Abstract
We have previously shown that the ability of overnight pretreatment with lipopolysaccharide (LPS) to suppress alveolar macrophage (AM) leukotrienes (LT) synthesis is explained by induction of nitric oxide (NO), and reactive oxygen intermediates (ROI). More recently we have demonstrated that the generation of peroxynitrite (ONOO−) from the combination of NO and ROI directly nitrotyrosinates the 5-lipoxygenase (5-LO) enzyme and reduces cell-free and intact AM 5-LO metabolism. This effect of ONOO− was associated with nitrotyrosination of the 5-LO enzyme in intact cells and after treatment of recombinant enzyme. We postulated that LPS treatment of cells resulted in activation of 5-LO with the generation of ROI, which in turn led to autoinactivation of the enzyme. In an effort to suppress ROI generated from activation of 5-LO we examined the effect of a direct 5-LO inhibitor on LPS-induced suppression of LT synthesis. Coincubation with the reversible 5-LO inhibitor zileuton during the LPS pretreatment of intact cells dose dependently blocked the inhibition of 5-LO metabolism by LPS. The effect of zileuton on LPS-induced suppression of LT synthesis was similar to that ofN-monomethyl-l-arginine. Zileuton had no effect on inducible nitric-oxide synthase induction. Interestingly, zileuton blocked ONOO−-induced nitrotyrosination of recombinant 5-LO in a cell-free system as well as of native enzyme in intact cells. Moreover, zileuton blocked the nitrotyrosination of other proteins. We conclude that the suppression of 5-LO activity occurring with LPS treatment can be blocked by zileuton. The mechanism by which zileuton is effective is in part explained by blocking nitrotyrosination of 5-LO.
Footnotes
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This work was supported by a grant from the General Clinical Research Center at University of Michigan (M01-RR00042). M.J.C. was the recipient of National Institutes of Health Grant R01-HL02810. M.P.-G. was supported by the National Institutes of Health Grants R01-HL58897 and P50 HL56402.
- Abbreviations:
- LT
- leukotrienes
- AA
- arachidonic acid
- 5-LO
- 5-lipoxygenase
- MAPK
- mitogen-activated protein kinase
- AM
- alveolar macrophage
- LPS
- lipopolysaccharide
- iNOS
- inducible nitric-oxide synthase
- NO
- nitric oxide
- ROI
- reactive oxygen intermediates
- ONOO−
- peroxynitrite
- DMEM
- Dulbecco's modified Eagle's medium
- EIA
- enzyme-linked immunoassay
- HPLC
- high-performance liquid chromatography
- l-NMMA
- N-monomethyl-l-arginine
- HETE
- hydroxyeicosatetraenoic acid
- NDGA
- nordihydroguaiaretic acid
- BSA
- bovine serum albumin
- Received April 26, 2001.
- Accepted June 28, 2001.
- The American Society for Pharmacology and Experimental Therapeutics
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