This work was initiated by the observation that treatment with PN inhibits PGIS with a concomitant appearance of a protein band that positively reacts with an antibody against NT. This study has confirmed tyrosine nitration of PGIS as the underlying mechanism of enzyme inhibition and has provided evidence for a broad physiological and pathophysiological significance of this new posttranslational protein modification. Our results focus on superoxide as a messenger, which combines with NO to exert tyrosine nitration. It turned out that the important process of endothelial activation is primarily based on superoxide generation, which can be mediated by xanthine oxidase (endotoxin), NADPH-oxidase (hyperglycemia/ diabetes), mitochondria (aging) or NO-synthase - oxidase (still under debate, e.g. aging). The results of this study are discussed under the aspects of1.) chemistry and biochemistry of PGIS nitration2.) superoxide as a messenger and3.) endothelial cell activation type IThe most important results are summarized as follows:1. Nitration of prostacyclin synthase· For this study two reliable and sensitive methods for the detection of nitrated PGIS and nitration in general have been established: The immunoprecipitation of nitrated PGIS and the total hydrolysis of microsomes and homogenates followed by HPLC analysis.· Both methods in combination with MALDI-TOF mass spectroscopy, confirmed PGIS nitration by peroxynitrite.· The applied methods confirm a high proteolytic stability of PGIS· Increased PGIS nitration was observed in different model systems, like hypoxia-reoxygenation, hyperglycemia and endotoxemia.2. Superoxide as a messenger· Our results demonstrate that superoxide in combination with NO generates peroxynitrite, which serves as a highly reactive derivate of the superoxide radical in the cellular system.· In the very early stage of inflammation in coronary arteries, xanthine oxidase was identified as major superoxide source.· In the aging process, mitochondria mainly contributed to the superoxide generation, partially caused by the reduction of functionally Mn-SOD· The superoxide generation under hyperglycaemic conditions was caused by the induction of NADPH-oxidase and further via the activation of the PKC cascade.3. Endothelial cell activation Type I· This work confirms, that different endogenous superoxide sources can lead into Phase 1 of endothelial cell activation (ECA-I) and therefore modulate endothelial function.· Endothelial properties are inverted within the first hour of superoxide formation, without affecting gene or protein expression.· The reaction of superoxide with NO leads to increased peroxynitrite generation, which nitrates and inactivates PGIS, thus strongly attenuating the two important signalling molecules NO and prostacyclin, which are essential for vascular homeostasis. In contrast, the substrate of PGIS, PGH2, with its vasoconstricting properties accumulates and therefore the balance of endothelial mediators is shifted towards vasoconstriction
