ADENOSINE A(2) RECEPTOR OCCUPANCY REGULATES STIMULATED NEUTROPHIL FUNCTION VIA ACTIVATION OF A SERINE THREONINE PROTEIN PHOSPHATASE/

Adenosine modulates generation of superoxide anion by neutrophils via occupancy of specific adenosine A, receptors, However, the intracellul ar signal transduction pathways by which occupancy of neutrophil aden osine A, receptors inhibits superoxide anion generation (O-2 (radical ion)) are not web understood. We, therefore, tested the hypothesis tha t signaling at polymorphonuclear leukocyte (PMN) adenosine receptors p roceeds via activation of a serine/threonine protein phosphatase (pp), Both the specific pp1 inhibitor calyculin A (10 nM) and the pp2A inhi bitor okadaic acid (10 mu M) enhanced O-2(radical ion) generation (185 +/- 24 and 189 +/- 35% of control, respectively, p < 0.0001 for both, n = 8), as reported previously, Calyculin A, but not okadaic acid, co mpletely reversed inhibition of stimulated O-2(radical ion) generation by the adenosine A2 receptor agonist 5'-N-ethylcarboxamidoadenosine ( NECA; IC50 = 30 nM ; p < 0.0001, analysis of variance). Calyculin A al so reversed the adenosine receptor-mediated desensitization of bound c hemoattractant receptors in neutrophils. Treatment of PMNs with NECA i ncreased the pp1 activity of crude membrane preparations in a time- an d dose-dependent fashion (EC(50) = 40 nM; p < 0.001, analysis of varia nce, n = 5). NECA inhibited cytosolic protein phosphatase activity by 78 +/- 12% (p < 0.003, n = 6) but did not shift pp1 catalytic subunit from cytosol to plasma membrane. Similar changes were observed in neut rophil cytoplasts depleted of organelles and nucleus. Moreover, the se lective protein kinase A inhibitor KT5720 (10 mu M) reversed the capac ity of dibutyryl cAMP but mt NECA to increase pp1 activity (p < 0.01, n = 5) in keeping with its effects on O-2 generation. Western blot ana lysis of PMN subcellular fractions demonstrated the presence of pp1 al pha and pp1 gamma 1 but not pp1 gamma(2) isotypes in both cytosol and plasma membrane but not in azurophil or specific granules. We conclude from these studies that signal transduction by adenosine in PMN proce eds via a novel pathway: cAMP-independent activation of a serine/threo nine protein phosphatase in the plasma membrane.