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), [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) ( approximately 50 kDa) and A(1) ( approximately 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 microm alloxazine, a specific A(2b) blocker. A(1) activation with 1 micromN(6)-CPA (a specific A(1) agonist) or 100 nm 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 microm), 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 microm adenosine, acts to decrease [cAMP](i) and A(2b), activated at >1 microm adenosine, increase [cAMP](i).
Tan-Allen
Kah Y
KY
School of Optometry, Indiana University, 800 East Atwater Avenue, Bloomington, IN 47405, USA.
Sun
Xing Cai
XC
Bonanno
Joseph A
JA
D000241:Adenosine / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000032:analysis; D002417:Cattle; D002463:Cell Membrane Permeability; D002478:Cells, Cultured; D002712:Chlorides / Q000378:metabolism; D000242:Cyclic AMP / Q000032:analysis; D004728:Endothelium, Corneal / Q000737:chemistry* / Q000378:metabolism; D005415:Flavins / Q000494:pharmacology; D019084:Fluorescent Antibody Technique, Indirect; D008564:Membrane Potentials; D058919:Purinergic P2 Receptor Antagonists; D012333:RNA, Messenger / Q000032:analysis; D011983:Receptors, Purinergic / Q000032:analysis* / Q000235:genetics; D018047:Receptors, Purinergic P1 / Q000032:analysis / Q000235:genetics; D018048:Receptors, Purinergic P2 / Q000032:analysis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction
D002712:Chlorides; D005415:Flavins; D058919:Purinergic P2 Receptor Antagonists; D012333:RNA, Messenger; D011983:Receptors, Purinergic; D018047:Receptors, Purinergic P1; D018048:Receptors, Purinergic P2; C008173:isoalloxazine; D000242:Cyclic AMP; D000241:Adenosine; D002118:Calcium