Adenosine receptor occupancy suppresses chemoattractant-induced phospholipase D activity by diminishing membrane recruitment of small GTPases

Adenosine (Ado) is an important autocrine modulator of neutrophil functions. In this study, we determined the effects of endogenous Ado on fMet-Leu-Phe (fMLP)induced phospholipase D (PLD) activity in neutrophils. The removal of extracellular Ado by Ado deaminase (ADA) or the blockade of its action by the A2a receptor antagonists 8-(3-chlorostyryl) caffeine (CSC) or CGS15943 markedly increased fMLP-induced PLD activation. The concentration-dependent stimulatory effects of CSC and CGS15943 were abolished by a pretreatment of neutrophil suspension-swith ADA, In contrast, the selective A2a receptor agonist CGS21680 suppressed fMLP-induced PLD activation. Furthermore, inhibition by CGS21680 of fMLP-induced PLD activity was reversed by CSC or CGS15943, The removal of Ado by ADA or the blockade of its action by CSC or CGS15943, markedly increased the membrane recruitment of cytosolic protein kinase C alpha (PKC alpha), RhoA, and ADP-ribosylation factor (ARF) in response to fMLP, As shown for PLD activity, the stimulatory effect of Ado receptor antagonists on PLD cofactors translocation was abolished by a pretreatment of the cells with ADA, Moreover, the membrane trans-location of both PKCa, RhoA, and ARF in response to fMLP was attenuated by CGS21680 and this effect of the A2a receptor agonist was antagonized by CSC or CGS15943, These data demonstrate that Ado released by neutrophils in the extracellular milieu inhibits PLD activation by blocking membrane association of ARF, RhoA, and PKC alpha through Ado A2a receptor occupancy. (Blood, 2000;95:519-527) (C) 2000 by The American Society of Hematology.