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L Al-Nakkash, M Coca-Prados, PS Reinach; Volume-activated Chloride Channel Gene Expression and Function in a Rabbit Corneal Epithelial Cell Line . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1677.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: It is unknown which chloride channels are involved in mediating the loss of intracellular chloride in rabbit corneal epithelial cells during a regulatory volume decrease (RVD) response to a hypotonic stress. The aim of this study was to determine whether stimulation of volume-activated chloride current (VACC) underlies such a response. We also probed for its gene expression. Methods: We used the whole-cell patch-clamp technique on SV40-immortalized rabbit corneal epithelial cells (tRCE), to examine the biophysical characteristics of the VACC. Evidence for its gene expression was sought based on RT-PCR and sequence validation. Results: Whole-cell patch-clamp studies demonstrated the presence of an outwardly rectifying VACC, induced by reducing the bath superfusate osmolality from 300 to 200 mOsm; at +100 mV the current increased from 6.35±0.94 pA/pF to 98.65±11.19 pA/pF (n=40). Time to reach peak steady-state current was 10-15 minutes. DIDS (500 µM) voltage-dependently inhibited VACC by 88% and 44% at +100 mV and -100 mV, respectively (n=7). NPPB (100 µM) inhibited VACC by 85% and 91% at +100 mV and -100 mV, respectively (n=6). There was no effect on VACC by either 250 µM glibenclamide (n=3) or 500 µM verapamil (n=4). Removal of calcium from the hypotonic bath solution prevented induction of the VACC. Consistent with the hypothesis that the VACC is ClC-3 , we found that PKC activation by PDBu (100 nM) prevented stimulation of VACC (n=2), but it had no effect once VACC was activated (n=7). Predicted cDNA products were obtained based on our primer design to selectively amplify ClC-3 message. Conclusion: These results show that the channel behavior underlying RVD responses in tRCE is associated with activation of VACC. Regulation of this chloride channel may be of importance in the maintenance of corneal epithelial barrier and dehydrating functions during an environmental stress.
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