Impaired cell volume regulation in Na(+)-H+ exchange-deficient mutants.
To elucidate the mechanism of regulatory volume increase (RVI) in Chinese hamster ovary cells, Na(+)-H+ exchange-deficient mutants, called AP-1, were derived from WT-5 cells, a wildtype subclone. The absence of functional antiports in AP-1 cells was established through measurements of intracellular pH (pHi) and Na+ uptake. Cells exposed to hypotonic medium initially swelled but regained near-normal volume within minutes. When isotonicity was then restored, WT-5 cells shrank immediately and then carried out RVI, which was inhibited by 0.1 mM amiloride. This amiloride-sensitive RVI was absent in the AP-1 mutants, suggesting involvement of Na(+)-H+ exchange. In some cell types, RVI is mediated by Na(+)-K(+)-2Cl- cotransport. Bumetanide-sensitive 86Rb+ (K+) influx was detectable in both WT-5 and AP-1 cells, suggesting the presence of Na(+)-K(+)-2Cl- cotransport. Bumetanide-sensitive influx was stimulated by osmotic shrinking in WT-5 cells, and only slightly in AP-1 cells. However, Na(+)-K(+)-2Cl- cotransport did not contribute to volume regulation, since bumetanide (50 microM) failed to inhibit RVI in osmotically shrunken WT-5 cells. The inability of cotransport to induce a volume gain in WT-5 cells was attributable to the simultaneous stimulation of Na(+)-K(+)-2Cl- efflux. The rate of efflux was similar in magnitude to the corresponding influx rate so that net Na(+)-K(+)-2Cl- cotransport was negligible. These results show that RVI in osmotically shrunken Chinese hamster ovary cells is mediated by the Na(+)-H+ antiport and that, although stimulated, Na(+)-K(+)-2Cl- cotransport does not contribute to anisosmotic volume regulation.
Rotin
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D
Division of Cell Biology, Hospital for Sick Children, Toronto, Ontario, Canada.
Grinstein
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S
D000818:Animals; D001826:Body Fluids / Q000378:metabolism*; D002034:Bumetanide / Q000494:pharmacology; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D006706:Homeostasis / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D007424:Intracellular Fluid / Q000187:drug effects / Q000378:metabolism*; D007700:Kinetics; D009154:Mutation; D011188:Potassium / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers; D028021:Sodium-Potassium-Chloride Symporters
D002352:Carrier Proteins; D017923:Sodium-Hydrogen Exchangers; D028021:Sodium-Potassium-Chloride Symporters; D002034:Bumetanide; D012964:Sodium; D011188:Potassium