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 A(1) and A(2b) adenosine receptors, while A(2a) and A(3) were negative. Western blot (WB) confirmed the presence of A(2b) (similar to 50 kDa) and A(1) (similar to 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 mu M alloxazine, a specific A2b blocker. A, activation with 1 mu m N-6-CPA (a specific A(1) agonist) or 100 nM adenosine decreased [cAMP](i) by 23 and 6%, respectively. Adenosine had no effect on [Ca2+](i) mobilization. Indirect immunofluorescence localized A(2b) receptors to the lateral membrane and A(1) 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 mu M), a general A(2) blocker. Adenosine-induced membrane depolarization was also inhibited by 33% (n = 6) in the presence of alloxazine. Bovine corneal endothelium expresses functional A(1) and A(2b) adenosine receptors. A(1), preferentially activated at < 1 mu m adenosine, acts to decrease [cAMP](i) and A(2b), activated at > 1 mu M adenosine, increase [cAMP](i). (c) 2004 Elsevier Ltd. All rights reserved.