Abstract
The effects of the trivalent arsenical phenylarsine oxide (PAO) on the activity of NADPH oxidase in human neutrophils were studied. PAO caused a rapid dose-dependent inhibition of superoxide generation which was maximal at a concentration of 1 microM, irrespective of the stimulating agent. This inhibitory effect was not due to impaired transduction of activation signals since neither degranulation nor phagocytosis were modified. When cytosolic and membrane fractions from resting neutrophils were combined to reconstitute the NADPH oxidase, O2-. generation was inhibited by PAO while translocation of the NADPH oxidase components to the plasma membrane fraction was not affected. The inhibition was completely and specifically reversed by 2,3-dimercaptopropanol, not by dithiothreitol or beta-mercaptoethanol, indicating that PAO binds covalently to spatially vicinal thiol groups. PAO inhibited the plasma membrane's capacity to initiate O2-. generation while it apparently did not affect the cytosol. When PAO was added subsequently to NADPH oxidase activation, no inhibition was observed, indicating that PAO cannot reach its target once the oxidase is functionally assembled. In conclusion, PAO is the first complete and reversible inhibitor of NADPH oxidase which could provide the basis for new therapeutical approaches in inflammatory diseases.
Highlights
:j: Supported by the French Ministery for Research and Teaching. §To whom correspondence should be addressed
phenylarsine oxide (PAO) cannot be considered as a specific inhibitor of tyrosine phosphatases since it affects glucose transport [32, 33], inhibits receptor endocytosis [34, 35], and blocks insulin-mediated activation ofp2Fas [36], we studied its effect on the 02 generation in human neutrophils
PAO Inhibits Superoxide Generation Induced by a Variety of Activators-Activation of neutrophils by a variety of stimuli elicits the assembly of an active NADPH oxidase which generates 02
Summary
Reagents and Antibodies-PAD was obtained from Aldrich-Chemie, Steinheim, Germany. Bovine erythrocyte superoxide dismutase, NADPH, ferricytochrome c, ATP, GTP, GTPyS, phorbol 12-myristate. Cell Free Assay of NADPH Oxidase-The activation ofNADPH oxidase was studied in a cell-free system using the membrane and cytosolic fractions isolated from unstimulated neutrophils after Percoll gradient, as described previously [41]. The plasma membrane-enriched fraction (25 fLg) and the cytosolic fraction (50 fLg) both suspended in PBS supplemented with 1 mM ATP, 1 mM MgCl2 , and 0.6 mM CaC12 in a final volume of 500 fLl were preincubated with arachidonic acid (final concentration 25 fLg/ml) for 2 min. The reconstitution assay was made by combining 0.5 ml of the eluted cytosolic proteins with 25 fLg of membrane proteins in the presence of ATP, MgCl2 , CaCl2 , NADPH, and arachidonic acid as described above. The nitrocellulose sheets were subsequently treated as described previously [42] with either antiphosphotyrosine antibodies or anti-p47phox, -p67phox, -p22phox, or -p91phox antibodies
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