INHIBITION OF NEUTROPHIL FUNCTION BY ASPIRIN-LIKE DRUGS (NSAIDS) - REQUIREMENT FOR ASSEMBLY OF HETEROTRIMERIC G-PROTEINS IN BILAYER PHOSPHOLIPID

Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit neutrophil func tions via mechanisms that are independent of their effects on prostagl andin biosynthesis. We examined the effects of sodium salicylate and p iroxicam on GTP/GDP exchange by a regulatory G protein (G alpha i). Pl asma membrane and cytosol of human neutrophils were prepared by nitrog en cavitation and discontinuous sucrose density centrifugation. Salicy late (3 mM) and piroxicam (50 mu M) reduced [S-35]GTP gamma S binding to purified plasma membranes [65 +/- 3.7 and 75 +/- 5.3% (P < 0.003) o f control, respectively]. Membrane-associated G alpha/beta gamma was s olubilized by treatment of plasma membranes with sodium cholate. NSAID s did not inhibit binding of GTP to solubilized G alpha/beta gamma der ived from detergent-treated plasma membranes. Lipid reconstitution was achieved by detergent dialysis followed by the addition of bilayer li posomes (phosphatidycholine). Salicylate and piroxicam inhibited GTP g amma S binding to G alpha/beta gamma derived from solubilized plasma m embranes reconstituted in phosphatidylcholine vesicles (bilayer struct ures) but had no effect when phosphatidylethanolamine (hexagonal phase II structure) was used for reconstitution. Salicylate and piroxicam h ad no effect on GTP binding to cytosolic fractions in which soluble G alpha i exists as a free subunit, suggesting that the effect required either assembly of G alpha i/beta gamma heterotrimer or the presence o f a lipid bilayer. Although the addition of purified bovine py subunit s to dialyzed cytosol increased both the total GIP binding capacity an d the pertussis toxin-dependent ADP-ribosylation of G alpha i, consist ent with assembly of a G protein heterotrimer, NSAIDs had no effect on GTP binding. In contrast, NSAIDs inhibited GTP binding to heterotrime ric G alpha(cytosol)/beta gamma(bovine) when the complex was inserted into bilayer liposomes. The data indicate that salicylate and piroxica m disrupt neutrophil function via their capacity to interfere with GTP /GDP exchange at an cu subunit of a regulatory G protein, an effect wh ich requires assembly of the active heterotrimer G alpha i/beta gamma in a phospholipid bilayer.