A Novel Approach for Characterization of Regulatory Volume Behavior in Human Corneal Epithelial Cells

Abstract

Abstract: : Purpose: To further clarify the plasma membrane ion transport pathways mediating regulatory volume decreases (RVD) to a hypotonic challenge, the ion transport mechanisms underlying this response were resolved in SV40–immortalized human corneal epithelial cells (HCEC). Methods: Two independent approaches at 37ºC were used to characterize RVD in calcein–AM loaded cells: 1) Cell fluorescence was recorded with a Fusion Universal Microplate Analyzer; 2) An inverted microscope coupled to a fluorescence imaging system monitored relative cell volume. Putative ion transport inhibitors and isosmotic ionic substitutions were used to resolve mechanisms underlying RVD during exposure to a 50% hypotonic challenge. Results: During exposure to this challenge, cell swelling was accompanied by RVD, which caused within ∼5 min recovery to their isotonic volume. Upon re–exposing the hypotonically–adapted cells to the isotonic solution, cell shrinkage occurred followed by regulatory volume increase (RVI). RVD could be inhibited by either K⁺ channel blockers, 100 µM glybenclamide, 10 mM tetraethylammonium (TEA), 1 mM 4–aminopyridine (4–AP) or 5 mM barium (Ba²⁺). Exposure to the isotonic solution containing elevated K⁺ (i.e. 20 mM) produced similar RVD suppression to these inhibitors. This correspondence further supports the role for the outward K⁺ movement during RVD. RVD was also partially inhibited by putative Cl^– channel blockers 100 µM niflumic acid and 1 mM 4,4’–diisothiocyanostilbene–2,2’disulfonic acid (DIDS), confirming that Cl^– is a counter ion for K⁺ egress. The KCl cotransporter (KCC) inhibitor 100 µM [(dihydroindenyl)oxy] alkanoic acid (DIOA), blocked RVD by 67%. KCC activity under the isotonic condition was confirmed based on cell shrinkage induced by 1 mM NEM, a KCC stimulator. Conclusions: This is the first report in HCEC documenting the involvement of KCC activity in the RVD response during a hypotonic challenge. Such activity stimulated in parallel with increases in KCl egress contributes to the RVD behavior elicited by this challenge.

Keywords: ion transporters • ion channels • cornea: epithelium