AJP - Cell Physiology, Vol 257, Issue 6 C1158-C1165, Copyright © 1989 by American Physiological Society
Impaired cell volume regulation in Na(+)-H+ exchange-deficient mutants
D. Rotin and S. Grinstein
Division of Cell Biology, Hospital for Sick Children, Toronto, Ontario, Canada.
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.
