ADA induced by hypoxia is enzymatically active. (A) To measure ADA enzyme activity on the endothelial surface, HMEC-1s were exposed over indicated time periods to hypoxia (pO₂ 20 mm Hg), 50 μM adenosine was added to the supernatant (HBSS) of intact HMEC-1s, and inosine generation was measured via HPLC. The HPLC tracing obtained from the supernatant is displayed and retention times for inosine (3.2 min) and for adenosine (3.7 min) are indicated (black line indicates HBSS alone; gray line, HBSS after addition of adenosine [50 μM]). (B) Induction of ADA activity in total-cell lysates of endothelia by hypoxia. To obtain an estimate of total ADA activity increase with hypoxia, the ADA activity in lysates of HMEC-1s was measured. (C) Induced, enzymatically active ADA is localized to the cell surface. ADA enzyme activity was measured in intact HMEC-1s by adding 50 μM adenosine to the supernatant (HBSS) and measuring inosine generation. All experiments were performed in the presence of 10 μM dipyridamole to prevent endothelial adenosine uptake. To confirm that the observed increase in inosine generation with hypoxia exposure reflects ADA activity, controls with 100 nM dCF were performed in normoxic (dCF [Norm]) or posthypoxic (dCF [Hyp 48 h]) endothelia. Note the robust hypoxia induction of functional ADA to the cell surface. (D) ADA release into the supernatant is increased with hypoxia. To measure ADA release into the supernatant, HMEC-1s were exposed to normoxia or hypoxia (HMEC-1, pO₂ 20 mm Hg, 48 hours). During the last 4 hours, the media was replaced with HBSS and ADA activity in the cell supernatant was measured. In controls, dCF (dCF [Hyp 48 h], 100 nM) inhibited soluble ADA activity in posthypoxic supernatants. In contrast, the HIV-1 envelope glycoprotein gp120 (gp120 [Hyp 48 h], 100 nM) did not affect ADA activity in the supernatant. Error bars indicate SD.