FIGURE 4.

Endothelial responses to ADP and S1P after inhibition of phospholipase C, calcineurin, or CaMKKβ. A, BAEC were treated with the PLC inhibitor U-73122 (10 μm) for 30 min and then stimulated for 5 min with ADP (50 μm) or S1P (100 nm). Immunoblots were probed with antibodies as indicated. Equal loading was confirmed using antibodies directed against total eNOS, AMPK, GSK3β, and ERK1/2. Shown is a representative immunoblot from four individual experiments that yielded similar results. Quantitative analysis of pooled data is shown for the relative phosphorylation of eNOS Ser¹¹⁷⁹ (B), GSK3β (C), and ERK1/2 (D). E, cell lysates were prepared from BAEC pretreated for 30 min with cyclosporine (100 nm) and then stimulated for the times indicated with epinephrine (1 μm), VEGF (20 ng/ml), or ADP (50 μm). Lysates from BAEC were resolved by SDS-PAGE and probed using antibodies directed against phospho-eNOS Ser¹¹⁶ and eNOS. Shown here is an immunoblot representing three individual experiments with equivalent results. Analysis of pooled data quantitating relative eNOS Ser¹¹⁶ phosphorylation after treatment with epinephrine, VEGF, or ADP is shown in F–H, respectively. * indicates p < 0.05 compared with agonist-mediated levels of eNOS Ser¹¹⁶ phosphorylation in the absence of cyclosporine. I, BAEC were incubated with STO-609 (10 μm) for 30 min and then treated with ADP (50 μm) or S1P (100 nm) for 5 min. Shown is a representative immunoblot from three individual experiments with similar results. Pooled quantitative data comparing relative phosphorylation of eNOS Ser¹¹⁷⁹ (J), eNOS Ser⁶³⁵ (K), AMPK (L), and ACC (M) are shown. Each bar in the graph represents the mean value ± S.E. * denotes p < 0.05, ** denotes p < 0.01, and *** denotes p < 0.001. p, phospho; veh, vehicle; NS, not significant; Epi, epinephrine.