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Yan Zhang, Qiang Xie, Xing Cai Sun, Joseph A. Bonanno; Enhancement of HCO3− Permeability across the Apical Membrane of Bovine Corneal Endothelium by Multiple Signaling Pathways. Invest. Ophthalmol. Vis. Sci. 2002;43(4):1146-1153.
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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 (pHi), as measured with a pH-sensitive dye. Ca2+-activated signaling was measured with the Ca2+-sensitive dye Fura-2. Qualitative changes in membrane potential (Em) 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 ATPγS, P2 receptor agonists that increased intracellular Ca2+ in corneal endothelium, enhanced HCO3 − permeability by 87% and 79%, respectively. ATPγS induced depolarization of the Em, consistent with anion channel activation, rather than activation of Ca2+-dependent K+ channels, which could secondarily increase extrusion of anions by Em 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 ATPγS. 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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