Rap activation and function in barrier integrity in endothelial cells through Epac. (A) Western blot analysis of cell lysates from HUVECs using an Epac1 or Epac2 antibody revealed a specific band at 110 kDa for Epac1, and 120 kDa and 79 kDa for Epac2, consistent with their predicted molecular weight and a described short isoform of Epac2 that has been shown to retain GEF activity toward Rap1.²²  (B) HUVECs were treated with forskolin and rolipram (F + R, 10 μM, and 20 μM, respectively) for 25 minutes or O-Me-cAMP (100 μM) for the times indicated. Pull-down assays to detect active Rap1 (Rap1-GTP) and Rap2 (Rap2-GTP) were then performed. Western blot for total Rap shows equal amount of Rap protein in all samples. The “fold induction” of Rap activation following the indicated treatments (numbers below Rap1-GTP and Rap2-GTP blots) was statistically significant for Rap1 at all time points (P < .05) and for Rap2 only at 15 minutes following O-Me-cAMP treatment (P < .05). (C) Analysis of PKA activation. HUVECs were treated with F + R, as in panel C, or O-Me-cAMP (500 μM) for the times indicated. Western blots for phospho-CREB (pCREB) or total CREB are shown and are representative of 4 independent experiments. (D) Permeability assays show HUVEC response with F + R or O-Me-cAMP at the doses indicated. Results are the normalized mean and standard error of 4 independent experiments (*P < .01 as compared to control). (E) Permeability was measured at 5 and 15 minutes following addition of F + R or O-Me-cAMP. cAMP elevation (F + R; □) and 100 μM (▦) and 500 μM of O-Me-cAMP (▪) significantly increased barrier function at both 5 and 15 minutes after their addition. Results are the normalized mean and standard error of 3 independent experiments (*P < .05, **P < .01 as compared to control [set at 1.0] for each time point).