Effects of ADA activity on endothelial adenosine signaling in vitro. (A) Modulation of adenosine signaling by hypoxia. Indicated concentrations of adenosine in HBSS were added to the apical surface of confluent normoxic (48-hour exposure to pO₂ 147) or posthypoxic (48-hour exposure to pO₂ 20 mm Hg) HMEC-1s and permeability to FITC-dextran (70 kDa) were quantified. Transendothelial flux was calculated by linear regression (3 samples over 60 minutes) and normalized as a percentage of control (HBSS). Data are derived from 6 monolayers in each condition. ^*Significant differences from baseline (P < .05). #Differences from baseline and from normoxia (P < .05). (B) Effect of extracellular ADA on paracellular permeability. Measurement of adenosine elicited barrier responses in normoxic endothelia (HMEC-1) with or without the addition of 0.1 nM bovine ADA. Note the dramatic decrease in adenosine-induced enhancement of endothelial barrier function in the presence of 0.1 nM ADA. ^*Significant differences from baseline (P < .05). #Differences from baseline and untreated controls (P < .05). (C) Effect of ADA inhibitor dCF on adenosine-elicited barrier responses. Posthypoxic endothelia (HMEC-1, pO₂ 20 mm Hg, 48 hours) were assessed for adenosine-elicited barrier responses in the presence of the highly specific ADA inhibitor dCF (100 nM). Note the dramatically increased adenosine elicited barrier responses with 100 nM dCF. ^*Significant differences from baseline (P < .05). #Differences from baseline and from untreated controls (P < .05). (D) Effect of inhibiting ADA binding to the ADA-complexing protein CD26 with gp120. Posthypoxic endothelia were washed with 100 nM gp120 in HBSS prior to measuring adenosine-elicited barrier function. Note the increased adenosine elicited barrier responses after gp120 treatment. ^*Significant differences from baseline (P < .05). #Differences from baseline and from untreated controls (P < .05). (A-D) Data are expressed as mean ± SD of percent control flux with HBSS only.