Fig. 4. Expression of adenosine receptor subtypes in Calu-3 cells. (A) Increase in the dye uptake rate induced by NECA (10 µM, 24 h) as quantified from dye uptake experiments. (B) RT-PCR analysis and western blot experiments showed expression of the adenosine receptor subtypes A_2A and A_2B. qRT-PCR analysis showed a remarkably higher expression of the A_2B adenosine receptor subtype. The results are given as average ± SEM from three cell culture passages. (C) NECA increased the intracellular cAMP concentration. The results are given as average ± SEM from three cell culture passages. (D) The increase in the dye uptake rate was attenuated by the AC inhibitor SQ22536 (SQ, 400 µM) and the PKA inhibitor Rp-cAMPs (Rp-c, 200 µM). (E) The NECA-induced increased dye uptake rate was not significantly affected by the A_2A adenosine receptor antagonist SCH58261 (SCH, 0.5 μM). The A_2B adenosine receptor antagonist MRS1754 (MRS, 0.5 μM) blocked the NECA-induced increase in the dye uptake rate. The A_2A adenosine receptor-specific agonist CGS21680 (CGS, 50 nM) did not change the dye uptake rate, while the A_2B adenosine receptor-specific agonist BAY60-6583 (BAY, 100 nM) significantly increased the dye uptake rate. (F) siRNA-knockdown of the A_2B adenosine receptor subtype blocked the NECA-induced increase in the dye uptake rate. The dye uptake rates are given as average ± SEM from respectively six experiments with three cell culture passages. The data were statistically compared to the control using student's t test (P<0.05 *, P<0.01 **, P<0.001 ***).