Figure 5.

Benidipine prevents R. parkeri-induced disruption of endothelial cell monolayer integrity (A, B, C) and an increase in cytoplasmic [Ca²⁺]_i (D, E) in tHBMECs. (A) Real-time traces of the concentration-dependent prevention by benidipine of the TEER drop across the microvascular monolayers induced by R. parkeri-infected tHBMECs. 96-well gold-electrode ECIS microarrays with stable tHBMEC monolayers were pretreated for 1 hour with 0.01–10 μM benidipine (RpTHB+Ben 0.01/0.1/1&10), M199 growth media only (M199), or 1 or 10 μM nifedipine (RpTHB+Nif 1&10) before addition of 2.4 × 10³ R. parkeri-infected (RpTHB) or uninfected control tHBMECs (THB). Vertical lines along the traces show S.E.M. of the corresponding ECIS traces. Higher MOIs of R. parkeri more rapidly disrupted monolayer integrity compared to a modestly lower R. parkeri MOI (1.6 × 10³ infected tHBMECs, [1.6K RpTHB]). A benidipine dose-dependent delay in TEER reduction was observed. (B and C) show TEER of tHBMEC monolayers induced by R. parkeri in the presence and absence of the Ca²⁺ channel blockers. Cell monolayers in 8-well gold-electrode ECIS arrays were pretreated for 1 hour with (a) 10 μM benidipine, (c) M199 growth media or (d) 10 μM nifedipine before addition of 3.0 × 10³ infected (a, d, e) or uninfected control tHBMECs (b). (C) Analysis of protective effects of benidipine against R. parkeri-induced disruption of tHBMEC monolayer integrity. For comparative statistical analysis the data were selected at 12 and 24h after the cell additions (arrows in B). Data are means ± SD, n=3. ***p< 0.001 for benidipine treatments against Rp-infected cells. (D) Representative traces of the intracellular [Ca²⁺]_i changes in Rp-infected tHBMECs, treated or non-treated with 10 μM benidipine. For comparative statistical analysis the kinetic data were selected at 50 min (white arrow on D), and shown in 6E. In 6E, 10 μM ATP was also used as a positive control to induce intracellular [Ca²⁺]_i rise. Data are means ± SEM, n=4, **p<0.01.