title: Role of vacuolar adenosine triphosphatase in the regulation of cytosolic pH in hepatocytes.
abstract: The responses of the cytosolic pH of hepatocytes in suspension to agents affecting the activity of vacuolar adenosine triphosphatase (V-ATPase) and Na/H exchange have been studied. Changes of cytosolic pH were determined both with dual-wavelength excitation (500/440 nm) of the fluorescence of 2',7'-bis-(2-carboxyethyl)-5(and 6)-carboxyfluorescein and from the distribution of 14C-dimethyloxazolidinedione; both methods gave very similar results. Changes of vesicular pH were determined by comparing the fluorescence of fluorescein isothiocyanate-dextran and rhodamine B isothiocyanate-dextran taken up by endocytosis. Nitrate, which inhibits V-ATPase in isolated organelles, induced a concentration-dependent acidification of the cytosol and alkalinization of vesicles, with maximal effects at 25-37.5 mM in each case, indicating that V-ATPase contributes to removal of cytosolic protons. On continued exposure to nitrate, the acidification underwent an amiloride-inhibitable reversal. At the higher concentrations of NO3-, both cytosolic acidification and vesicular alkalinization were reduced or absent. Bafilomycin A1 caused alkalinization of vesicular pH; cytosolic acidification was not observed, possibly because of other ionic exchanges. Recovery of cytosolic pH from an acid load (2 min exposure to 5% CO2) was sensitive to both 25 mM NO3- and to ouabain. The pH dependence of the nitrate effect was tested with media of different pH; the activity was negligible at cytosolic pH 6.2 and rose to a maximum at cytosolic pH 7.3. Treatment of hepatocytes with 0.5-1.0 mM ouabain resulted in an initial alkalinization (0.5-2 min duration) of the cytosol, followed by a spontaneous reversal and, on occasion, further acidification. The alkalinization was blocked by 25 mM NO3-, but not by 25 mM gluconate. The results suggest that the cytosolic alkalinization is caused by a stimulation of H+ uptake by V-ATPase activity. We conclude that V-ATPase make an important contribution to the regulation of the cytosolic pH of hepatocytes.
authors.affiliation: Department of Pharmacology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140.
authors.forename: S J
authors.initials: SJ
authors.lastname: Wadsworth
authors.forename: G D
authors.initials: GD
authors.lastname: van Rossum
mesh_terms: D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002245:Carbon Dioxide / Q000494:pharmacology; D018118:Chloride Channels / Q000187:drug effects / Q000378:metabolism; D002712:Chlorides / Q000378:metabolism / Q000494:pharmacology; D004705:Endocytosis; D005456:Fluorescent Dyes; D006863:Hydrogen-Ion Concentration; D007424:Intracellular Fluid / Q000737:chemistry*; D007425:Intracellular Membranes / Q000378:metabolism; D008099:Liver / Q000378:metabolism*; D018942:Macrolides; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects; D009566:Nitrates / Q000494:pharmacology; D010042:Ouabain / Q000494:pharmacology; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000502:physiology*; D011522:Protons; D051381:Rats; D017207:Rats, Sprague-Dawley; D017923:Sodium-Hydrogen Exchangers / Q000187:drug effects / Q000502:physiology; D014617:Vacuoles / Q000201:enzymology
chemical_list: D000900:Anti-Bacterial Agents; D018118:Chloride Channels; D002712:Chlorides; D005456:Fluorescent Dyes; D018942:Macrolides; D009566:Nitrates; D011522:Protons; D017923:Sodium-Hydrogen Exchangers; D002245:Carbon Dioxide; D010042:Ouabain; D000584:Amiloride; C040929:bafilomycin A1; D006180:Proton-Translocating ATPases