pyrophosphate and sphingosine-1-phosphate

To determine whether lysophosphatidic acid (LPA) or sphingosine-1-phosphate (S1P) affects transcellular resistance across cultured rabbit corneal epithelial and endothelial cells.. 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.. 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.. 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.

Topics: Actins; Animals; Cell Culture Techniques; Diphosphates; Electric Impedance; Endothelium, Corneal; Epithelium, Corneal; Fluorescent Antibody Technique, Indirect; Glycerol; GTP-Binding Protein alpha Subunits, Gi-Go; Lysophospholipids; Pertussis Toxin; Rabbits; Receptors, Lysophosphatidic Acid; Sphingosine; Tetradecanoylphorbol Acetate

Topics: Cell Differentiation; Cells, Cultured; Diphosphates; Endothelium, Vascular; Glycerol; Humans; Ligands; Lysophospholipids; Receptors, Cell Surface; Receptors, G-Protein-Coupled; Receptors, Lysophosphatidic Acid; Sphingosine