Elsevier

Biochemical Pharmacology

Volume 47, Issue 3, 9 February 1994, Pages 563-572
Biochemical Pharmacology

Inhibition of neutrophil function by aspirin-like drugs (NSAIDS): Requirement for assembly of heterotrimeric G proteins in bilayer phospholipid

https://doi.org/10.1016/0006-2952(94)90189-9Get rights and content

Abstract

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 (Gαi). Plasma membrane and cytosol of human neutrophils were prepared by nitrogen eavitation and discontinuous sucrose density centrifugation. Salicylate (3 mM) and piroxicam (50 μM) reduced [35S]GTPγS binding to purified plasma membranes [65 ± 3.7 and 75 ± 5.3% (P < 0.003) of control, respectively]. Membrane-associated βγ was solubilized by treatment of plasma membranes with sodium cholate. NSAIDs did not inhibit binding of GTP to solubilized βγ 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 GTPγS binding to βγ 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 Gαi exists as a free subunit, suggesting that the effect required either assembly of Gαiβγ heterotrimer or the presence of a lipid bilayer. Although the addition of purified bovine βγ subunits to dialyzed cytosol increased both the total GIP binding capacity and the pertussis toxin-dependent ADP-ribosylation of Gαi, consistent with assembly of a G protein heterotrimer, NSAIDs had no effect on GTP binding. In contrast, NSAIDs inhibited GTP binding to heterotrimeric cytosolβγbovine 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 α subunit of a regulatory G protein, an effect which requires assembly of the active heterotrimer Gαiβγ in a phospholipid bilayer.

References (39)

Cited by (0)

View full text