Autocatalytic nitration of prostaglandin endoperoxide synthase-2 by nitrite inhibits prostanoid formation in rat alveolar macrophages

摘要

Aims: Prostaglandin endoperoxide H2 synthase (PGHS) is a well-known target for peroxynitrite-mediated nitration. In several experimental macrophage models, however, the relatively late onset of nitration failed to coincide with the early peak of endogenous peroxynitrite formation. In the present work,weaimed to identify an alternative, peroxynitrite-independent mechanism, responsible for the observed nitration and inactivation of PGHS-2 in an inflammatory cell model. Results: In primary rat alveolar macrophages stimulated with lipopolysaccharide (LPS), PGHS-2 activity was suppressed after 12 h, although the prostaglandin endoperoxide H2 synthase (PGHS-2) protein was still present. This coincided with a nitration of the enzyme. Coincubation with a nitric oxide synthase-2 (NOS- 2) inhibitor preserved PGHS-2 nitration and at the same time restored thromboxane A2 (TxA2) synthesis in the cells. Formation of reactive oxygen species (ROS) was maximal at 4 h and then returned to baseline levels. Nitrite (NO2 -) production occurred later than ROS generation. This rendered generation of peroxynitrite and the nitration of PGHS-2 unlikely. We found that the nitrating agent was formed from NO2-, independent from superoxide (?O2 - ). Purified PGHS-2 treated with NO2 - was selectively nitrated on the active site Tyr371, as identified by mass spectrometry (MS). Exposure to peroxynitrite resulted in the nitration not only of Tyr 371, but also of other tyrosines (Tyr). Innovation and Conclusion: The data presented here point to an autocatalytic nitration of PGHS-2 byNO 2 - , catalyzed by the enzyme's endogenous peroxidase activity and indicate a potential involvement of this mechanism in the termination of prostanoid formation under inflammatory conditions. ? 2012 Mary Ann Liebert, Inc.