Abstract
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.
Footnotes
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This work was supported by Grant HL37556 from the National Institutes of Health.
- Abbreviations:
- D609
- tricyclodecan-9-yl-xanthate
- PC-PLC
- phosphatidylcholine-specific phospholipase C
- [Ca2+]c
- cytosolic concentration of Ca2+
- U73122
- 1-(6-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione
- PI-PLC
- phosphatidylinositol-specific phospholipase
- PBS
- phosphate-buffered saline
- BSA
- bovine serum albumin
- KRP
- Krebs-Ringer phosphate buffer
- NBT
- nitro blue tetrazolium
- PMA
- phorbol-12-myristate-13-acetate
- Received September 7, 2001.
- Accepted December 5, 2001.
- The American Society for Pharmacology and Experimental Therapeutics
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