Fei Yin1, Mitchell A Watsky. 1. Department of Physiology, University of Tennessee Health Science Center, Memphis, 38106, USA.
Abstract
PURPOSE: To determine whether lysophosphatidic acid (LPA) or sphingosine-1-phosphate (S1P) affects transcellular resistance across cultured rabbit corneal epithelial and endothelial cells. METHODS: Electric cell-substrate impedance sensing (ECIS) was used to measure electrical resistance across cultured rabbit corneal epithelial and endothelial monolayers. After a 1-hour equilibration period, different concentrations of LPA or S1P were added to each well, and the effect observed for 4 hours. For cells significantly affected by LPA or S1P, pertussis toxin (PTX) or dioctyl-glycerol pyrophosphate (DGPP 8:0) was added along with LPA or S1P in separate experiments. Cells were also treated with phorbol 12-myristate 13-acetate (PMA) in the presence of LPA or S1P in different tests. The influence of LPA and S1P on epithelial and endothelial cell F-actin was determined with immunohistochemistry. RESULTS: LPA significantly increased the resistance of both the epithelial and endothelial monolayers, whereas S1P increased the resistance in only the endothelial cells. PTX blocked both the LPA- and S1P-induced increases in resistance, and DGPP (8:0) inhibited LPA-induced transcellular resistance in both the epithelium and endothelium. LPA and S1P prevented PMA-induced resistance decreases across epithelial and endothelial cells. F-actin staining around cell borders was more intense in both LPA- and S1P-treated cells. CONCLUSIONS: LPA increases transcellular resistance across cultured rabbit corneal epithelial and endothelial cell monolayers, and the effect is mediated through the LPA(1) receptor and signaled through Galpha(i/o). S1P-stimulated increases in endothelial resistance are also signaled through Galpha(i/o). Both LPA and S1P prevented increased transcellular permeabilities induced by PMA, and increased actin stress fiber formation in epithelial and endothelial cells.
PURPOSE: To determine whether lysophosphatidic acid (LPA) or sphingosine-1-phosphate (S1P) affects transcellular resistance across cultured rabbit corneal epithelial and endothelial cells. METHODS: Electric cell-substrate impedance sensing (ECIS) was used to measure electrical resistance across cultured rabbit corneal epithelial and endothelial monolayers. After a 1-hour equilibration period, different concentrations of LPA or S1P were added to each well, and the effect observed for 4 hours. For cells significantly affected by LPA or S1P, pertussis toxin (PTX) or dioctyl-glycerol pyrophosphate (DGPP 8:0) was added along with LPA or S1P in separate experiments. Cells were also treated with phorbol 12-myristate 13-acetate (PMA) in the presence of LPA or S1P in different tests. The influence of LPA and S1P on epithelial and endothelial cell F-actin was determined with immunohistochemistry. RESULTS:LPA significantly increased the resistance of both the epithelial and endothelial monolayers, whereas S1P increased the resistance in only the endothelial cells. PTX blocked both the LPA- and S1P-induced increases in resistance, and DGPP (8:0) inhibited LPA-induced transcellular resistance in both the epithelium and endothelium. LPA and S1P prevented PMA-induced resistance decreases across epithelial and endothelial cells. F-actin staining around cell borders was more intense in both LPA- and S1P-treated cells. CONCLUSIONS:LPA increases transcellular resistance across cultured rabbit corneal epithelial and endothelial cell monolayers, and the effect is mediated through the LPA(1) receptor and signaled through Galpha(i/o). S1P-stimulated increases in endothelial resistance are also signaled through Galpha(i/o). Both LPA and S1P prevented increased transcellular permeabilities induced by PMA, and increased actin stress fiber formation in epithelial and endothelial cells.
Authors: Lynn W Maines; Kevin J French; Ellen B Wolpert; David A Antonetti; Charles D Smith Journal: Invest Ophthalmol Vis Sci Date: 2006-11 Impact factor: 4.799