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  • Title: The regulation of cytoplasmic pH in human fibroblasts.
    Author: Moolenaar WH, Tertoolen LG, de Laat SW.
    Journal: J Biol Chem; 1984 Jun 25; 259(12):7563-9. PubMed ID: 6330063.
    Abstract:
    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.
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