Molecular Mechanisms of Nitric Oxide-Induced Intestinal Epithelial Barrier Defects: Role of Tight Junctional Disruption and Myosin Light Chain Phosphorylation

• Main Authors: Hsin-Da Chiu, 邱新達
• Other Authors: 余佳慧
• Format: Others
• Language: zh-TW
• Published: 2009
• Online Access: http://ndltd.ncl.edu.tw/handle/83958019030416345587

碩士 === 國立臺灣大學 === 生理學研究所 === 97 === Physiological level of nitric oxide (NO) is crucial for maintaining gastrointestinal homeostasis, yet excessive production of mucosal NO is associated with gut inflammation and barrier defects. Intestinal barrier is composed of a monolayer of enterocytes linked by tight junctional proteins, e.g. ZO-1 and occludin, and perijunctional actinomyosin ring (PAMR). Disruption of TJs or contraction of PAMR leads to the increase of epithelial permeability. The mechanism of actinomyosin contraction was similar between epithelial and smooth muscle cells, which involved the phosphorylation of myosin light chain (MLC) by kinases, e.g. myosin light chain kinase (MLCK) and Rho-associated kinase (ROCK). Activation of PKCzeta (a membrane-associated atypical PKC) played a role in tight junctional remodeling in epithelial cells and was found associated with Rho signaling in smooth muscle cells. The aim was to investigate whether MLC phosphorylation and TJ disruption were involved in NO-induced intestinal epithelial barrier defects. Human colonic carcinoma Caco-2 cells were exposed to 0.5-5 mM S-Nitroso-N-acetylpenicillamine (SNAP, a NO donor) for 24 hr, and the transepithelial resistance (TER) and apical-to-basolateral flux rate of dextran-FITC (MW3000) were used as indicators of paracellular permeability. Post-confluent Caco-2 cells grown on transwells for 14-21 days established TJ and TER of 250-300 Ω*cm2. Exposure to SNAP increased epithelial permeability in a dose-dependent manner. SNAP concentration at 1 mM induced a 69% drop of TER and an 11-fold increase of epithelial permeability that were apoptosis-independent. The loss of epithelial barrier triggered by 1 mM SNAP was not blocked by pretreatment with a pancaspase or caspase-3 inhibitor. Densitometric analysis of western blots showed that 1 mM SNAP caused PKCzeta phosphorylation at 15 min post-challenge, as well as MLC phosphorylation, occludin cleavage and ZO-1 decrease at 24 hr post-challenge. Disorganization of TJs and cytoskeleton was associated with cell rounding in SNAP-exposed cells. Inhibitors to ROCK (Y-27632, 20 μM) and to MLCK (a novel membrane-permeant inhibitor of MLCK (PIK), 125-175 μM) reduced the level of MLC phosphorylation caused by SNAP challenge. Pretreatment with a ROCK inhibitor (Y-27632, 20-50 μM) attenuated SNAP-induced barrier defects whereas MLCK inhibitors, i.e. PIK (125-175 μM) or ML-7 (20 μM) had no effect. Moreover, pretreatment with PKCzeta inhibitory pseudosubstrate (20 μM) blocked SNAP-induced permeability rise and TJ disruption, but did not decrease the MLC phosphorylation. These findings suggest that exposure to NO induced PKCzeta-dependent TJ disruption and ROCK-dependent MLC phosphorylation, resulting in an increase of epithelial permeability.