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

Departments of ¹Molecular Cell Physiology and ²Surgery, Division of Digestive Surgery, ³Department of Respiratory Molecular Medicine, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto, Japan

Submitted 30 June 2006 ; accepted in final form 16 January 2007

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 (-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/kgH₂O), 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.

A6 epithelial cell; cell volume; N-(6-methoxyquinolyl) acetoethyl ester; high-resolution flow cytometer; Cell Lab Quanta