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-Ph e (fMLP)induced phospholipase D (PLD) activity in neutrophils. The removal of extracellular Ado by Ado deaminase (ADA) or the blockade of its action b y the A2a receptor antagonists 8-(3-chlorostyryl) caffeine (CSC) or CGS1594 3 markedly increased fMLP-induced PLD activation. The concentration-depende nt stimulatory effects of CSC and CGS15943 were abolished by a pretreatment of neutrophil suspension-swith ADA, In contrast, the selective A2a recepto r agonist CGS21680 suppressed fMLP-induced PLD activation. Furthermore, inh ibition by CGS21680 of fMLP-induced PLD activity was reversed by CSC or CGS 15943, The removal of Ado by ADA or the blockade of its action by CSC or CG S15943, markedly increased the membrane recruitment of cytosolic protein ki nase C alpha (PKC alpha), RhoA, and ADP-ribosylation factor (ARF) in respon se to fMLP, As shown for PLD activity, the stimulatory effect of Ado recept or antagonists on PLD cofactors translocation was abolished by a pretreatme nt of the cells with ADA, Moreover, the membrane trans-location of both PKC a, RhoA, and ARF in response to fMLP was attenuated by CGS21680 and this ef fect of the A2a receptor agonist was antagonized by CSC or CGS15943, These data demonstrate that Ado released by neutrophils in the extracellular mili eu inhibits PLD activation by blocking membrane association of ARF, RhoA, a nd PKC alpha through Ado A2a receptor occupancy. (Blood, 2000;95:519-527) ( C) 2000 by The American Society of Hematology.