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 H(2)O, 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.
Miyazaki
Hiroaki
H
Departments of Molecular Cell Physiology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto, Japan.
Shiozaki
Atsushi
A
Niisato
Naomi
N
Marunaka
Yoshinori
Y
D000818:Animals; D001834:Body Water / Q000378:metabolism; D002460:Cell Line; D048429:Cell Size / Q000187:drug effects*; D018118:Chloride Channels / Q000037:antagonists & inhibitors; D002712:Chlorides / Q000378:metabolism*; D005434:Flow Cytometry; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000493:pharmacokinetics; D006982:Hypertonic Solutions / Q000494:pharmacology*; D007425:Intracellular Membranes / Q000378:metabolism*; D007668:Kidney / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D009579:Nitrobenzoates / Q000494:pharmacology; D009994:Osmolar Concentration; D011803:Quinine / Q000494:pharmacology; D011804:Quinolines / Q000493:pharmacokinetics; D015398:Signal Transduction; D014982:Xenopus laevis
D018118:Chloride Channels; D002712:Chlorides; D005456:Fluorescent Dyes; D006982:Hypertonic Solutions; D009579:Nitrobenzoates; D011804:Quinolines; C078229:N-(6-methoxyquinolyl)acetoethyl ester; C058176:5-nitro-2-(3-phenylpropylamino)benzoic acid; D011803:Quinine