Biphasic regulation of leukocyte superoxide generation by nitric oxide and peroxynitrite

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.) with the formation of peroxynitrite (ONOO-); however, questions remain regarding the effects and mechanisms by which NO. and ONOO- modulate the PMN oxidative burst. Therefore, we directly measured the dose-dependent effects of NO. and ONOO- on O-2(.-) generation from human PMNs stimulated with phorbol 12-myristate 13-acetate using EPR spin trapping. Pretreatment with low physiological (muM) concentrations of NO. from NO. gas had no effect on PMN O-2(.-) generation, whereas high levels (greater than or equal to 50 muM) exerted inhibition. With ONOO- pretreatment, however, a biphasic modulation of O-2(.-) generation was seen with stimulation by muM levels, but inhibition at higher levels. With the NO. donor NOR-1, which provides more sustained release of NO. 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 ERR MAPKs and was blocked by their inhibition, Thus, low physiological levels of NO. 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. or ONOO- feed back and inhibit O-2(.-) generation. This biphasic concentration-dependent regulation of the PMN oxidant burst by NO.-derived ONOO- may be of critical importance in regulating the process of inflammation.