Effects of aniso-osmolarity and hydroperoxides on intracellular pH in isolated rat hepatocytes as assessed by (2',7')-bis(carboxyethyl)-5(6)-carboxyfluorescein and fluorescein isothiocyanate-dextran fluorescence.
Freshly isolated rat hepatocytes were plated for 4-6 h and either loaded with (2',7)-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) or allowed to endocytose fluorescein isothiocyanate (FITC)-coupled dextran in order to study the effects of aniso-osmotic exposure and oxidative stress on cytosolic (pHcyt) and apparent vesicular pH (pHves) by single-cell fluorescence recordings. In the presence of normo-osmotic (305 mosmol/l) medium pHcyt was 7.23 +/- 0.03 (n = 108), whereas an apparent pH of 6.07 +/- 0.02 (n = 156) was found in the vesicular compartment accessible to endocytosed FITC-dextran. Substitution of 60 mM NaCl against 120 mM raffinose had no effect on pHcyt or apparent pHves, whereas addition of NH4Cl increased both pHcyt and apparent pHves. Hypo-osmotic cell swelling lowered pHcyt, whereas simultaneously apparent pHves increased. These effects were rapidly reversible upon re-institution of normo-osmotic media. Similarly, an increase of apparent pHves was observed when cell swelling was induced by Ba2+, glutamine or histidine. Conversely, hyperosmotic cell shrinkage due to addition of NaCl or raffinose led to a cytosolic alkalinization and a vesicular acidification. Both, H2O2 (0.2 mmol/l) and t-butyl-hydroperoxide (0.2 mmol/l) were without effect on pHcyt, but lowered apparent pHves by about 0.2 pH units. Ba2+ (1 mmol/l) diminished the acidifying effect of the hydroperoxides by about 50%. Pretreatment of the cells with colchicine, but not with lumicolchicine, largely abolished the effects of aniso-osmolarity and hydroperoxides on pHves. The data suggest that hepatocellular hydration affects the proton gradients built up across the membranes of endocytotic FITC-dextran-accessible compartments in a microtubule-dependent way. They further suggest that hydroperoxides induce vesicular acidification in a colchicine- and Ba(2+)-sensitive way. Because hydroperoxides induce Ba(2+)-sensitive cell shrinkage [Hallbrucker, Ritter, Lang, Gerok and Häussinger (1992) Eur. J. Biochem. 211, 449-458], the results are compatible with the view that hydroperoxide-induced cell shrinkage mediates vesicular acidification. It is concluded that modulation of vesicular pH by the hepatocellular hydration state may play a role in triggering some metabolic changes in response to cell swelling/shrinkage.
Schreiber
R
R
Medizinische Universitätsklinik Freiburg, Germany.
Stoll
B
B
Lang
F
F
Häussinger
D
D
D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D017609:Barium Compounds / Q000494:pharmacology; D002478:Cells, Cultured; D002712:Chlorides / Q000494:pharmacology; D003078:Colchicine / Q000494:pharmacology; D003911:Dextrans; D004705:Endocytosis; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives; D005452:Fluoresceins; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes; D005973:Glutamine / Q000494:pharmacology; D006639:Histidine / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D008099:Liver / Q000378:metabolism*; D008162:Lumicolchicines / Q000494:pharmacology; D008297:Male; D009994:Osmolar Concentration; D010545:Peroxides / Q000494:pharmacology*; D011887:Raffinose / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D017382:Reactive Oxygen Species / Q000494:pharmacology; D012965:Sodium Chloride / Q000494:pharmacology; D013050:Spectrometry, Fluorescence / Q000379:methods; D020122:tert-Butylhydroperoxide
D017609:Barium Compounds; D002712:Chlorides; D003911:Dextrans; D005452:Fluoresceins; D005456:Fluorescent Dyes; D008162:Lumicolchicines; D010545:Peroxides; D017382:Reactive Oxygen Species; C015219:fluorescein isothiocyanate dextran; D000643:Ammonium Chloride; D005973:Glutamine; C024986:barium chloride; D012965:Sodium Chloride; C042109:lumicolchicine; D006639:Histidine; C043829:2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein; D020122:tert-Butylhydroperoxide; D006861:Hydrogen Peroxide; D016650:Fluorescein-5-isothiocyanate; D011887:Raffinose; D003078:Colchicine