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H Liu, SP Srinivas, M Satpathy, R Mitra, MA Watsky; Volume Regulated Anion Channels (VRAC) In Cultured Rabbit Corneal Keratocytes . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1699.
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Purpose: Blockers of VRAC inhibit proliferation of vascular endothelial cells and also prevent angiogenesis (Nilius et al., Gen Pharmacol, 27: 67-77, 1996). In this study, we have characterized VRAC in corneal keratocytes to facilitate identification of agents that might inhibit excessive stromal cell proliferation and potentially stromal/haze and/or scar formation during the corneal wound healing response. Methods: Cultured keratocytes were grown to confluence on glass coverslips. Dynamic fluorescence quenching (DFQ) (Srinivas and Bonanno. Am J Physiol. 272:C1405-14, 1997) was employed to measure changes in cell volume using the halide-sensitive fluorescent dye MQAE under Cl--free conditions; Cl- was replaced by NO3-. The same dye was employed to assess the relative permeability of I- and NO3- quantitatively under isosmotic and hyposmotic conditions (Srinivas et al., Biophys J. 75(1):115-23,1998). All experiments were conducted at 37°C under HCO3--free conditions. RT-PCR was employed to determine expression of CLC homologues, candidates for VRAC activity. Results: Hyposmotic and hyperosmotic shocks caused increase and decrease in MQAE fluorescence, respectively (n=8). Exposure to Gramicidin (1-2 µM) known to form Na+ channels, under hyposmotic conditions caused an increase in MQAE fluorescence indicating secondary cell swelling (n=4). RT-PCR results indicated positive bands for CLC-3 and CLC-5, but expression of CLC-2 and CFTR could not be detected. Under hyposmotic conditions (200-220 mosM), both inward and outward permeability of I- were significantly enhanced (≷ 200%; n = 4) relative to isosmotic conditions. Conclusion:(1). Rapid changes in cell volume of keratocytes can be measured using the DFQ method. (2). GC induced secondary swelling is consistent with Na+-NO3- influx; the cation influx being mediated by GC conductance for Na+ and that of the anion by VRAC activity, presumably CLC-3 which has been suggested to possess VRAC characteristics. (3) Increased I- permeability is consistent with selectivity of VRAC for their relatively higher permeability to I-.
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