Characterization of adenosine receptors in bovine corneal endothelium

Abstract

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] 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 μM alloxazine, a specific A 2b blocker. A 1 activation with 1 μM 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 [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 μM), 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 μM adenosine, acts to decrease [cAMP] i and A 2b , activated at >1 μM adenosine, increase [cAMP] i .

Adenosine receptor expression and function in bladder uroepithelium

“…The A 2b receptor antibody recognized a single molecular mass species of 52 kDa, which is greater than the calculated mass of 36.3 kDa (Fig. 1C) but is identical to the reported mass of A 2b receptors detected in bovine corneal endothelium and kidney vasculature (20,34). The A 3 -specific antibody detected a protein species of 52 kDa, consistent with previous reports (20), and a species of 36.2 kDa, the nominal molecular mass of the protein.…”

“…1). As previously reported (20,34), a predominant molecular mass species of ϳ38 kDa was detected with an anti-A 1 receptor antibody (Fig. 1A), which is close to the calculated molecular mass of the A 1 receptor (36.5 kDa).…”

Role of Carbonic Anhydrase IV in Corneal Endothelial HCO3−Transport

“…36 Cultured and fresh bovine corneal endothelial surface proteins were labeled with 200 g EZ-link sulfo-NHS-biotin (Pierce) per milliliter of bicarbonatefree Ringer solution, pH 7.5, at room temperature for 30 minutes. Cells were lysed (50 mM Tris base, 150 mM NaCl, 0.5% deoxycholic acidsodium salt, 2% SDS, and 1% NP-40, pH 7.5, protease inhibitor cocktail) and then sonicated.…”

Molecular expression and functional involvement of the bovine calcium-activated chloride channel 1 (bCLCA1) in apical HCO3− permeability of bovine corneal endothelium

“…ATP is the most likely ligand for P2 receptors and since release of ATP by endothelium is accelerated by cell swelling (Srinivas et al, 2001), it is likely that trauma or similar perturbations would stimulate transport in response to a break in barrier integrity. Moreover, ATP released by endothelium is converted to adenosine, which acitivates A2 B receptors leading to increased [cAMP] (Tan-Allen et al, 2005). Activation of PKA increases apical CFTR permeability and dephosphorylates MLC leading to increased barrier resistance (Srinivas et al, 2004).…”