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

Abstract

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 suspensionswith 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 (PKC), 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 translocation of both PKC, 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 through Ado A2a receptor occupancy.

Autocrine activation of adenosine A1 receptors blocks D1A but not D1B dopamine receptor desensitization

“…Taken together, these data indicate firstly that adenosine receptors in COS-7 cells are permanently activated by adenosine released by the cells, and secondly that they attenuate agonist-induced desensitization of D 1A but not D 1B dopamine receptors. The autocrine role of adenosine previously been observed in GH4 pituitary cells (Zapata et al 1997), neutrophils (Thibault et al 2000) and epithelial cells (Musante et al 1999). These data further support the role of adenosine as a tonic modulator of cell responses.…”

Exercise, Immunity, and Illness

“…Although considered to be a major factor, activation of the HPA axis alone cannot wholly account for changes in the neutrophil function (Laing et al, 2008), as some of the noted inhibitory effects of cortisol on receptor mediated responses (e.g., fMLP) would not explain decreased responses to stimulants that activate neutrophils independent of surface receptors (e.g., PMA) (O'Flaherty et al, 1991;Tomchek et al, 1991;Peake, 2002). Thus other mechanisms have also been suggested to be involved in neutrophil dysfunction, where elevations in catecholamines, cyclic adenosine monophosphate, complement proteins (C5a), direct cellular oxidative damage and growth hormone may occur with the inflammatory response to prolonged exercise and have been shown to interfere with calcium signaling or other intermediates of intracellular pathways (Henson et al, 1978;Hack et al, 1994;Suzuki et al, 1999;Thibault et al, 2000;Tintinger et al, 2001;Robson et al, 2003;Laing et al, 2008).…”

Cytohesin-1 regulates fMLF-mediated activation and functions of the β2 integrin Mac-1 in human neutrophils

“…PLB-985 (4ϫ10 7 cells/ml) in RPMI 1640 supplemented with 10 mM NaCl was incubated with 20 M SecinH3 or an equal volume of DMSO for 1 h at 37°C prior to treatment with 1 mM DFP for 10 min at room temperature. The cells were warmed for 5 min at 37°C, incubated 5 min with 10 M CB and 0.1 U/ml ADA to eliminate adenosine [27,28], and stimulated with 100 nM fMLF for 2 min. Incubations were stopped by diluting the cells fivefold with ice-cold HBSS and centrifuging (700 g, 7 min at 4°C).…”