Role of a Na+-dependent Cl-/HCO3- exchange in regulation of intracellular pH in fibroblasts.
We previously reported that, in a HCO3(-)-free medium, cytoplasmic pH (pHi) of hamster fibroblasts (CCL39) is primarily regulated by an amiloride-sensitive Na+/H+ antiport (L'Allemain, G., Paris, S., and Pouysségur, J. (1984) J. Biol. Chem. 259, 5809-5815). Here we demonstrate the existence of an additional pHi-regulating mechanism in CCL39 cells, namely a Na+-dependent HCO3-/Cl- exchange. Evidence for this system is based on 36Cl- influx studies and on pHi measurements in PS120, a CCL39-derived mutant lacking the Na+/H+ antiport activity. 36Cl- influx rate is a saturable function of external [Cl-] (apparent Km approximately equal to 7 mM), is competitively inhibited by external HCO3- (KI approximately equal to 3 mM), and by stilbene derivatives (KI approximately equal to 20 microM for 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid). Measurements of pHi recovery after an acute acid load indicate that PS120 cells possess an acid-extruding mechanism dependent on external HCO3-, which is inhibited by stilbene derivatives and requires external Na+. Since 22Na+ influx is stimulated upon addition of HCO3- to acid-loaded cells and this effect is completely abolished by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, we conclude that Na+ is co-transported with HCO3-, in exchange for intracellular Cl-. In a HCO3(-)-containing medium, this pHi-regulating mechanism appears to have two essential physiological functions for the Na+/H+ antiport-deficient mutant: protection of the cells against excessive cytoplasmic acidification and establishment of a steady-state pHi permissive for growth, at neutral or slightly acidic pHo values (6.6-7.2).
L'Allemain
G
G
Paris
S
S
Pouysségur
J
J
D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D001639:Bicarbonates / Q000378:metabolism*; D001826:Body Fluids / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism; D002460:Cell Line; D002712:Chlorides / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D005347:Fibroblasts / Q000166:cytology; D006863:Hydrogen-Ion Concentration; D007424:Intracellular Fluid / Q000378:metabolism*; D008168:Lung / Q000166:cytology; D012964:Sodium / Q000378:metabolism*; D017923:Sodium-Hydrogen Exchangers
D001639:Bicarbonates; D002352:Carrier Proteins; D002712:Chlorides; D017923:Sodium-Hydrogen Exchangers; D000584:Amiloride; D012964:Sodium