A previous study on BCECs has shown expression of A1- and A2B-receptors by RT-PCR and immunocytochemistry.
49 To investigate the identity of the receptors involved in the adenosine-mediated suppression of the effect of thrombin on the Ca
2+ wave, we used adenosine receptor agonists. As shown in
Figure 3 , in cells pretreated with CPA (selective A1 receptor agonist; 100 μM for 30 minutes), thrombin reduced the AA from 35,000 ± 3,700 (
N = 25) under control conditions to 17,000 ± 1,900 (
P < 0.05;
N = 25;
Fig. 3 ). It can also be noted that CPA itself had no significant effect on the AA compared with control conditions (
N = 25;
Fig. 3 ). These experiments suggest that the effect of adenosine on the suppression of thrombin-mediated inhibition of the Ca
2+ wave is not through A1 receptors. In contrast to CPA, NECA is known to enhance cAMP significantly in corneal endothelial cells,
49 consistent with its potent activation of A2B receptors, known to be coupled to adenylate cyclase via G
αS. Pretreatment with NECA (100 μM for 30 minutes) overcame the reduction of the AA of the Ca
2+ wave by thrombin (
P < 0.05;
N = 25). Specifically, the AA in the presence of the combination of NECA and thrombin did not differ significantly from the AA in the presence of NECA alone (31,100 ± 5,100 in NECA plus thrombin vs. 33,900 ± 4,700 in NECA;
N = 25;
Fig. 3 ).
To further establish that the inhibition of the effect of thrombin by adenosine is mediated through cAMP, we used forskolin to activate adenylate cyclase directly and also used the cAMP-dependent phosphodiesterase inhibitor, rolipram (selective for the PDE4 subtype). Pretreatment with forskolin (10 μM for 2 minutes) or the combination of forskolin (10 μM for 2 minutes) and rolipram (200 μM for 30 minutes) significantly inhibited the effect of thrombin when compared with thrombin alone (AA 23,100 ± 4,200 in forskolin plus thrombin and 38,000 ± 6,500 in forskolin plus rolipram plus thrombin versus 9,700 ± 1,800 in thrombin;
N = 25;
P < 0.005 and
P < 0.001, respectively;
Fig. 4 ).