Biochemical characterization of reactive nitrogen species by eosinophil peroxidase in tyrosine nitration

Abstract

It is well known that eosinophils are involved in tyrosine nitration. In this study, we evaluated tyrosine nitration by rat eosinophils isolated from peritoneal fl uid and constituent eosinophils in the stomach. Rat peritoneal eosinophils activated with 1 &#956;M phorbol myristate acetate (PMA) and 50 &#956;M NO2 &#65437; showed immunostaining for nitrotyrosine only in smaller cells, despite the fact that eosinophils are capable of producing superoxide (O2·&#65437;). Free tyrosine nitrating capacity after incubation with PMA and NO2 &#65437; was 4-fold higher in eosinophils than in neutrophils. Catalase and &#65400;- and &#65402; -tocopherol inhibited free tyrosine nitration by reactive nitrogen species from eosinophils but not that by peroxynitrite. Superoxide dismutase augmented free tyrosine nitration by activated eosinophils and peroxynitrite. The concentration of nitric oxide released from eosinophils was relatively low (0.32 &#956;M/106 cells/h) and did not contribute to the formation of nitrotyrosine. On the other hand, most constituent eosinophils constituent in the rat stomach stimulated by PMA and NO2 &#65437; showed tyrosine nitration capacity. These results suggest that intact cells other than apoptotic-like eosinophils eluted in the intraperitoneal cavity could not generate reactive species responsible for nitration by a peroxidase-dependent mechanism. In contrast, normal eosinophils in the stomach were capable of nitration, suggesting that the characteristics of eosinophils in gastric mucosa are diff erent from those eluted in the peritoneal cavity.</p