The technique for the simultaneous recording of cell volume changes and pH(i) in single cells was used to study the role of HCO3/- in regulatory volume decrease (RVD) by the osteosarcoma cells UMR-106-01. In the presence of HCO3/-, steady state pH(i) is regulated by Na+/H+ exchange, Na+(HCO3/-)3 cotransport and Na+-independent Cl-/HCO3/- exchange. Following swelling in hypotonic medium, pH(i) was reduced from 7.16 ± 0.02 to 6.48 ± 0.02 within 3.4 ± 0.28 min. During this period of time, the cells performed RVD until cell volume was decreased by 31 ± 5% beyond that of control cells (RVD overshoot). Subsequently, while the cells were still in hypotonic medium, pH(i) slowly increased from 6.48 ± 0.02 to 6.75 ± 0.02. This increase in pH(i) coincided with an increase in cell volume back to normal (recovery from RVD overshoot or hypotonic regulatory volume increase (RVI). The same profound changes in cell volume and pH(i) after cell swelling were observed in the complete absence of Cl- or Na+, providing HCO3/- was present. On the other hand, depolarizing the cells by increasing external K+ or by inhibition of K+ channels with quinidine, Ba2+ or tetraethylammonium prevented the changes in pH(i) and RVD. These findings suggest that in the presence of HCO3/-, RVD in UMR-106-01 cells is largely mediated by the conductive efflux of K+ and HCO3/-. Removal of external Na+ but not Cl- prevented the hypotonic RVI that occurred after the overshoot in RVD. Amiloride had no effect, whereas pretreatment with 4,4'- diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) strongly inhibited hypotonic RVI. Thus, hypotonic RVI is mediated by a Na(out)/+-dependent, Cl--independent and DIDS-inhibitable mechanism, which is indicative of a Na+(HCO3/-)3 cotransporter. This is the first evidence for the involvement of this transporter in cell volume regulation. The present results also stress the power of the new technique used in delineating complicated cell volume regulatory mechanisms in attached single cells.
|Original language||English (US)|
|Number of pages||5|
|Journal||Journal of Biological Chemistry|
|Publication status||Published - Sep 25 1992|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology