Biphasic Regulation of Leukocyte Superoxide Generation by Nitric Oxide and Peroxynitrite

doi:10.1074/jbc.M006341200

Biphasic Regulation of Leukocyte Superoxide Generation by Nitric Oxide and Peroxynitrite*

From the ^‡Molecular and Cellular Biophysics Laboratories, Department of Medicine, Division of Cardiology and the Electron Paramagnetic Resonance Center and the ^¶Department of Medicine, Division of Clinical Immunology, The Johns Hopkins Medical Institutions, Baltimore, Maryland 21224 and the ^§Department of Pharmacology and the Electron Spin Resonance Center, Kanagawa Dental College, 82 Inaoka-cho Yokosuka, Kanagawa 238-0003, Japan

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^⋅) 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 (μm) concentrations of NO^⋅ from NO^⋅ gas had no effect on PMN O⨪₂ generation, whereas high levels (≥50 μm) exerted inhibition. With ONOO⁻ pretreatment, however, a biphasic modulation of O⨪₂ generation was seen with stimulation by μm 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 ERK 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.

Footnotes

↵* This work was supported by National Institutes of Health Grants HL-38324 and HL-63744.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵‖ To whom correspondence should be addressed: EPR Center, Johns Hopkins Asthma and Allergy Center, 5501 Johns Hopkins Bayview Circle, Baltimore, MD 21224. Tel.: 410-550-0339; Fax: 410-550-2448; E-mail: jzweier@welch.jhu.edu.
Published, JBC Papers in Press, September 6, 2000, DOI 10.1074/jbc.M006341200
Abbreviations:

PMNs

polymorphonuclear leukocytes

O⨪₂

superoxide

NO^⋅

nitric oxide

ONOO⁻

peroxynitrite

MAPK

mitogen-activated protein kinase

ERK

extracellular signal-regulated kinase

MEK

MAPK/ERK kinase

DEPMPO

5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide

PIPES

1,4-piperazinediethanesulfonic acid

PMA

phorbol 12-myristate 13-acetate

FeTMPS

5,10,15,20-tetrakis(2,4,6-trimethyl-3,3-disulfonatophenyl)porphyrinato iron(III)
- Received July 18, 2000.
- Revision received September 5, 2000.
The American Society for Biochemistry and Molecular Biology, Inc.