Figure 3.

¹⁴C-Sucrose transport through the bEnd.3 monolayer was increased after the blockade of DAGLα. The bEnD.3 endothelial cells were cultured on a trans-well insert, then treated with an increasing amount of 2-AG (0–10 nmole). In a separate experiment, cells were treated with LEI-106 (1.3 mM) or vehicle (0.9% DMSO in media) with or without 2-AG (600 pmol) for 15 min. KCl pulse (100 mM, 5 min) was used as a positive control. Following treatment, luminal media was replaced with fresh media containing ¹⁴C-sucrose (0.25 µCi/mL). Abluminal media was collected at 5 min and 30 min post-treatment and subjected to measure radioactivity with a liquid scintillation counter. ¹⁴C-sucrose uptake assay was performed 72 h after transfection of DAGLα siRNA or non-targeting control. ¹⁴C-sucrose (0.25 µCi/mL) was added to the luminal side, abluminal media was collected at two time-points (5 min and 30 min), and then radioactivity was measured. (A) Schema of experimental setting. (B) 2-AG treatment at any dose did not cause a significant change in ¹⁴C-sucrose uptake at 5 min time-point compared to vehicle control (2-AG at any dose vs. vehicle: p > 0.05, F(5,28) = 4.010, as assessed by one-way ANOVA with Bartlett’s test) All data represent the % of vehicle-treated ± SEM (n = 3–10/group). ns = non-significant. (C) A significant increase in ¹⁴C-sucrose uptake after 300 pmol of 2-AG treatment was observed at 6–30 min time-point compared to vehicle control (2-AG-300 pmol vs. vehicle: p = 0.0172, 2-AG at other doses vs. vehicle: p > 0.05, F(5,20) = 3.879, as assessed by one-way ANOVA with Bartlett’s test) All data represent the % of vehicle-treated ± SEM (n = 3–10/group). ns = non-significant, * p < 0.05. (D) LEI-106 (1.3 mM) increased the ¹⁴C-sucrose uptake at 5 min time-point compared to vehicle control, indicating the presence of paracellular leak (LEI-106 vs. vehicle: p = 0.0033, t(9) = 3.965 as assessed by unpaired t-test). The application of 2-AG did not significantly reduce the elevated sucrose uptake induced by LEI-106 (LEI-106 vs. LEI-106 + 2-AG: p = 0.0797, t(7) = 2.049 as assessed by unpaired t-test). All data represent the % of vehicle-treated ± SEM from three-four independent experiments using 4 trans-well inserts/group. ** p < 0.01 compared to vehicle control. ns = non-significant. (E) Increased ¹⁴C-sucrose uptake was also observed at the 30 min time-point after LEI-106 treatment (LEI-106 vs. vehicle: p = 0.0092, t(8) = 3.415 as assessed by unpaired t-test). 2-AG treatment significantly mitigated the increase in sucrose uptake caused by DAGLα inhibition (LEI-106 vs. LEI-106 + 2-AG: p = 0.05, t(7) = 2.273 as assessed by unpaired t-test). All data represent the % of vehicle-treated ± SEM from three-four independent experiments using 4 trans-well inserts/group. * p < 0.05, ** p < 0.01 compared to corresponding controls. (F) Silencing of DAGLα significantly increased the sucrose uptake in the first 5 min, compared to non-targeting control, suggesting reduced barrier integrity caused by genetic inhibition of DAGLα (DAGLα siRNA vs. non-targeting siRNA: p = 0.0022, t(8) = 4.420 as assessed by unpaired t-test, n = 5). Data are shown mean ± SEM. ** p < 0.01, compared to the non-targeting control. (G) No significant difference between DAGLα siRNA and control was observed at the later time-point (6–30 min) (DAGLα siRNA vs. non-targeting siRNA: p = 0.8204, t(8) = 0.2347 as assessed by unpaired t-test, n = 5). Data are shown mean ± SEM. ns = non-significant, compared to the non-targeting control.