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J.P. Vessey, C. Shi, L. Mao, T.M. Maillet, S. Barnes, M.E. Kelly; Osmoregulation of Membrane Trafficking and Ion Conductance in Human Non-Pigmented Ciliary Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2003;44(13):3466.
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Purpose: To evaluate cellular strategies that allow epithelial cells to respond to osmotic stress, we investigated membrane turnover and changes in membrane ion permeability in non-pigmented ciliary epithelial cells following hyposmotic stimulation. Methods: We used a human SV40 transformed NPCE cell line. Membrane turnover was measured in real time using the fluorescent probe, FM1-43 and whole-cell patch-clamp techniques were used to measure changes in membrane conductance and currents. Cells were exposed to a decrease in external osmolarity of 100 mosm-1 to initiate cell swelling. Results: Hyposmotic stimulation and cell swelling increased membrane trafficking in human NPCE cells by 27.8% over the basal rate of constitutive membrane turnover. This included increases in both endocytosis and exocytosis, as evident by an increase in FM1-43 fluorescence on hyposmotic exposure in the presence of the dye and an increase in the time-constant for dye loss after wash-out. The increase in membrane turnover with hyposmotic stimulation was accompanied by an increase in membrane conductance from 2.9 to 14.2 nS at +75 mV and 0.136 to 1.04 nS at -75 mV (n=4). Treatment of cells with the Cl channel blocker, niflumic acid, or the K channel blocker, TEACl, revealed that >90% of the increase in whole-cell conductance was due to Cl- conductance. The hyposmotic increase in Cl- conductance was reduced in cells pre-treated with antisense to either ClC-3 or ClC-4 subtypes of Cl channels, by dialysis with Cl channel antibodies and by the PKC activator, phorbol dibutyryl ester. Conclusion: Our data indicate that hyposmotic stimulation recruits new membrane and enhances the rate of membrane turnover in NPCE cells. The accompanying increase in Cl- conductance involves at least two different types of Cl channel, ClC-3 and ClC-4, the activity of which is also modulated by protein phosphorylation. These results suggest that the effect of osmotic stress on membrane trafficking may provide a mechanism whereby ion channels and signaling complexes become redistributed among cellular compartments.
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