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

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 (alpha-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-](i) during 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+](i) change during RVD by using a high-resolution flow cytometer with a halide-specific fluorescent dye, N-(6-methoxyquinolyl) acetoethyl ester. The [Cl+] i in A6 cells in an isotonic medium was 43.6 +/- 3.1 mM. After hypotonic shock (268 to 134 mosmol/ kgH(2)O), a rapid increase of cell volume followed by RVD occurred. The RVD caused drastic diminution of [Cl-] i from 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-] i and that RVD shows its action via reduction of [Cl-] i acting as an intracellular signal regulating cellular physiological functions.