TY - JOUR T1 - The δ-Isoform of Protein Kinase C Causes Inducible Nitric-Oxide Synthase and Nitric Oxide Up-Regulation: Key Mechanism for Oxidant-Induced Carbonylation, Nitration, and Disassembly of the Microtubule Cytoskeleton and Hyperpermeability of Barrier of Intestinal Epithelia JF - Journal of Pharmacology and Experimental Therapeutics JO - J Pharmacol Exp Ther SP - 482 LP - 494 DO - 10.1124/jpet.102.047308 VL - 305 IS - 2 AU - A. Banan AU - A. Farhadi AU - J. Z. Fields AU - L. J. Zhang AU - M. Shaikh AU - A. Keshavarzian Y1 - 2003/05/01 UR - http://jpet.aspetjournals.org/content/305/2/482.abstract N2 - Using intestinal (Caco-2) cells, we found that oxidant-induced disruption of barrier integrity requires microtubule disassembly. Protein kinase C (PKC)-δ isoform seems to be essential for disruption, but the mechanism is unknown. Because inducible nitric-oxide synthase (iNOS) is key to oxidant stress, we hypothesized that PKC-δ activation is essential in oxidant-induced iNOS up-regulation and the consequent cytoskeletal oxidation and disarray and monolayer barrier dysfunction. Cells were transfected with an inducible plasmid to overexpress native PKC-δ or with a dominant-negative to inhibit the activity of native PKC-δ. Clones were then incubated with oxidant (H2O2) ± modulators. Parental cells were treated similarly. Exposure to oxidant-disrupted monolayers by increasing native PKC-δ activity, increasing six iNOS-related variables (iNOS activity and protein, nitric oxide, oxidative stress, tubulin oxidation and nitration), decreasing polymerized tubulin, disrupting the cytoarchitecture of microtubules, and causing monolayer dysfunction. Induction of PKC-δ overexpression by itself (3.5-fold) led to oxidant-like disruptive effects, including activation of the iNOS-driven pathway. Overexpression-induced up-regulation of iNOS was potentiated by oxidants. iNOS inhibitors or oxidant scavengers were protective. Dominant inhibition of native PKC-δ activity (99.5%) prevented all measures of oxidant-induced iNOS up-regulation and protected the monolayer barrier. The conclusions are as follows. 1) Oxidants induce loss of epithelial barrier integrity by oxidizing and disassembling the cytoskeleton, in part, through the activation of PKC-δ and up-regulation of iNOS. 2) Overexpression and activation of PKC-δ are by themselves key for cellular injury by oxidative stress of iNOS. 3) We thus report a pathophysiological mechanism, activation of iNOS pathway and its injurious consequences to the cytoskeleton, including oxidation and nitration, among the “novel” subfamily of PKC isoforms. The American Society for Pharmacology and Experimental Therapeutics ER -