Physiological significance of hypotonicity-induced regulatory volume decrease: reduction in intracellular Cl−concentration acting as an intracellular signaling

Abstract

Regulatory volume decrease (RVD) occurs after hypotonicity-caused cell swelling. RVD is caused by activation of ion channels and transporters, which cause effluxes of K+, Cl−, and H2O, leading to cell shrinkage. Recently, we showed that hypotonicity stimulated transepithelial Na+reabsorption via elevation of epithelial Na+channel (α-ENaC) expression in renal epithelia A6 cells in an RVD-dependent manner and that reduction of intracellular Cl−concentration ([Cl−]i) stimulated the Na+reabsorption. These suggest that RVD would reveal its stimulatory action on the Na+reabsorption by reducing [Cl−]i. However, the reduction of [Cl−]iduring RVD has not been definitely analyzed due to technical difficulties involved in halide-sensitive fluorescent dyes. In the present study, we developed a new method for the measurement of [Cl−]ichange during RVD by using a high-resolution flow cytometer with a halide-specific fluorescent dye, N-(6-methoxyquinolyl) acetoethyl ester. The [Cl−]iin A6 cells in an isotonic medium was 43.6 ± 3.1 mM. After hypotonic shock (268 to 134 mosmol/kgH2O), a rapid increase of cell volume followed by RVD occurred. The RVD caused drastic diminution of [Cl−]ifrom 43.6 to 10.8 mM. Under an RVD-blocked condition with NPPB (Cl−channel blocker) or quinine (K+channel blocker), we did not detect the reduction of [Cl−]i. Based on these observations, we conclude that one of the physiological significances of RVD is the reduction of [Cl−]iand that RVD shows its action via reduction of [Cl−]iacting as an intracellular signal regulating cellular physiological functions.