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J.E. Capo–Aponte, V. Bildin, P. Iserovich, Z. Pan, J.W. Du, F. Zhang, P.S. Reinach; Functional and Molecular Characterization of K–Cl Cotransporter Isoform Expression in Corneal Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2912.
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© ARVO (1962-2015); The Authors (2016-present)
The potassium–chloride co–transporter (KCC) plays a crucial role in inducing volume regulation and cell proliferation in many different tissues. Four KCC isoforms have been characterized, KCC1–KCC4. In this study, we sought to determine the presence in corneal epithelial cells isolated from different species whether any of these isoforms are expressed and their contribution to regulatory volume decrease (RVD) activity. This assessment was done on primary cultured human corneal epithelial cells (HCEC), and SV40–immortalized human (iHCEC) and rabbit corneal epithelial (iRCEC) cells.
Cell volume changes, induced by a 50% hypotonic challenge in calcein–loaded cells, were characterized using a fluorescence microplate analyzer. The presence of multiple KCC isoforms was determined using Western blot analysis.
Hypotonic challenge induced cell swelling, which was followed by a rapid RVD (τc = 2.5 min) in iHCEC. In contrast, iRCEC displayed a much slower RVD (τc = 20 min) after exposure to a similar challenge. The KCC inhibitor [(dihydroindenyl)oxy] alkanoic acid (DIOA), at a concentration of 100 µM, blocked RVD by 34±2 and 30±6% in iHCEC and iRCEC. The presence of KCC in iHCEC and iRCEC was also validated based on cell shrinkage (39±4 and 34±7% in iHCEC and iRCEC, respectively) induced by 1 mM N–ethylmalemide (NEM), a KCC stimulator, under isotonic conditions. KCC1, KCC3, and KCC4, with molecular weights of ∼135, 120, and 130 kDa, respectively, were all expressed in iHCEC, iRCEC, and HCEC.
KCC activity is required for volume regulation in iHCEC and iRCEC. HCEC, iHCEC, and iRCEC all express three of the KCC isoforms: KCC1, KCC3, and KCC4.
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