Autocatalytic Nitration of Prostaglandin Endoperoxide Synthase-2 by Nitrite Inhibits Prostanoid Formation in Rat Alveolar Macrophages

Abstract

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, 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 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 Tyr₃₇₁, but also of other tyrosines (Tyr). Innovation and Conclusion: The data presented here point to an autocatalytic nitration of PGHS-2 by NO₂ ⁻, 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. Antioxid. Redox Signal. 17, 1393–1406.