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Am. J. Respir. Cell Mol. Biol., Volume 23, Number 6, December, 2000 748-754

Phospholipase D and Priming of the Respiratory Burst by H2O2 in NR8383 Alveolar Macrophages

Julio Girón-Calle and Henry Jay Forman

Department of Environmental Health Sciences, School of Public Health, and Center for Free Radical Biology, University of Alabama at Birmingham, Birmingham, Alabama

Previous investigation showed that preincubation within a range of nontoxic H2O2 concentrations enhanced subsequently stimulated superoxide production by rat alveolar macrophages in response to various stimuli. In the present study, the NR8383 rat alveolar macrophage cell line was used to further investigate the priming effect of H2O2. Using nitroblue tetrazolium, which formed an insoluble formazan when reduced by superoxide, modulation of the respiratory burst was visualized in a cell population exposed to a concentration gradient of H2O2 before stimulation. This model system illustrates how H2O2 may constitute a signaling molecule for a feed-forward regulation of the respiratory burst during inflammation. n-Butanol, which allows consumption of phosphatidic acid by the transphosphatidylation reaction, and propanolol, which inhibits phosphatidic acid phosphohydrolase, were used to investigate the possible involvement of phospholipase D in this phenomenon. These two agents were found to inhibit the basal adenosine diphosphate-stimulated respiratory burst. Inhibition of the H2O2-enhanced respiratory burst was equally or slightly less effective when expressed as percentage of controls. Furthermore, phospholipase D was not activated by H2O2 concentrations that enhance superoxide production. Thus, phospholipase D does not mediate the enhancement of the respiratory burst by H2O2, although it may be activated by high concentrations of this hydroperoxide.




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