Abstract

Activation of the NADPH oxidase-derived oxidant burst of polymorphonuclear leukocytes (PMNs) is of critical importance in inflammatory disease. PMN-derived superoxide (O(2)) can be scavenged by nitric oxide (NO( small middle dot)) with the formation of peroxynitrite (ONOO(-)); however, questions remain regarding the effects and mechanisms by which NO( small middle dot) and ONOO(-) modulate the PMN oxidative burst. Therefore, we directly measured the dose-dependent effects of NO( small middle dot) and ONOO(-) on O(2) generation from human PMNs stimulated with phorbol 12-myristate 13-acetate using EPR spin trapping. Pretreatment with low physiological (microm) concentrations of NO( small middle dot) from NO( small middle dot) gas had no effect on PMN O(2) generation, whereas high levels (> or =50 microm) exerted inhibition. With ONOO(-) pretreatment, however, a biphasic modulation of O(2) generation was seen with stimulation by microm levels, but inhibition at higher levels. With the NO( small middle dot) donor NOR-1, which provides more sustained release of NO( small middle dot) persisting at the time of O(2) generation, a similar biphasic modulation of O(2) generation was seen, and this was inhibited by ONOO(-) scavengers. The enhancement of O(2) generation by low concentrations of ONOO(-) or NOR-1 was associated with activation of the ERK MAPKs and was blocked by their inhibition. Thus, low physiological levels of NO( small middle dot) present following PMN activation are converted to ONOO(-), which enhances O(2) generation through activation of the ERK MAPK pathway, whereas higher levels of NO( small middle dot) or ONOO(-) feed back and inhibit O(2) generation. This biphasic concentration-dependent regulation of the PMN oxidant burst by NO( small middle dot)-derived ONOO(-) may be of critical importance in regulating the process of inflammation.

Highlights

  • Superoxide (O2.) can with the formation of be scavenged peroxynitrite by nitric oxide (ONOO؊); questions remain regarding the effects and mechanisms by which NO1⁄7 and ONOO؊ modulate the polymorphonuclear leukocytes (PMNs) oxidative burst

  • To confirm that the ONOOϪ formation was the basis by which low doses of NOR-1 stimulated PMN O2. generation, we investigated the effects of the ONOOϪ scavenger urate and the ONOOϪ decomposition catalyst FeTMPS on this process

  • We examined the effects of inhibiting the MEK/ ERK pathway on the increase in PMN O2. generation mediated by extrinsic ONOOϪ or PMN-derived ONOOϪ formed from NOR-1

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Summary

Introduction

Superoxide (O2.) can with the formation of be scavenged peroxynitrite by nitric oxide (ONOO؊); questions remain regarding the effects and mechanisms by which NO1⁄7 and ONOO؊ modulate the PMN oxidative burst. It was observed that ONOOϪ, formed from the reaction of NO1⁄7 gered MAPK-mediated increases with PMN-derived in O2. Generation from PMA-stimulated PMNs were measured by EPR spin trapping with DEPMPO.

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