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B. J. Himpens, R. Ponsaerts, C. D'hondt, S. P. Srinivas, J. Vereecke; Visualization of Thrombin-Induced Myosin Light Chain Phosphorylation in Bovine Corneal Endothelial Cells. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2693.
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Thrombin inhibits Ca2+ wave propagation in bovine corneal endothelial cells (BCEC). Although this thrombin effect is dependent on myosin light chain (MLC) phosphorylation, the mechanisms underlying the block of hemichannels and gap junctions involved in intercellular communication are not clear. In order to characterize the role of actin cytoskeleton on the thrombin effect, we have visualized phosphorylated MLC (pMLC) in cultured BCEC.
Confluent BCEC monolayers, 10 days after isolation, were exposed to thrombin (2 U/ml) for 5 min. pMLC was confocally visualized by immunocytochemistry (100 X objective) using a rabbit polyclonal antibody directed against pMLC-2 (Ser19) (Cell Signaling TechnologyTM, #3671, MA). A goat anti-rabbit IgG antibody conjugated to FITC was used as secondary antibody. Phalloidin, conjugated to Alexa Fluor 546, was used to stain the actin cytoskeleton.
Thrombin induced a significant increase in pMLC as indicated by an increase in the absolute area under the peak of the intensity histogram of pMLC positive pixels from 7684 ± 82 µm2 in control conditions to 8055 ± 80 µm2 (n=10) (P = 0.003) (1024x1024 image resolution with a total area of 8487.50 µm2). On an fluorescence intensity scale from 0 to 255, the peak of the distribution shifted to higher intensity values from 57.3 ± 1.9 in control conditions (n=9) to 68.7 ± 3.8 after thrombin stimulation (n=10) (P = 0.015). F-actin mainly appears as a cortical band in control conditions. Upon thrombin stimulation, the appearance of this cortical band was altered due to formation of more condensed F-actin bundles (n=10).
We visualized MLCp, and our analysis showed a direct increase of MLCp and reorganization of the actin cytoskeleton in BCEC upon thrombin stimulation. We speculate that thrombin-induced reduction of intercellular communication in BCEC is due to reorganization of the actin cytoskeleton by altered MLC phosphorylation status.
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