Back to Search
Start Over
Enhancement of HCO(3)(-) permeability across the apical membrane of bovine corneal endothelium by multiple signaling pathways.
- Source :
-
Investigative ophthalmology & visual science [Invest Ophthalmol Vis Sci] 2002 Apr; Vol. 43 (4), pp. 1146-53. - Publication Year :
- 2002
-
Abstract
- Purpose: 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.<br />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.<br />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.<br />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.
- Subjects :
- Animals
Calcium metabolism
Calcium-Calmodulin-Dependent Protein Kinase Type 2
Calcium-Calmodulin-Dependent Protein Kinases antagonists & inhibitors
Calcium-Calmodulin-Dependent Protein Kinases physiology
Cattle
Cell Membrane metabolism
Cells, Cultured
Chloride Channels genetics
Chloride Channels metabolism
Cyclic AMP-Dependent Protein Kinases antagonists & inhibitors
Cyclic AMP-Dependent Protein Kinases physiology
Enzyme Inhibitors pharmacology
Hydrogen-Ion Concentration
Isotonic Solutions
Membrane Potentials physiology
Protein Kinase C antagonists & inhibitors
Protein Kinase C physiology
Receptors, Adrenergic metabolism
Receptors, Purinergic metabolism
Reverse Transcriptase Polymerase Chain Reaction
Ringer's Solution
Bicarbonates metabolism
Cell Membrane Permeability physiology
Endothelium, Corneal metabolism
Signal Transduction physiology
Subjects
Details
- Language :
- English
- ISSN :
- 0146-0404
- Volume :
- 43
- Issue :
- 4
- Database :
- MEDLINE
- Journal :
- Investigative ophthalmology & visual science
- Publication Type :
- Academic Journal
- Accession number :
- 11923259