Enhancement of HCO(3)(-) permeability across the apical membrane of bovine corneal endothelium by multiple signaling pathways.
OBJECTIVE
In this study, the involvement of signaling pathways in the regulation of HCO(3)(-) 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 HCO(3)(-)-rich Cl(-)-free Ringer's solution (28.5 mM; pH 7.5). Relative changes in apical HCO(3)(-) permeability were assayed by pulsing the apical perfusion bath with a low-HCO(3)(-) 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. Ca(2+)-activated signaling was measured with the Ca(2+)-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 M) enhanced HCO(3)(-) permeability by a factor of 2. Forskolin (40 microM) exerted a 6.3-fold increase of HCO(3)(-) permeability, which was inhibited by the Cl(-) channel blockers, glibenclamide (50 microM) and niflumic acid (100 microM). Adenosine triphosphate (ATP) and ATPgammaS, P(2) receptor agonists that increased intracellular Ca(2+) in corneal endothelium, enhanced HCO(3)(-) permeability by 87% and 79%, respectively. ATPgammaS induced depolarization of the E(m), consistent with anion channel activation, rather than activation of Ca(2+)-dependent K(+) channels, which could secondarily increase extrusion of anions by E(m) hyperpolarization. Cyclopiazonic acid (CPA), an endoplasmic reticulum (ER) Ca(2+)-pump inhibitor that increased [Ca(2+)](i), also enhanced HCO(3)(-) permeability by 95%. Both the calmodulin kinase II (CaMKII) inhibitor KN-62 and the PKC inhibitor bisindolylmaleimide I (BIMI), decreased HCO(3)(-) permeability induced by ATPgammaS. The PKC activator PMA also increased HCO(3)(-) 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 HCO(3)(-) permeability across the apical membrane of the cultured corneal endothelium. Multiple signaling pathways (PKA, PKC, and Ca(2+)/CaMKII) contribute to the HCO(3)(-) transport in cultured corneal endothelium. Both cAMP and Ca(2+)-activated Cl(-) channels (possibly CLCA) may be involved in HCO(3)(-) transport.
Zhang
Yan
Y
School of Optometry, Indiana University, Bloomington, Indiana 47405, USA.
Xie
Qiang
Q
Sun
Xing Cai
XC
Bonanno
Joseph A
JA
D000818:Animals; D001639:Bicarbonates / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000502:physiology; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D002463:Cell Membrane Permeability / Q000502:physiology*; D002478:Cells, Cultured; D018118:Chloride Channels / Q000235:genetics / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000502:physiology; D004728:Endothelium, Corneal / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007552:Isotonic Solutions; D008564:Membrane Potentials / Q000502:physiology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000502:physiology; D011941:Receptors, Adrenergic / Q000378:metabolism; D011983:Receptors, Purinergic / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000077331:Ringer's Solution; D015398:Signal Transduction / Q000502:physiology*
D001639:Bicarbonates; D018118:Chloride Channels; D004791:Enzyme Inhibitors; D007552:Isotonic Solutions; D011941:Receptors, Adrenergic; D011983:Receptors, Purinergic; D000077331:Ringer's Solution; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002118:Calcium