Characterization of expression and function of adenosine receptors in mouse neutrophils

Abstract

Cardioprotective effects of adenosine are thought to be due, in part, to stimulation of adenosine receptors (ARs) on inflammatory cells. Aims of this study were to characterize the expression of ARs and to determine their regulatory roles in mouse neutrophils. Real-time PCR analysis revealed similar levels of A2AAR and A3AR transcripts, followed by A2BAR and negligible amounts of A1AR. Radioligand binding using the selective A2AAR antagonist [125I]ZM241385 and A3AR agonist [125I]AB-MECA also suggested that A2AAR and A3AR proteins are expressed at comparable levels. To determine the effect of AR activation on superoxide (O2.-) production by neutrophils, cells were treated with AR agonists for 30 minutes prior to addition of stimuli and O2.- produced was measured using the chemiluminescent probe MCLA. The non-selective AR agonist NECA, A2AAR agonist CGS21680 and A3AR agonist CP-532,903 inhibited O2.- production by wild type (WT) neutrophils stimulated with N-formyl peptide fMLP, complement C5a or platelet activating factor but not with phorbol myristate acetate. With fMLP stimulation, both CGS21680 and CP-532,903 inhibited O2.- production with similar potency (EC50 values of 16.43 ± 2.07 and 38.10 ± 1.60 nM respectively). CP-532,903 also inhibited directed migration of WT neutrophils towards high concentrations of fMLP. Specificity of the agonists was confirmed by repeating the assays with neutrophils from A2AAR or A3AR gene knock-out mice. In conclusion, while activation of A2AAR leads to inhibition of superoxide production, A3AR activation inhibits both superoxide production as well as migration of activated mouse neutrophils. This work was funded by the NIH