Abstract
Our recent study indicates that hypotonicity-induced decreases in intracellular Cl− concentration ([Cl−]i) could act as a signal to regulate Na+ reabsorption through changes in αENaC mRNA expression in renal epithelial A6 cells. This result suggests that the change of [Cl−]i is one of the important signals to cell function. However, currently reported techniques for the measurement of [Cl−]i by using halide-specific fluorescent dyes lack sufficient sensitivity and accuracy. One reason for problems in the use of these dyes for measurement of [Cl−]i during hypotonicity-induced regulatory volume decrease (RVD) is a change of intracellular dye concentration during RVD, since a fluorescent intensity of these indicators depends not only on [Cl−]i but also on intracellular concentration of dyes. In this study, we have developed a new method for measuring [Cl−]i by using a cell analyzer Quanta. This flow cytometer can simultaneously measure the exact cell volume by Coulter principle and the fluorescent intensity. The concentration of Cl− in A6 cells diminished during RVD by 72% (from 47.3 mM to 13.3 mM). This reduction of [Cl−]i was blocked by inhibition of RVD with quinine (K+ channel blocker) or NPPB (Cl− channel blocker). These results suggest that a change in external osmolality is converted into the change in [Cl−]i, and that the change of [Cl−]i is the primary hypotonic signal in A6 cells. This work was supported by Grants-in-Aid from JSPS (17390057, 17590191 and 17790154). [J Physiol Sci. 2006;56 Suppl:S66]
