Adenosine A2 receptor occupancy regulates stimulated neutrophil function via activation of a serine/threonine protein phosphatase

Abstract

Adenosine modulates generation of superoxide anion by neutrophils via occupancy of specific adenosine A(2A) receptors. However, the intracellular signal transduction pathways by which occupancy of neutrophil adenosine A(2A) receptors inhibits superoxide anion generation (O2/.) are not well understood. We, therefore, tested the hypothesis that signaling at polymorphonuclear leukocyte (PMN) adenosine receptors proceeds via activation of a serine/threonine protein phosphatase (pp). Both the specific pp1 inhibitor calyculin A (10 nM) and the pp2A inhibitor okadaic acid (10 μM) enhanced O2/. 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, completely reversed inhibition of stimulated O2/-. generation by the adenosine A2 receptor agonist 5'-N- ethylcarboxamidoadenosine (NECA; IC50 = 30 nM; p < 0.0001, analysis of variance). Calyculin A also reversed the adenosine receptor-mediated desensitization of bound chemoattractant receptors in neutrophils. Treatment of PMNs with NECA increased the pp1 activity of crude membrane preparations in a time- and dose-dependent fashion (EC50 = 40 nM; p < 0.001, analysis of variance, 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 neutrophil cytoplasts depleted of organelles and nucleus. Moreover, the selective protein kinase A inhibitor KT5720 (10 μM) reversed the capacity of dibutyryl cAMP but not NECA to increase pp1 activity (p < 0.01, n = 5) in keeping with its effects on O2/. generation. Western blot analysis of PMN subcellular fractions demonstrated the presence of pp1α and pp1γ1 but not pp1γ2 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 proceeds via a novel pathway: cAMP-independent activation of a serine/threonine protein phosphatase in the plasma membrane.