(A) C57BL/6 mice were mechanically ventilated (inspiratory pressure of 35 mbar, 100% oxygen). After the indicated time periods, lungs were harvested, total RNA was isolated, and A1AR, A2AAR, A2BAR, and A3AR mRNA levels were determined by real-time RT-PCR. Data were calculated relative to the internal housekeeping gene (β-actin) and are expressed as mean fold change compared with control (0 min ventilation) ± SD at each indicated time (n = 4). Note selective induction of the A2BAR gene during high-pressure ventilation (10-fold, P < 0.01; n = 4). (B) Comparative gene expression of pulmonary ARs using real-time PCR. Relative expression levels in untreated controls or in mice after 180 min mechanical ventilation (inspiratory pressure of 35 mbar, 100% oxygen) are shown. Values are expressed as mean ± SEM (n = 4). *P < 0.05 compared with A2AAR. (C) Mice were mechanically ventilated (35 mbar inspiratory pressure, 100% oxygen), and lungs were harvested at the indicated time points, shock frozen, and lysed, and proteins were resolved by SDS-PAGE. Resultant western blots were probed with anti-A2BAR antibodies. To control for loading conditions, blots were stripped and reprobed for actin expression. One representative experiment of 3 is shown. (D) To examine the influence of mechanical ventilation on pulmonary A2BAR expression patterns, C57BL/6 mice were ventilated in a pressure-controlled setting over 0 h or 3 h (35 mbar inspiratory pressure, 100% inspired oxygen concentration). Lungs were stained with antibodies for A2BAR. IgG controls were used at identical concentrations and staining conditions as the target primary antibodies (original magnification, ×400; n = 4).