Abstract

We have previously shown that the biologically important reactive oxygen metabolite hydrogen peroxide (H2O2) stimulates arachidonic acid (AA) release and thromboxane A2 synthesis in the rat alveolar macrophage. We have now investigated the effects of H2O2 on alveolar macrophage 5-lipoxygenase metabolism. H2O2 failed to stimulate detectable synthesis of leukotriene B4, leukotriene C4, or 5-hydroxyeicosatetraenoic acid (5-HETE) as determined by reverse-phase high performance liquid chromatography (RP-HPLC) and sensitive radioimmunoassays (RIAs). This was not explained by oxidative degradation of leukotrienes by H2O2 at the concentrations used. Moreover, RIA and RP-HPLC analyses demonstrated that H2O2 dose-dependently inhibited synthesis of leukotriene B4, leukotriene C4, and 5-HETE induced by the agonists A23187 (10 microM) and zymosan (100 micrograms/ml), over the same concentration range at which it augmented synthesis of the cyclooxygenase products thromboxane A2 and 12-hydroxy-5,8,10-heptadecatrienoic acid. Four lines of evidence suggested that H2O2 inhibited alveolar macrophage leukotriene and 5-HETE synthesis by depleting cellular ATP, a cofactor for 5-lipoxygenase. 1) H2O2 depleted ATP in A23187- and zymosan-stimulated alveolar macrophages with a dose dependence very similar to that for inhibition of agonist-induced leukotriene synthesis. 2) The time courses of ATP depletion and inhibition of leukotriene B4 synthesis by H2O2 were compatible with a rate-limiting effect of ATP on leukotriene synthesis in H2O2-exposed cultures. 3) Treatment of alveolar macrophages with the electron transport inhibitor antimycin A prior to A23187 stimulation depleted ATP and inhibited leukotriene B4 and C4 synthesis to equivalent degrees, while thromboxane A2 production was spared. 4) Incubation with the ATP precursors inosine plus phosphate attenuated both ATP depletion and inhibition of leukotriene B4 and C4 synthesis in alveolar macrophages stimulated with A23187 in the presence of H2O2. Our results show that H2O2 has the capacity to act both as an agonist for macrophage AA metabolism, and as a selective inhibitor of the 5-lipoxygenase pathway, probably as a result of its ability to deplete ATP. Depletion of cellular energy stores by oxidants generated during inflammation in vivo may be a means by which the inflammatory response is self-limited.

Highlights

  • From theDivision of Pulmonary andCritical Care Medicine, Department of Internal Medicine, University of Michigan and Veterans Administration Medical Centers, Ann Arbor, Michigan 48109-0360

  • Reverse-phase HPLC analysis of media from [14C]arachidonic acid (AA)-prelabeledalveolar macrophage cultures exposed for 30 min to 0.15 mM H202confirmed the production of [‘4C]thromboxane BQ,and further showed release of [“CIHHTand [14C]AA (Fig. 1).no measurable 14C-labeledleukotrienes or mono-HETEs were detected following this H202exposure

  • In order to examine whether depletion of adenosine triphosphate (ATP) and inhibition of 5-lipoxygenase metabolism were causally related, we sought to augment ATP levels in H202-exposedalveolar macrophage cultures and determine the effect of such a manipulation on 5-lipoxygenase metabolite production

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Summary

THEJOURNAL OF BIOLOGICACLHEMISTRY

Vol 263,No.29,Issue ofOctober 15,pp. 14776-14783.1988 Printed in U.S.A. Hydrogen Peroxide Inhibits Alveolar Macrophage 5-Lipoxygenase Metabolism in Association with Depletion of ATP*. 4) Incubation with the ATP precursors inosine plus phosphate attenuatedboth ATP depletion and inhibitioonf leukotriene B4 and C4 synthesis in alveolar macrophages stimulated with A23187 in the presence of has been less well-studied, Burghuber and associates [15] provided evidence for 5-lipoxygenase product formation in isolated perfused lungs injured with hydrogen peroxide (H202),and Taniguchi and co-workers [16] found increased leukotriene B4 concentrationsin bronchoalveolar lavage fluid of rats maintained in a hyperoxic environment. Following adherence and overnight culture, alveolar macrophage monolayers were incubated with added HZOZ(0.15 mM) in Dulbecco’s phosphate-buffered saline containing 0.1%glucose (1 ml/plate), fromwhich duplicate 50-pl aliquots were taken for assay at time points up to 1 h. The total radioactivity (minus background), given in cpm, contained within each peak is thromboxane B,, 362; HHT, 258; AA, 251

RESULTS
To further evaluate the importance of ATP depletion in
DISCUSSION
Methods
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