Vol. 301, Issue 1, 87-94, April 2002

Priming of Alveolar Macrophage Respiratory Burst by H₂O₂ Is Prevented by Phosphatidylcholine-Specific Phospholipase C Inhibitor Tricyclodecan-9-yl-xanthate (D609)

Julio Girón-Calle, Kousthub Srivatsa and Henry Jay Forman

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

The respiratory burst in alveolar macrophages is enhanced in vitro by pre-exposure to nontoxic concentrations of hydroperoxides before stimulation by an agonist, which may represent a feed-forward regulatory mechanism. Tricyclodecan-9-yl-xanthate (D609), an inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC), suppresses this priming of the respiratory burst by pre-exposure to H₂O₂ in NR8383 alveolar macrophages (up to 100 µM D609, 400 nmol of H₂O₂ added to 5 × 10⁶ cells 15 min before stimulation with ADP). D609 has potential as an antioxidant due to its dithiocarbonate functional group that allows it to slowly react with H₂O₂ and rapidly reduce cytochrome c, which interferes with a common assay for the respiratory burst. Nonetheless, the antioxidant properties of D609 do not account for its inhibition of priming of the respiratory burst by H₂O₂. Reduction of nitro blue tetrazolium is the basis for an assay for superoxide production with which D609 does not interfere. With this assay, it was found that D609 does not inhibit the respiratory burst per se, but prevents its enhancement by pre-exposure to H₂O₂. Consistent with a role of diacylglycerol generation by phospholipase C, this enhancement was mimicked by pre-exposure to phorbol ester. In contrast with priming, receptor-mediated stimulation of the respiratory burst depends on the better characterized phosphatidylinositol-specific phospholipase C. Priming of the respiratory burst by H₂O₂ joins the list of inflammatory responses that are inhibited by D609. Nevertheless, the results herein indicate that caution should be exercised in the interpretation of the effects of D609 to consider both antioxidant effects and inhibition of PC-PLC.