Fig. 7.

Effect of (S)- and (R)-FTY720 regioisomers on PP2A activity and induced barrier disruption. (a) Bar graphs depict pooled PP2A activity data quantified as the amount of free PO₄ cleaved from pT substrate relative to a standard curve for methanol vehicle (white), ethanol vehicle (grey), FTY720 (5 µM) (green), (S)-FTY720 regioisomer (5 µM) (blue), or (R)-FTY720 regioisomer (5 µM) (purple) (±S.D.). n = 3 independent experiments per condition; *p < 0.05 agonist versus ethanol vehicle alone, p < 0.05 agonist versus methanol vehicle alone. (b) HPAEC were plated on gold microelectrodes for TER measurements as described in the Methods section. Bar graphs depict pooled TER data from HPAEC pre-treated for 1 h with no inhibitor (grey), CAL inhibitor (calyculin A, PP1/2A-C inhibitor, 2 nM, green), or OKA inhibitor (okadaic acid, PP2A inhibitor, 50 nM, blue), then stimulated with (S)-FTY720 regioisomer (10 µM), (R)-FTY720 regioisomer (10 µM), or S1P (1 µM) as indicated. The data are expressed as change in TER, compared to normalized unstimulated or inhibitor only controls, at 4 h ((S)- and (R)-FTY720 regioisomers) or 10 min (S1P) after agonist stimulation (±S.E.M.). Normalized resistance values over 1 indicate EC barrier enhancement. Normalized resistance values under 1 indicate EC barrier disruption. n = 3 independent experiments per condition; *p < 0.01 agonist alone versus unstimulated cells.

S1P: sphingosine-1-phosphate; FTY720: 2-amino-2-(2-[4-octylphenyl]ethyl)-1,3-propanediol.