Figure 6.

Adenosine A_2B receptor (AdoRA_2B) induction by hypoxia enhances barrier response. (A) Microarray analysis of individual adenosine receptors in response to hypoxia (AdoRA₁, AdoRA_2A, AdoRA_2B, and AdoRA₃). Confluent HMEC-1 were exposed to normoxia or hypoxia (12 h exposure to pO₂ 20 torr) and the relative expression of individual adenosine receptors was quantified from total RNA by microarray analysis. Data are expressed as fold change ± SD relative to normoxia. (B) Real-time PCR analysis was employed to confirm hypoxia-regulated expression of individual adenosine receptors (AdoRA₁, AdoRA_2A, AdoRA_2B, and AdoRA₃) in HMEC-1. Data were calculated relative to internal control (β-actin) and are expressed as fold increase ± SD over normoxia at indicated time points. Results are derived from three experiments in each condition (*, P < 0.01 compared with normoxia). (C) Increased AdoRA_2B surface protein with hypoxia. Confluent HMEC-1 monolayers were exposed to indicated periods of hypoxia, monolayers were washed, surface proteins were biotinylated, and cells were lysed. AdoRA_2B was immunoprecipitated with mAb to human AdoRA_2B and resolved by SDS-PAGE, and resultant Western blots were probed with avidin-peroxidase. A representative experiment of three is shown. (D) Hypoxia induction of AdoRA_2B enhances barrier response of HMEC-1 to adenosine. Indicated concentrations of adenosine were added to HMEC-1 monolayers preexposed to normoxia or hypoxia (48 h). Addition of the specific AdoRA_2B-antagonist MRS 1754 (100 nM) significantly shifted the adenosine dose response in posthypoxic endothelium (P < 0.01 by ANOVA). Data are derived from 6 monolayers in each condition. Data are expressed as mean ± SD of percent control flux.