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G. M. Sumida, W. D. Stamer; S1P Receptor-Mediated Effects in Human Schlemm’s Canal Endothelial Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(13):682. doi: https://doi.org/.
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The lipid mediator sphingosine 1-phosphate (S1P) decreases outflow facility in perfused human eyes. In addition to expression by trabecular meshwork (TM) cells, S1P receptors are present in Schlemm’s canal (SC) endothelial cells. We hypothesize that S1P effects on outflow facility are due to increased cell-cell junction assembly at the level of the inner wall of SC. The aim of the present study was to determine the time course of S1P receptor activation and its participation in junction assembly in cultured SC cells.
The phosphorylation status of myosin light chain (MLC) was used to determine the time course of S1P receptor activation in primary cultures of SC and TM cells (SC46, SC53, TM87, TM89) treated with 1µM S1P or vehicle for 1, 3, 5, 30, and 60 minutes. Relative MLC and phospho-MLC levels were obtained through western blot analysis of whole cell lysates. To study junctional organization, SC and human umbilical vein endothelial cells (HUVEC) grown on polyethylene terephthalate membrane inserts were treated with 1µM S1P or vehicle and immunostained for cortactin or β-catenin. Cells were then counterstained with phalloidin and viewed using confocal immunofluorescence microscopy.
Baseline levels of MLC phosphorylation in vehicle-treated SC cells were at or below the threshold of detection. Within 5 minutes after S1P treatment, however, SC cells reached maximal levels of MLC phosphorylation. Detectable changes of β-catenin and filamentous actin redistribution to the cell periphery were observed following S1P treatment in both SC and HUVEC cells compared to vehicle controls. Interestingly, no significant difference in cortactin localization was observed in S1P versus vehicle-treated SC cells, unlike that seen in the positive control (HUVECs).
Functional S1P receptors mediate rapid phosphorylation of MLC in human SC cells. Concurrently, actin reorganization and recruitment of β-catenin to sites of intercellular junctions following S1P treatment may facilitate a decrease in permeability of the inner wall of SC (and associated decrease in outflow facility) by increasing junctional organization.
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