Colon epithelial cell death in 2,4,6-trinitrobenzenesulfonic acid-induced colitis is associated with increased inducible nitric-oxide synthase expression and peroxynitrite production

Peroxynitrite, derived from the reaction of nitric oxide (NO.) with superox ide (O-2(.)), is a potent nitrating and oxidizing agent that can induce apo ptosis in a variety of different cell types. In the present study, we inves tigated the possible role of peroxynitrite as a mediator of colon epithelia l cell death in rat colitis. Rat colon inflammation was induced by intracol onic administration of 2,4.6-trinitrobenzenesulfonic acid (TNBS) and rats w ere sacrificed 24 h after TNBS administration. Expression of inducible nitr ic-oxide synthase (iNOS) was detected by reverse transcription-polymerase c hain reaction and immunohistochemistry. The enzymatic activities of Ca2+-in dependent iNOS and Ca2+-dependent constitutive nitric-oxide synthase were d etermined biochemically. Evidence of peroxynitrite-mediated cell injury was detected by immunostaining of nitrotyrosine, Apoptosis was examined by in situ terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) a ssay and DNA gel electrophoresis, To evaluate the specific contribution of peroxynitrite to the observed cell injury, a selective iNOS inhibitor, L-N- G-[1-iminoethyl]lysine (L-NIL), was administered after TNBS induction. Morp hological examination and analysis of TUNEL/cytokeratin double immunofluore scence revealed significant apoptosis in mucosal epithelial cells. Nitrotyr osine was colocalized with TUNEL, strongly demonstrating the association of peroxynitrite with the apoptotic death of colon epithelial cells. The admi nistration of L-NIL reduced iNOS activity in 24-h lesions by 92% and also s ignificantly attenuated both nitrotyrosine staining and apoptotic cell coun ts in the colon epithelium, These results strongly suggest that local eleva ted level of peroxynitrite produced from increased iNOS expression and acti vity is a major contributor to colon epithelial apoptosis during colon infl ammation.