Nitric Oxide Inactivates NADPH Oxidase in Pig Neutrophils by Inhibiting Its Assembling Process

Hirotada Fujii,Kohji Ichimori,Kiyotaka Hoshiai,Hiroe Nakazawa

doi:10.1074/jbc.272.52.32773

Hirotada Fujii, Kohji Ichimori + Show 2 more

Open Access

https://doi.org/10.1074/jbc.272.52.32773

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Abstract

The effects of nitric oxide (NO) on superoxide (O-2) generation of the NADPH oxidase in pig neutrophils were studied. NO dose-dependently suppressed O-2 generation of both neutrophil NADPH oxidase and reconstituted NADPH oxidase. Effects of NO on NADPH-binding site and the redox centers including FAD and low spin heme in cytochrome b558 and the electron transfer rates from NADPH to heme via FAD were examined under anaerobic conditions. Both reaction rates and the Km value for NADPH were unchanged by NO. Visible and EPR spectra of cytochrome b558 showed that the structure of heme was unchanged by NO, indicating that NO does not affect the redox centers of the oxidase. In reconstituted NADPH oxidase system, NO did not inhibit O-2 generation of the oxidase when added after activation. The addition of NO to the membrane component or the cytosol component inhibited the activity by 24.0 +/- 5.3 or 37.4 +/- 7.1%, respectively. The addition of NO during the activation process or to the cytosol component simultaneously with myristate inhibited the activity by 74.0 +/- 5.2 or 70.0 +/- 8.3%, respectively, suggesting that cytosol protein(s) treated with myristate becomes susceptible to NO. Peroxynitrite did not interfere with O-2 generation.

Highlights

Nitric oxide (NO) is recognized as one of the key mediators in many physiological and pathological processes
We studied the effects of peroxynitrite (ONOOϪ) on NADPH oxidase to confirm that the results obtained above were caused by NO because addition of NO in the presence of O2. produces ONOOϪ at nearly diffusion-limited rates [13] and because ONOOϪ is known to be a potent oxidant
The measurements were performed in neutrophils, reconstituted NADPH oxidase, and solubilized NADPH oxidase obtained from stimulated cells in the presence or the absence of NO or ONOOϪ

Summary

EXPERIMENTAL PROCEDURES

Materials—Myristic acid and arachidonic acid from Wako Pure Chemical (Tokyo, Japan) were dissolved in ethanol. The membrane component of the NADPH oxidase was solubilized from the membrane vesicle with heptylthioglucoside [19] and used for further purification of cytochrome b558. The reaction was started by adding PMA for cells (37 °C) or 0.1 mM NADPH for both the stimulated and reconstituted NADPH oxidase (25 °C) as reported previously [22]. The measurements were performed in neutrophils, reconstituted NADPH oxidase, and solubilized NADPH oxidase obtained from stimulated cells in the presence or the absence of NO or ONOOϪ. The electron flux from NADPH to FAD was evaluated by measuring the reduction rate of cytochrome c, which was utilized as an exogenous electron acceptor (reaction 2) according to the previously reported method [19]. Typical EPR conditions were: microwave power, 5 mW; modulation amplitude, 10 gauss at 100 kHz; response, 0.3 s; sweep time, 4 min

RESULTS AND DISCUSSION

TABLE I

ϪNO ϩNO

Cytochrome c reduction