Role of Edg family receptors and heterotrimeric G proteins in Sph-1-P–mediated endothelial cell barrier regulation. (a) Subconfluent bovine endothelial cells were incubated with Edg-1 antisense or their respective control scrambled oligonucleotides (10 μg/ml, 48 hours). This treatment reduced Edg expression by approximately 50%, whereas control oligonucleotide was without effect. (b) In similar experiments, Edg-1 and Edg-3 antisense (AS) oligonucleotides significantly attenuated the Sph-1-P–induced TER response. Sph-1-P–induced TER increased from baseline normalized resistance level of 1.0 to a level of 1.5 to 1.75 in control oligo-treated cells. Data are presented as percentages of Sph-1-P–induced TER in AS-treated cells to Sph-1-P–induced TER in control oligonucleotide–treated cells (mean ± SE, n = 3, P < 0.05). Inset: ECIS tracing reflecting the reduction of the Sph-1-P response in Edg-1–AS treated cells. Tracing number 1, cells with control oligo; number 2, cells with control oligo and stimulated with Sph-1-P; number 3, cells with Edg-1 antisense; and number 4, cells with Edg-1 antisense and stimulated with Sph-1-P. (c) Bovine endothelial cells were cotransfected with pEGFP and a plasmid encoding either Edg-1, Edg-3, Edg-5, or the empty vector. GFP-positive cells were isolated by flow cytometry (13) and subjected to TER measurements. The percentage of changes of resistance in Edg-transfected cells relative to the vector-transfected cells were calculated. Overexpression of Edg-5, but not Edg-1 or Edg-3, augmented Sph-1-P–induced increases in TER. Data are presented as percentage of Sph-1-P–induced TER in cells overexpressing Edg receptors to Sph-1-P–induced TER in cells transfected with vector control (mean ± SE, n = 3, P < 0.05). Inset: ECIS tracing to show the augmentation of Sph-1-P response in cells expressing Edg-5. In tracing number 1 cells are transfected with the empty vector; in tracing number 2 cells are transfected with the empty vector and stimulated with Sph-1-P; in tracing number 3 cells are transfected with Edg-5; and in tracing number 4 cells are transfected with Edg-5 and stimulated with Sph-1-P. (d) Bovine endothelium were either incubated with PTX (200 ng/ml, 2 hours) or transfected with plasmids encoding G protein inhibitory peptides βARK or G_iα2 using pEGFP cotransfection and cell-sorting strategies described for c. Each G protein manipulation, that is, PTX, βARK, and G_iα1/2 expression, significantly attenuated the Sph-1-P response (mean ± SE, n = 3, P < 0.05). Inset: Time-dependent ADP ribosylation of G protein in bovine endothelial monolayers incubated with PTX followed by in vitro ADP-ribosylation. PTX treatment before cell lysis completely inactivated G_i protein via ADP-ribosylation as we have described previously (27, 28).