Long-term expression of c-H-ras stimulates Na-H and Na(+)-dependent Cl-HCO3 exchange in NIH-3T3 fibroblasts.
In a study of intracellular pH (pHi) regulation, we compared c-H-ras-transformed fibroblasts (FT9s) with parental NIH 3T3 cells. Cells were grown on a coverslip and placed in a flow-through cuvette; pHi was monitored using a fluorescent dye. Whether cells were in a CO2-free or CO2-containing solution, steady-state pHi was approximately 0.5 higher in FT9s than in 3T3s. Rates of pHi recovery from acid loads in the presence of CO2 indicate that total net acid extrusion (Jtotal) was far greater in FT9s than 3T3s in the pHi range 6.3-7.4. In both FT9s and 3T3s, pHi recovery was blocked by removing Na+. In FT9s, most of Jtotal was blocked by ethyl isopropyl amiloride (EIPA) and was probably due to Na-H exchange. A smaller component was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) and required Cl- and was probably due to Na(+)-dependent Cl-HCO3 exchange. In 3T3s, the DIDS-sensitive and Cl(-)-dependent components of Jtotal were very small. The dominant acid-extruder in 3T3s appears to be a Na-H exchanger insensitive to 50 microM EIPA. We determined the flux-pHi relationships for both the Na-H and Na(+)-dependent Cl-HCO3 exchangers in both FT9s and 3T3s. p21ras alkaline shifts the pHi profile of each transporter by approximately 0.7, without affecting the Vmax. These shifts in the flux-pHi relationships provide a mechanism for the large increase in steady-state pHi produced by long-term expression of p21ras.
Kaplan
D L
DL
Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06510.
Boron
W F
WF
D016475:3T3 Cells / Q000378:metabolism; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D017920:Antiporters / Q000378:metabolism*; D001639:Bicarbonates / Q000378:metabolism*; D001693:Biological Transport, Active; D002712:Chlorides / Q000378:metabolism*; D011905:Genes, ras; D006133:Growth Substances / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D051379:Mice; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism
D017920:Antiporters; D001639:Bicarbonates; D002712:Chlorides; D006133:Growth Substances; D000584:Amiloride; D012964:Sodium; D016283:Proto-Oncogene Proteins p21(ras); D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; C039614:ethylisopropylamiloride