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 (O 2 . ) 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 subcellular fractions demonstrated the presence of pp1␣ and pp1␥1 but not pp1␥ 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 proceeds via a novel pathway: cAMP-independent activation of a serine/threonine protein phosphatase in the plasma membrane.Adenosine, an autacoid released by many different cell types, regulates a variety of stimulated neutrophil functions including production of superoxide anion generation (1, 2), ␤ 2 -integrin-and L-selectin-mediated adhesion to endothelial cells (3, 4), and phagocytosis (5). Adenosine regulates these neutrophil functions by coupling with specific cell surface receptors (2) on the neutrophil (reviewed in Ref. 6).Four different types of adenosine receptors (A 1 , A 2A , A 2B , and A 3 ) have been described, both at the molecular level and by pharmacological analyses (7). Based on pharmacologic data, it appears that the A 2A receptor on neutrophils mediates the inhibition of neutrophil superoxide anion generation, adhesion, and phagocytosis (6). Before their resolution at the molecular level, adenosine A 2 receptors were believed to modulate cellular function via activation of adenylyl cyclase, with cAMP as their intracellular messenger. As expected, occupancy of adenosine A 2 receptors stimulated the accumulation of cAMP in neutrophils, but unexpectedly, cAMP proved not to be the second messenger for inhibition of stimulated superoxide anion generation (8 -10).Because adenosine A 2 receptor occupancy had previously been shown to increase cytosolic protein phosphatase activity in bovine adrenal chromaffin (PC12) cells (11), we tested the hypothesis that occupancy of neutrophil adenosine receptors stimulates an increase in the serine/threonine protein phosphatase activity of the plasma membrane where it would be situated to modulate function of the neutrophil NADPH-oxidase. We found that the protein phosphatase 1 (pp1) 1 inhibitor calyculin A completely reversed the effects of adenosine receptor occupancy on stimulated neutrophil generation of superoxide anion. Moreover, adenosine receptor occupancy stimulated an increase in plasma membrane-associated protein phosphatase activity, most likely pp1, and that activation of this phosphatase is independent of cAMP. , N-formylmethionyl-leucyl-phenylalanine (FMLP), ATP, phosphorylase b, phosphorylase kinase, bovine serum albumin, Ficoll type 70, cytochalasin B, caffeine, cytochrome c, superoxide dismutase, and Brij were supplied by Sigma. K...

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