Characterization of adenosine receptors in bovine corneal endothelium

• Published in: Experimental eye research Vol. 80; no. 5; pp. 687 - 696
• Main Authors: Tan-Allen, Kah Y., Sun, Xing Cai, Bonanno, Joseph A.
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 01.05.2005
• Subjects: Adenosine - pharmacology; adenosine receptors; Animals; Blotting, Western; Calcium - analysis; cAMP; Cattle; Cell Membrane Permeability; Cells, Cultured; Chlorides - metabolism; Cl − permeability; corneal endothelium; Cyclic AMP - analysis; Endothelium, Corneal - chemistry; Endothelium, Corneal - metabolism; Flavins - pharmacology; Fluorescent Antibody Technique, Indirect; Membrane Potentials; Purinergic P2 Receptor Antagonists; Receptors, Purinergic - analysis; Receptors, Purinergic - genetics; Receptors, Purinergic P1 - analysis; Receptors, Purinergic P1 - genetics; Receptors, Purinergic P2 - analysis; Receptors, Purinergic P2 - genetics; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger - analysis; corneal endothelium; cAMP; Cl − permeability; adenosine receptors
• ISSN: 0014-4835; 1096-0007
• DOI: 10.1016/j.exer.2004.12.002

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, [Ca 2+] 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 [Ca 2+] 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 (∼50 kDa) and A 1 (∼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 μ m alloxazine, a specific A 2b blocker. A 1 activation with 1 μ m N 6-CPA (a specific A 1 agonist) or 100 n m adenosine decreased [cAMP] i by 23 and 6%, respectively. Adenosine had no effect on [Ca 2+] 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 μ 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 μ m adenosine, acts to decrease [cAMP] i and A 2b, activated at >1 μ m adenosine, increase [cAMP] i.