Characterization of adenosine receptors in bovine corneal endothelium

Previous studies indicated that adenosine can increase [cAMP]i and stimulate fluid transport by corneal endothelium. The purpose of this study was to determine which adenosine receptor subtype(s) are expressed and to examine their functional roles in modulating [cAMP]i, [Ca2+]i and effects on Cl− permeability in corneal endothelium. We screened bovine corneal endothelium (BCE) for adenosine receptor subtypes by RT-PCR and immunoblotting, and examined the effects of pharmacological agents on adenosine stimulated Cl− transport, [cAMP]i and [Ca2+]i. RT-PCR indicated the presence of A1 and A2b adenosine receptors, while A2a and A3 were negative. Western blot (WB) confirmed the presence of A2b (∼50 kDa) and A1 (∼40 kDa) in fresh and cultured BCE. Ten micromolar adenosine increased [cAMP]i by 2.7-fold over control and this was inhibited 66% by 10 μm alloxazine, a specific A2b blocker. A1 activation with 1 μmN6-CPA (a specific A1 agonist) or 100 nm adenosine decreased [cAMP]i by 23 and 6%, respectively. Adenosine had no effect on [Ca2+]i mobilization. Indirect immunofluorescence localized A2b receptors to the lateral membrane and A1 to the apical surface in cultured BCE. Adenosine significantly increased apical Cl− permeability by 2.2 times and this effect was nearly abolished by DMPX (10 μm), a general A2 blocker. Adenosine-induced membrane depolarization was also inhibited by 33% (n=6) in the presence of alloxazine. Bovine corneal endothelium expresses functional A1 and A2b adenosine receptors. A1, preferentially activated at <1 μm adenosine, acts to decrease [cAMP]i and A2b, activated at >1 μm adenosine, increase [cAMP]i.