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Fig. 2. Activation of the A_2A receptor specifically potentiates the expression of COX-2 from human neutrophils. (A) Human PMNs were stimulated for 60 minutes with indicated agonists, alone or in the presence of adenosine deaminase (ADA; 0.1 U/ml), or with ADA and the A_2AR agonist CGS 21680 (1 µM). Reactions were stopped and samples were processed for the determination of COX-2 by western immunoblotting. Shown are immunoblots from one experiment, typical of at least n=3 experiments performed in identical conditions with separate donors. fMLP, formyl-methionyl-leucyl-phenylalanine (100 nM); GM/TNF, mixture of granulocyte-macrophage colony-stimulating factor and tumor necrosis factor- (1 nM and 100 nM, respectively); PMA (10 nM), phorbol 12-myristate 13-acetate; LPS, lipopolysaccharide (100 ng/ml); Zop, opsonized zymosan (300 µg/ml); Zno, nonopsonized zymosan (300 µg/ml); Bop, opsonized bacteria (10⁸ bacteria/ml). (B) Human leukocytes were stimulated with fMLP or LPS, for 1 hour (PMNs) or 4 hours (Monocytes) alone or in the presence of ADA or with ADA and CGS 21680; samples were processed for the determination of COX-2 by western immunoblotting. In each condition, one typical immunoblot is shown and is representative of at least n=3 experiments performed in identical conditions with different donors. Bar graphs depict the expression of COX-2 protein levels, relative to those observed in leukocytes stimulated in the absence of ADA and CGS 21680, as determined by densitometric analysis of the bands in three independent experiments (mean±s.e.m.; n=3). *Statistically different from diluent-treated cells.

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