A2B adenosine receptor (A2BAR) on HMEC-1 cells promotes endothelial barrier function in vitro. Inhibition of A2BAR mRNA expression by stable expression of A2BAR siRNA. HMEC-1 cells were stably transfected with a plasmid expressing A2BAR siRNA or a nonspecific siRNA (Control Transfected, SCR). Cells were exposed to hypoxia (pO₂ = 20 torr) and real-time RT-PCR was used to confirm repression of the A2BAR (A) Data were calculated relative to β-actin and are expressed as fold change over control plus or minus SD after indicated time periods of hypoxia exposure. Results are derived from 3 experiments (*P < .05, different from normoxia; #P < .01, compared with normoxia and control transected; **P < .01 different from control transfected). Western blot analysis was used to confirm siRNA repression of the A2BAR protein (B). A representative blot of 3 is shown (a vertical line has been inserted to indicate a repositioned gel lane.), in addition to densitometric analysis of A2BAR protein levels relative to β-actin (*P < .01, different from normoxia; #P < .05, compared with normoxia and control transected; **P < .05 different from control transfected, n = 3). Repression of A2BAR expression inhibits adenosine-mediated enhancement of endothelial barrier function. Indicated concentrations of adenosine in HBSS were added to the apical surface of confluent normoxic (48-hour exposure to pO₂ = 147 torr) or hypoxic (48-hour exposure to pO₂ = 20 torr) HMEC-1 that were stably transfected with A2BAR (D) or control (C) siRNA. Permeability to FITC-dextran (70 kDa) was quantified by measuring transendothelial flux (3 samples over 60 minutes) and normalized as a percentage of control (HBSS). Baseline flux rates for normoxic conditions where approximately 200 to 250 pM/min per cm² and 800 to 1000 pM/min per cm² after hypoxia exposure (less than 5% of tracer). Data are derived from 6 monolayers in each condition (*P < .05, compared with baseline; #P < .05, compared with baseline and normoxia). (E) The A2BAR antagonist PSB1115 inhibits adenosine-mediated enhancement of endothelial barrier function. Adenosine elicited barrier responses were measured in normoxic endothelia (HMEC-1) with or without the addition of 1 μM PSB1115 (*P < .05, compared with baseline). (F) The A2BAR agonist BAY 60-6583 promotes barrier responses. Hypoxic endothelia (48-hour exposure to pO₂ = 20 torr) that were stably transfected with A2BAR or control siRNA were assessed for BAY 60-6583–elicited barrier responses. Data are expressed as means plus or minus SD (*P < .05, compared with baseline. #P < .05, compared with baseline and untreated controls, n = 6).