Functional increase in CD73 surface activity by hypoxia. (a) Epithelial monolayers were exposed to indicated periods of hypoxia and washed, and surface CD73 activity was determined by HPLC analysis of E-AMP conversion to E-ADO (black bars). To determine specificity, a similar analysis was performed in the presence of the CD73 inhibitor APCP (white bars). Data are derived from five to seven monolayers in each condition, and results are expressed as mean percent E-AMP conversion ± SEM. The inset is a representative HPLC tracing demonstrating peak resolution of E-AMP and E-ADO. mAu, milli-absorbance unit. (b) Confluent T84 monolayers were subjected to hypoxia for 24 hours and treated with 2 mM EDTA for 5 minutes, followed by incubation in cell culture media with normo-calcium in the presence of indicated concentrations of 5′-AMP. TER was monitored over time, and the results shown represent the percent recovery of TER over 2 hours relative to no 5′-AMP. Also shown are plots of monolayers coincubated with the adenosine A_2B receptor antagonist alloxazine (allox; 10 μM). Data are mean ± SEM from three separate experiments. *P < 0.025 compared with normoxia.