title: Ionic mechanisms of regulatory volume increase (RVI) in the human hepatoma cell-line HepG2.
abstract: We studied the effects of hypertonic stress on ion transport and cell volume regulation (regulatory volume increase; RVI) in the human tumor cell-line HepG2. Ion conductances were monitored in intracellular current-clamp measurements with rapid ion-substitutions and in whole-cell patch-clamp recordings; intracellular pH buffering capacity and activation of Na(+)/H(+) antiport were determined fluorometrically; the rates of Na(+)-K(+)-2Cl(-) symport and Na(+)/K(+)-ATPase were quantified on the basis of time-dependent and furosemide- or ouabain-sensitive (86)Rb(+) uptake, respectively; changes in cell volume were recorded by means of confocal laser-scanning microscopy. It was found that hypertonic conditions led to the activation of a cation conductance that was inhibited by Gd(3+), flufenamate as well as amiloride, but not by benzamil or ethyl-isopropyl-amiloride (EIPA). Most likely, this cation conductance was non-selective for Na(+) over K(+). Hypertonic stress did not change K(+) conductance, whereas possible changes in Cl(-) conductance remain ambiguous. The contribution of Na(+)/H(+)antiport to the RVI process appeared to be minor. Under hypertonic conditions an approximately 3.5-fold stimulation of Na(+)-K(+)-2Cl(-)symport was observed but this transporter did not significantly contribute to the overall RVI process. Hypertonic stress did not increase the activity of Na(+)/K(+)-ATPase, which even under isotonic conditions appeared to be working at its limit. It is concluded that the main mechanism in the RVI of HepG2 cells is the activation of a novel non-selective cation conductance. In contrast, there is little if any contribution of K(+) conductance, Na(+)/H(+) antiport, Na(+)-K(+)-2Cl(-) symport, and Na(+)/K(+)-ATPase to this process.
authors.affiliation: Max-Planck-Institut für molekulare Physiologie, Abteilung Epithelphysiologie, Postfach 500247, 44202 Dortmund, Germany, frank.wehner@mpi-dortmund.mpg.de
authors.forename: Frank
authors.initials: F
authors.lastname: Wehner
authors.forename: Peter
authors.initials: P
authors.lastname: Lawonn
authors.forename: Hanna
authors.initials: H
authors.lastname: Tinel
mesh_terms: D000468:Alkalies / Q000378:metabolism; D000584:Amiloride / Q000494:pharmacology; D006528:Carcinoma, Hepatocellular; D004232:Diuretics / Q000494:pharmacology; D022781:Hepatocytes / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006982:Hypertonic Solutions / Q000494:pharmacology; D008113:Liver Neoplasms; D009997:Osmotic Pressure; D018408:Patch-Clamp Techniques; D011188:Potassium / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism; D028021:Sodium-Potassium-Chloride Symporters / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism; D014407:Tumor Cells, Cultured; D014882:Water-Electrolyte Balance / Q000187:drug effects / Q000502:physiology*
chemical_list: D000468:Alkalies; D004232:Diuretics; D006982:Hypertonic Solutions; D017923:Sodium-Hydrogen Exchangers; D028021:Sodium-Potassium-Chloride Symporters; D000584:Amiloride; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium