iNOS upregulation mediates oxidant-induced disruption of F-actin and barrier of intestinal monolayers

Using oxidant-induced hyperpermeability of monolayers of intestinal (Caco-2 ) cells as a model for the pathophysiology of inflammatory bowel disease (I BD), we previously showed that oxidative injury to the F-actin cytoskeleton is necessary for the disruption of monolayer barrier integrity. We hypothe sized that this cytoskeletal damage is caused by upregulation of an inducib le nitric oxide (NO) synthase (iNOS)-driven pathway that overproduces react ive nitrogen metabolites (RNMs) such as NO and peroxynitrite (OONO-), which cause actin nitration and disassembly. Monolayers were exposed to H2O2 or to RNMs with and without pretreatment with antioxidants or iNOS inhibitors. H2O2 concentrations that disassembled and/or disrupted the F-actin cytoske leton and barrier integrity also caused rapid iNOS activation, NO over-prod uction, and actin nitration. Added OONO- mimicked H2O2; iNOS inhibitors and RNM scavengers were protective. Our results show that oxidant-induced F-ac tin and intestinal barrier disruption are caused by rapid iNOS upregulation that further increases oxidant levels; a similar positive feedback mechani sm may underlie the episodic recurrence of the acute IBD attack. Confirming these mechanisms in vivo would provide a rationale for developing novel an ti-RNM therapies for IBD.