Two Pathways of Activation of the Superoxide-Generating NADPH Oxidase of Phagocytes In Vitro—Distinctive Effects of Inhibitors

Abstract

The NADPH oxidase complex of phagocytes comprises a membrane-associated flavocytochrome b ₅₅₉, and 4 cytosolic components: p47^phox, p67^phox, p40^phox, and the small GTPase Rac. Activation of the oxidase in vivo is the result of assembly of the cytosolic components with cytochrome b ₅₅₉ and is mimicked in vitro by a cell-free system consisting of membranes, p47^phox, p67^phox, nonprenylated or prenylated Rac, and an anionic amphiphile as activator (defined as “p47^phox and amphiphile-dependent” or canonical pathway). We reported that prenylated Rac1 is capable of activating the NADPH oxidase in vitro in the absence of p47^phox and amphiphile (defined as “p47^phox and amphiphile-independent” pathway). We now demonstrate that the 2 pathways exhibit distinctive susceptibilities to inhibitors: 1) The anionic amphiphile lithium dodecyl sulfate, an activator of the canonical pathway, has the opposite effect (inhibition) on oxidase activation by prenylated Rac and p67^phox; 2) GDP and, paradoxically, GTP (but not GMP, ATP, ADP, and AMP) prevent oxidase activation by the p47^phox and amphiphile-independent pathway but do not affect activation by the canonical pathway; 3) The Rac-binding domain of p21-activated kinase is a potent inhibitor of activation by the p47^phox and amphiphile-independent pathway while exerting a milder inhibitory effect on the canonical pathway; 4) The C-terminal polybasic Rac1 peptide 177–191 and the cationic antibiotic neomycin sulfate inhibit activation by the canonical pathway but do not affect activation by the p47^phox and amphiphile-independent pathway; 5) Binding of prenylated Rac1 to membrane-mimicking phospholipid vesicles is, nevertheless, enhanced when these contain negatively charged lipids. It is proposed that preferential inhibition of oxidase activation, via the p47^phox and amphiphile-independent pathway, is a reflection of interference by the inhibitors with Rac-dependent recruitment of p67^phox to the membrane.