Characterization of Regulatory Volume Behavior by Fluorescence Quenching in Human Corneal Epithelial Cells

An in-depth understanding of the mechanisms underlying regulatory volume behavior in corneal epithelial cells has been in part hampered by the lack of adequate methodology for characterizing this phenomenon. Accordingly, we developed a novel approach to characterize time-dependent changes in relative cell volume induced by anisosmotic challenges in calcein-loaded SV40-immortalized human corneal epithelial (HCE) cells with a fluorescence microplate analyzer. During a hypertonic challenge, cells shrank rapidly, followed by a temperature-dependent regulatory volume increase (RVI), τ_c = 19 min. In contrast, a hypotonic challenge induced a rapid (τ_c = 2.5 min) regulatory volume decrease (RVD). Temperature decline from 37 to 24°C reduced RVI by 59%, but did not affect RVD. Bumetanide (50 μM), ouabain (1 mM), DIDS (1 mM), EIPA (100 μM), or Na⁺-free solution reduced the RVI by 60, 61, 39, 32, and 69%, respectively. K⁺, Cl⁻ channel and K⁺-Cl⁻ cotransporter (KCC) inhibition obtained with either 4-AP (1 mM), DIDS (1 mM), DIOA (100 μM), high K⁺ (20 mM) or Cl⁻-free solution, suppressed RVD by 42, 47, 34, 52 and 58%, respectively. KCC activity also affects steady-state cell volume, since its inhibition or stimulation induced relative volume alterations under isotonic conditions. Taken together, K⁺ and Cl⁻ channels in parallel with KCC activity are important mediators of RVD, whereas RVI is temperature-dependent and is essentially mediated by the Na⁺-K⁺-2Cl⁻ cotransporter (Na⁺-K⁺-2Cl⁻) and the Na⁺-K⁺ pump. Inhibition of K⁺ and Cl⁻ channels and KCC but not Na⁺-K⁺-2Cl⁻ affect steady-state cell volume under isotonic conditions. This is the first report that KCC activity is required for HCE cell volume regulation and maintenance of steady-state cell volume.