Figure 3.
Signaling events involved in Sph-1-P–mediated endothelial cell barrier enhancement. (a) Bovine endothelial cells were incubated with tyrosine kinase inhibitors genistein (Gen; 100 μM, 30 minutes), herbimycin A (Herb; 5 μM, 8 hours), or erbstatin analog (Erb; 20 μM, 1 hour); the PI-3′ kinase inhibitor LY294002 (LY; 25 μM, 30 minutes), p42/p44 MAP kinase inhibitor UO126 (UO; 10 μM, 30 minutes), the p38 MAP kinase inhibitor SB203580 (SB; 20 μM, 30 minutes), or p60 src inhibitor PP2 (10 μM, 30 minutes), followed by stimulation with Sph-1-P (1 μM). TER was continuously measured for 3 hours and the maximal Sph-1-P response (10–15 minutes after stimulation) were employed for determining the effect of the inhibitors on Sph-1-P–induced barrier enhancement. Data are presented as mean ± SE, n = 3, P < 0.05. Genistein, herbimycin A, and erbstatin analogues did not significantly alter the basal TER, but inhibited Sph-1-P–induced increases in TER, indicating the involvement of tyrosine kinases in Sph-1-P–mediated modulation of endothelial cell barrier integrity. (b) Endothelial cells were incubated with Sph-1-P (1 μM) for indicated times, followed by Western analysis using an phosphotyrosine Ab. Sph-1-P stimulated tyrosine phosphorylation of multiple proteins as indicated by arrows.