Abstract
Abstract: :
Purpose: : To characterize BCE A2b Receptor (A2bR) and determine its functional role(s) in ion transport. Methods: We screened for A1, A2a, A2b and A3 adenosine receptors using RT–PCR. Protein detection using Western Blot (WB) and localization with immunostaining were performed to localize the A2b receptor. Pharmacological studies were used to assess functional roles of A2b receptor in BCE fluid transport. Results: Preliminary RT–PCR results suggested presence of A1 and A2b receptors. Because adenosine and other agents that increase [cAMP] are known to stimulate fluid transport by BCE, we hypothesized that A2b receptors are likely to be present. A2b immunostaining from cultured BCE and fresh tissue sections from bovine corneas indicated that A2b receptor localization is predominantly basolateral. Our A2b WB from sulfo–NHS–biotin membrane preparation showed a 50 kDa band for both fresh and cultured BCE, with mouse brain as our positive control. Previous cAMP assay with 10 µM adenosine, a general A1 and A2 receptor agonist, produced a 3–5 fold increase in [cAMP]i , consistent with the presence of the A2b subtype. Fura–2 measurement of [Ca]i failed to detect a change in cell [Ca2+] upon adenosine application, indicating the lack of significant A1 receptor activation. Past studies indicated that adenosine and forskolin increased chloride permeability. Our initial pharmacological studies using alloxazine (a specific A2bR blocker) and DMPX (a general A2R antagonist) showed that chloride permeability in the presence of adenosine is inhibited by 34% and 90%, respectively. A secondary effect of adenosine on increased chloride permeability is membrane depolarization from chloride efflux. An increase in K+ permeability could sustain this chloride efflux. RT–PCR screening suggests the presence of KCNQ1, a cAMP–activated K+ channel. The secondary effect of A2bR activation on K+ channel permeability is currently being investigated. Conclusions: Our preliminary results support the presence of A2bR in BCE. Immunostaining results indicated that the A2bR localization is basolateral. Pharmacological results are consistent with a functional role of A2bR in ion transport.
Keywords: cornea: endothelium • signal transduction: pharmacology/physiology • immunohistochemistry