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Enhancement of HCO₃^- Permeability across the Apical Membrane of Bovine Corneal Endothelium by Multiple Signaling Pathways

¹ From the School of Optometry, Indiana University, Bloomington, Indiana.

PURPOSE. In this study, the involvement of signaling pathways in the regulation of HCO₃^- permeability across the apical membrane of the corneal endothelium was examined.

METHODS. Cultured bovine corneal endothelial cells (CBCECs) were grown to confluence on permeable membranes. Apical and basolateral sides were perfused with a HCO₃^--rich Cl^--free Ringer’s solution (28.5 mM; pH 7.5). Relative changes in apical HCO₃^- permeability were assayed by pulsing the apical perfusion bath with a low-HCO₃^- Cl^--free Ringer’s solution (2.85 mM; pH 6.5), in the presence or absence of agonists or inhibitors, and comparing the rates of change in intracellular pH (pH_i), as measured with a pH-sensitive dye. Ca²⁺-activated signaling was measured with the Ca²⁺-sensitive dye Fura-2. Qualitative changes in membrane potential (E_m) were measured with a voltage-sensitive dye. RT-PCR using calcium–activated chloride channel (CLCA)–specific primers was used to examine the expression of CLCA in the corneal endothelium.

RESULTS. The adenoceptor agonist adenosine (20 µM) enhanced HCO₃^- permeability by a factor of 2. Forskolin (40 µM) exerted a 6.3-fold increase of HCO₃^- permeability, which was inhibited by the Cl^- channel blockers, glibenclamide (50 µM) and niflumic acid (100 µM). Adenosine triphosphate (ATP) and ATPS, P₂ receptor agonists that increased intracellular Ca²⁺ in corneal endothelium, enhanced HCO₃^- permeability by 87% and 79%, respectively. ATPS induced depolarization of the E_m, consistent with anion channel activation, rather than activation of Ca²⁺-dependent K⁺ channels, which could secondarily increase extrusion of anions by E_m hyperpolarization. Cyclopiazonic acid (CPA), an endoplasmic reticulum (ER) Ca²⁺-pump inhibitor that increased [Ca²⁺]_i, also enhanced HCO₃^- permeability by 95%. Both the calmodulin kinase II (CaMKII) inhibitor KN-62 and the PKC inhibitor bisindolylmaleimide I (BIMI), decreased HCO₃^- permeability induced by ATPS. The PKC activator PMA also increased HCO₃^- permeability by a factor of 1.8. RT-PCR using CLCA-specific primers showed the expression of CLCA1 in both fresh and cultured BCECs.

CONCLUSIONS. Activation of adenoceptors and purinoceptors enhances HCO₃^- permeability across the apical membrane of the cultured corneal endothelium. Multiple signaling pathways (PKA, PKC, and Ca²⁺/CaMKII) contribute to the HCO₃^- transport in cultured corneal endothelium. Both cAMP and Ca²⁺-activated Cl^- channels (possibly CLCA) may be involved in HCO₃^- transport.

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