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 functions via mechanisms that are independent of their effects on prostaglandin biosynthesis. We examined the effects of sodium salicylate and piroxicam on GTP/GDP exchange by a regulatory G protein (G alpha i). Plasma membrane and cytosol of human neutrophils were prepared by nitrogen cavitation and discontinuous sucrose density centrifugation. Salicylate (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) of control, respectively]. Membrane-associated G alpha/beta gamma was solubilized by treatment of plasma membranes with sodium cholate. NSAIDs did not inhibit binding of GTP to solubilized G alpha/beta gamma derived from detergent-treated plasma membranes. Lipid reconstitution was achieved by detergent dialysis followed by the addition of bilayer liposomes (phosphatidycholine). Salicylate and piroxicam inhibited GTP gamma S binding to G alpha/beta gamma derived from solubilized plasma membranes reconstituted in phosphatidylcholine vesicles (bilayer structures) but had no effect when phosphatidylethanolamine (hexagonal phase II structure) was used for reconstitution. Salicylate and piroxicam had 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 of a lipid bilayer. Although the addition of purified bovine py subunits to dialyzed cytosol increased both the total GIP binding capacity and the pertussis toxin-dependent ADP-ribosylation of G alpha i, consistent with assembly of a G protein heterotrimer, NSAIDs had no effect on GTP binding. In contrast, NSAIDs inhibited GTP binding to heterotrimeric G alpha(cytosol)/beta gamma(bovine) when the complex was inserted into bilayer liposomes. The data indicate that salicylate and piroxicam disrupt neutrophil function via their capacity to interfere with GTP/GDP exchange at an cu subunit of a regulatory G protein, an effect which requires assembly of the active heterotrimer G alpha i/beta gamma in a phospholipid bilayer.