The regulation of cytoplasmic pH in human fibroblasts

• Published in: The Journal of biological chemistry 1984-06, Vol.259 (12), p.7563-7569
• Main Authors: Moolenaar, W H, Tertoolen, L G, de Laat, S W
• Format: Article
• Language: English
• Subjects: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid - pharmacology; Amiloride - pharmacology; Ammonia - pharmacology; Bicarbonates - pharmacology; Biological and medical sciences; Carrier Proteins - metabolism; Cell physiology; Cytoplasm - physiology; Fibroblasts - cytology; Fluorometry; Fundamental and applied biological sciences. Psychology; Humans; Hydrogen-Ion Concentration; Kinetics; Male; Membrane and intracellular transports; Molecular and cellular biology; Sodium - pharmacology; Sodium-Hydrogen Exchangers
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(17)42827-4

The regulation of cytoplasmic pH (pHi) has been examined in normal human foreskin fibroblasts (HF cells) using a fluorometric technique for continuously monitoring rapid pHi transients. We previously reported that pHi in HF cells is rapidly raised by growth factors due to activation of a Na+/H+ exchange mechanism in the plasma membrane ( Moolenaar , W. H., Tsien , R. Y., van der Saag , P. T., and de Laat , S. W. (1983) Nature (Lond.) 304, 645-648). Here we characterize the ionic basis of pHi homeostasis in quiescent HF cells. When HF cells are acid-loaded by externally applied weak acids or by pretreatment with NH4+, pHi immediately recovers toward its resting value (approximately 7.05). pHi recovery follows an exponential time course and is accompanied by enhanced Na+ influx and net H+ extrusion. Recovery of pHi and concomitant Na+/H+ fluxes are reversibly inhibited by amiloride (half-maximal effect at approximately 0.1 mM). The rate of pHi recovery from an acid load depends on external Na+ (half-maximal rate at approximately 35 mM), but is independent of external anions (HCO3-, Cl-) and is not affected by membrane depolarization. Li+ can substitute for Na+ in pHi recovery. In Na+-free media, pHi spontaneously falls to a new resting value, from which it rapidly recovers after readdition of Na+. A stepwise increase in external pH (pHo) accelerates pHi recovery from an acid load and raises the resting pHi by approximately 50% of the pHo shift. The response of pHi to alkaline pHo shifts is abolished by amiloride and by Na+ removal. It is concluded that pHi in HF cells is closely regulated by an amiloride-sensitive, reversible Na+/H+ exchanger, which is driven by the transmembrane concentration gradients for Na+ and H+. Under normal conditions, the exchanger appears to be relatively inactive, while its rate is increasingly stimulated by lowering pHi or by raising pHo.