低浸透圧刺激シグナルとしての細胞内クロライドイオン濃度の役割

抄録

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]