title: Autocatalytic nitration of prostaglandin endoperoxide synthase-2 by nitrite inhibits prostanoid formation in rat alveolar macrophages.
abstract: OBJECTIVE
  Prostaglandin endoperoxide H(2) 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, we aimed 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 H(2) 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 A(2) (TxA(2)) synthesis in the cells. Formation of reactive oxygen species (ROS) was maximal at 4 h and then returned to baseline levels. Nitrite (NO(2)(-)) 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 NO(2)(-), independent from superoxide ((•)O(2)(-)). Purified PGHS-2 treated with NO(2)(-) was selectively nitrated on the active site Tyr(371), as identified by mass spectrometry (MS). Exposure to peroxynitrite resulted in the nitration not only of Tyr(371), but also of other tyrosines (Tyr).
  
  
  CONCLUSIONS
  The data presented here point to an autocatalytic nitration of PGHS-2 by NO(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.
authors.affiliation: Department of Biology, University of Konstanz, Konstanz, Germany. stefan.schildknecht@uni-konstanz.de
authors.forename: Stefan
authors.initials: S
authors.lastname: Schildknecht
authors.forename: Christiaan
authors.initials: C
authors.lastname: Karreman
authors.forename: Andreas
authors.initials: A
authors.lastname: Daiber
authors.forename: Cheng
authors.initials: C
authors.lastname: Zhao
authors.forename: Jürg
authors.initials: J
authors.lastname: Hamacher
authors.forename: David
authors.initials: D
authors.lastname: Perlman
authors.forename: Birgit
authors.initials: B
authors.lastname: Jung
authors.forename: Bernd
authors.initials: B
authors.lastname: van der Loo
authors.forename: Peter
authors.initials: P
authors.lastname: O'Connor
authors.forename: Marcel
authors.initials: M
authors.lastname: Leist
authors.forename: Volker
authors.initials: V
authors.lastname: Ullrich
authors.forename: Markus Michael
authors.initials: MM
authors.lastname: Bachschmid
mesh_terms: D000818:Animals; D016676:Macrophages, Alveolar / Q000378:metabolism*; D009566:Nitrates / Q000378:metabolism*; D009573:Nitrites / Q000378:metabolism*; D011451:Prostaglandin-Endoperoxide Synthases / Q000378:metabolism*; D011453:Prostaglandins / Q000378:metabolism*; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism
chemical_list: D009566:Nitrates; D009573:Nitrites; D011453:Prostaglandins; D017382:Reactive Oxygen Species; D011451:Prostaglandin-Endoperoxide Synthases