ascorbic-acid and 2--7--bis(carboxyethyl)-5(6)-carboxyfluorescein

The regulation of intracellular pH (pHi) was examined in newborn rat skin basal cells. When basal cells were acid-loaded by externally applied weak acid or by pretreatment with NH4+, the pHi recovered toward its resting value. This recovery was accompanied by Na+ influx and H+ efflux. The recovery of pHi and concomitant Na+/H+ fluxes were reversibly inhibited by amiloride. Other ionic flux inhibitors 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and furosemide slightly inhibited the pHi recovery from an acid load. The recovery rate from an acid load depended on the concentration of the external Na+.Li+ could substitute for Na+ in the pHi recovery, but K+ had no effect on the recovery. The keratin synthesis of basal cells was decreased by amiloride, and this decrement was deduced to be due to the acidification of the intracellular space. Although ascorbic acid had no effect on the pHi recovery of basal cells, it enhanced the keratin synthesis of the cells. Moreover, an inhibitor of the collagen synthesis, cis-hydroxyproline, suppressed the keratin synthesis but had no effect on the pHi recovery. We therefore believe that the keratin synthesis of the cells was also regulated by the environment, which was formed by the synthesized collagen. Thus, the pHi in the skin basal cells was regulated by the amiloride-sensitive exchange system, and this system seems to be involved in the keratin synthesis.

Topics: Animals; Animals, Newborn; Ascorbic Acid; Blotting, Western; Cells, Cultured; Fluoresceins; Fluorescent Dyes; Fluorometry; Hydrogen-Ion Concentration; Hydroxyproline; Keratins; Kinetics; Rats; Rats, Wistar; Skin; Sodium

Regulation of the initial rate of uptake and steady-state concentration of ascorbate (reduced vitamin C) was investigated in rat cerebral astrocytes. Although these cells did not synthesize vitamin C, they accumulated millimolar concentrations of ascorbate when incubated with medium containing the vitamin at a level (200 microM) typical of brain extracellular fluid. Initial rate of [14C]-ascorbate uptake and intracellular ascorbate concentration were dependent on extracellular Na+ and sensitive to the anion transport inhibitor sulfinpyrazone. Comparison of the efflux profiles of ascorbate and 2',7'-bis(carboxyethyl)-5 (or -6)-carboxyfluorescein from astrocytes permeabilized with digitonin localized most intracellular ascorbate to the cytosol. Pretreatment of astrocytes with dibutyryl cyclic AMP (dBcAMP) doubled their initial rate of sulfinpyrazone-sensitive [14C]ascorbate uptake compared with cells treated with either n-butyric acid or vehicle. dBcAMP also increased steady-state intracellular ascorbate concentration by 39%. The relatively small size of the change in astrocytic ascorbate concentration was explained by the finding that dBcAMP increased the rate of efflux of the vitamin from ascorbate-loaded cells. These results indicate that uptake and efflux pathways are stimulated by cyclic AMP-dependent mechanisms and that they regulate the cytosolic concentration of ascorbate in astrocytes.

Topics: Animals; Ascorbic Acid; Astrocytes; Biological Transport; Brain; Bucladesine; Fluoresceins; Fluorescent Dyes; Intracellular Membranes; Osmolar Concentration; Rats; Rats, Wistar