu M), an inhibitor of the endo-membrane Ca2+-ATPase. ZM 241385 also neutralized the cyclic AMP-elevating and anti-inflammatory interactions of CGS 21680 with neutrophils. 6 We conclude that A(2A) receptors regulate the pro-inflammatory activities of human neutrophils by promoting cyclic AMP-dependent sequestration of cytosolic Ca2+.