Two pathways of activation of the superoxide-generating NADPH oxidase of phagocytes in vitro - Distinctive effects of inhibitors

Natalia Sigal, Yara Gorzalczany, Edgar Pick

Research output: Contribution to journalArticlepeer-review

Abstract

The NADPH oxidase complex of phagocytes comprises a membrane-associated flavocytochrome b559, and 4 cytosolic components: p47phox, p67phox, p40phox, and the small GTPase Rac. Activation of the oxidase in vivo is the result of assembly of the cytosolic components with cytochrome b559 and is mimicked in vitro by a cell-free system consisting of membranes, p47phox, p67phox, nonprenylated or prenylated Rac, and an anionic amphiphile as activator (defined as "p47phox and amphiphile-dependent" or canonical pathway). We reported that prenylated Rac1 is capable of activating the NADPH oxidase in vitro in the absence of p47phox and amphiphile (defined as "p47phox 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 p67phox; 2) GDP and, paradoxically, GTP (but not GMP, ATP, ADP, and AMP) prevent oxidase activation by the p47phox 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 p47phox 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 p47phox 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 p47phox and amphiphile-independent pathway, is a reflection of interference by the inhibitors with Rac-dependent recruitment of p67phox to the membrane.

Original languageEnglish
Pages (from-to)147-159
Number of pages13
JournalInflammation
Volume27
Issue number3
DOIs
StatePublished - Jun 2003

Keywords

  • NADPH oxidase
  • Phagocytes
  • Rac1
  • Superoxide
  • p47
  • p67

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