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Vol. 305, Issue 2, 482-494, May 2003
-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
Department of Internal Medicine, Section of Gastroenterology and
Nutrition, Pharmacology, and Molecular Physiology, Rush University
Medical Center, Chicago, Illinois
Using intestinal (Caco-2) cells, we found that oxidant-induced
disruption of barrier integrity requires microtubule disassembly. Protein kinase C (PKC)-
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- 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- or with a
dominant-negative to inhibit the activity of native PKC-. Clones
were then incubated with oxidant (H2O2) ± modulators. Parental cells were treated similarly. Exposure to
oxidant-disrupted monolayers by increasing native PKC- 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- 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- 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- and up-regulation of iNOS. 2) Overexpression and activation of
PKC- 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.
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