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

Gang Yue, Pi Shiang Lai, Kingsley Yin, Frank F. Sun, Robert G. Nagele, Xiao Liu, Kersti K. Linask, Congli Wang, King Teh Lin, Patrick Y.K. Wong

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57 Scopus citations

Abstract

Peroxynitrite, derived from the reaction of nitric oxide (NO) with superoxide (O2), is a potent nitrating and oxidizing agent that can induce apoptosis in a variety of different cell types. In the present study, we investigated the possible role of peroxynitrite as a mediator of colon epithelial cell death in rat colitis. Rat colon inflammation was induced by intracolonic administration of 2,4,6-trinitrobenzenesuffonic acid (TNBS) and rats were sacrificed 24 h after TNBS administration. Expression of inducible nitric-oxide synthase (iNOS) was detected by reverse transcription-polymerase chain reaction and immunohistochemistry. The enzymatic activities of Ca2+-independent iNOS and Ca2+-dependent constitutive nitric-oxide synthase were determined 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) assay and DNA gel electrophoresis. To evaluate the specific contribution of peroxynitrite to the observed cell injury, a selective iNOS inhibitor, L-NG-[1-iminoethyl] lysine (L-NIL), was administered after TNBS induction. Morphological examination and analysis of TUNEL/ cytokeratin double immunofluorescence revealed significant apoptosis in mucosal epithelial cells. Nitrotyrosine was colocalized with TUNEL, strongly demonstrating the association of peroxynitrite with the apoptotic death of colon epithelial cells. The administration of L-NIL reduced iNOS activity in 24-h lesions by 92% and also significantly attenuated both nitrotyrosine staining and apoptotic cell counts in the colon epithelium. These results strongly suggest that local elevated level of peroxynitrite produced from increased iNOS expression and activity is a major contributor to colon epithelial apoptosis during colon inflammation.

Original languageEnglish (US)
Pages (from-to)915-925
Number of pages11
JournalJournal of Pharmacology and Experimental Therapeutics
Volume297
Issue number3
StatePublished - Jun 1 2001
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Molecular Medicine
  • Pharmacology

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