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J Wang, MA Watsky; Corneal Keratocyte Ion Channels are Dependent on Cell Phenotype and Culture Conditions . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1698.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: We have previously demonstrated that corneal keratocytes in the process of healing a wound lose many of the ion channel subtypes found in quiescent cells, while gaining an LPA (10 µM) activated Cl - current (LPACl). The purpose of this study was to characterize the ionic currents present in rabbit corneal keratocytes cultured under conditions designed to promote the keratocyte and myofibroblast phenotypes. Methods: Rabbit corneal keratocytes were cultured at high and low density in a 14% serum containing medium, and at low density in Keratinocyte serum-free medium (KSFM; Gibco). Cells were patch clamped using the amphotericin perforated-patch technique. Phenotype was determined using αSM-actin immunostaining. Results: Keratocytes cultured in serum at high density were αSM-actin negative, spindle shaped, and apparently of the keratocyte phenotype. Cells cultured in serum at low density were αSM-actin positive with stress fibers, flat with processes, and of the myofibroblast phenotype. Cells cultured in KSFM seemed to have focal αSM-actin staining and were also flat with processes, apparently of an intermediate (possibly fibroblast) phenotype. The high density cells (keratocytes) had a high frequency of delayed rectifying K + current (19/33 or 58%; DRK) and volume-activated Cl - current (8/11 or 73%; VACl) expression, and a low frequency of LPACl (2/15 or 13%) and inward Na + current (2/30 or 7%; NaI) expression. Low density cells (myofibroblasts) had a high frequency of both VACl (15/16 or 94%) and LPACl (44/52 or 85%) currents, and a low frequency of DRK (69/231 or 30%) expression and no NaI (0/7) current expression. KSFM cells (intermediate phenotype) showed minimal current expression, with only 24% (5/21) of cells expressing DRK and 11% (1/9) expressing VACl, with no evidence of any of the other currents. Conclusion: Cultured rabbit corneal keratocytes express different ionic currents that are dependent on their phenotype.
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