Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1994 Oct 1;303(Pt 1):113–120. doi: 10.1042/bj3030113

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.

R Schreiber 1, B Stoll 1, F Lang 1, D Häussinger 1
PMCID: PMC1137564  PMID: 7524479

Abstract

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.

Full text

PDF
113

Images in this article

114Figure 2
on p.114

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Aplin A., Jasionowski T., Tuttle D. L., Lenk S. E., Dunn W. A., Jr Cytoskeletal elements are required for the formation and maturation of autophagic vacuoles. J Cell Physiol. 1992 Sep;152(3):458–466. doi: 10.1002/jcp.1041520304. [DOI] [PubMed] [Google Scholar]
  2. Bode B. P., Kilberg M. S. Amino acid-dependent increase in hepatic system N activity is linked to cell swelling. J Biol Chem. 1991 Apr 25;266(12):7376–7381. [PubMed] [Google Scholar]
  3. Corasanti J. G., Gleeson D., Boyer J. L. Effects of osmotic stresses on isolated rat hepatocytes. I. Ionic mechanisms of cell volume regulation. Am J Physiol. 1990 Feb;258(2 Pt 1):G290–G298. doi: 10.1152/ajpgi.1990.258.2.G290. [DOI] [PubMed] [Google Scholar]
  4. Dumont M., D'Hont C., Durand-Schneider A. M., Legrand-Defretin V. L., Feldmann G., Erlinger S. Inhibition by colchicine of biliary secretion of diethylmaleate in the rat: evidence for microtubule-dependent vesicular transport. Hepatology. 1991 Jul;14(1):10–15. doi: 10.1002/hep.1840140103. [DOI] [PubMed] [Google Scholar]
  5. Gleeson D., Corasanti J. G., Boyer J. L. Effects of osmotic stresses on isolated rat hepatocytes. II. Modulation of intracellular pH. Am J Physiol. 1990 Feb;258(2 Pt 1):G299–G307. doi: 10.1152/ajpgi.1990.258.2.G299. [DOI] [PubMed] [Google Scholar]
  6. Graf J., Haddad P., Haeussinger D., Lang F. Cell volume regulation in liver. Ren Physiol Biochem. 1988 May-Oct;11(3-5):202–220. doi: 10.1159/000173163. [DOI] [PubMed] [Google Scholar]
  7. Hallbrucker C., Ritter M., Lang F., Gerok W., Häussinger D. Hydroperoxide metabolism in rat liver. K+ channel activation, cell volume changes and eicosanoid formation. Eur J Biochem. 1993 Feb 1;211(3):449–458. doi: 10.1111/j.1432-1033.1993.tb17570.x. [DOI] [PubMed] [Google Scholar]
  8. Hallbrucker C., vom Dahl S., Lang F., Gerok W., Häussinger D. Modification of liver cell volume by insulin and glucagon. Pflugers Arch. 1991 Jun;418(5):519–521. doi: 10.1007/BF00497781. [DOI] [PubMed] [Google Scholar]
  9. Haüssinger D. Nitrogen metabolism in liver: structural and functional organization and physiological relevance. Biochem J. 1990 Apr 15;267(2):281–290. doi: 10.1042/bj2670281. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Häussinger D., Lang F. Cell volume and hormone action. Trends Pharmacol Sci. 1992 Oct;13(10):371–373. doi: 10.1016/0165-6147(92)90114-l. [DOI] [PubMed] [Google Scholar]
  11. Häussinger D., Lang F. Cell volume in the regulation of hepatic function: a mechanism for metabolic control. Biochim Biophys Acta. 1991 Dec 12;1071(4):331–350. doi: 10.1016/0304-4157(91)90001-d. [DOI] [PubMed] [Google Scholar]
  12. Häussinger D., Saha N., Hallbrucker C., Lang F., Gerok W. Involvement of microtubules in the swelling-induced stimulation of transcellular taurocholate transport in perfused rat liver. Biochem J. 1993 Apr 15;291(Pt 2):355–360. doi: 10.1042/bj2910355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Häussinger D., Stehle T., Lang F. Volume regulation in liver: further characterization by inhibitors and ionic substitutions. Hepatology. 1990 Feb;11(2):243–254. doi: 10.1002/hep.1840110214. [DOI] [PubMed] [Google Scholar]
  14. Häussinger D., Stoll B., vom Dahl S., Theodoropoulos P. A., Markogiannakis E., Gravanis A., Lang F., Stournaras C. Effect of hepatocyte swelling on microtubule stability and tubulin mRNA levels. Biochem Cell Biol. 1994 Jan-Feb;72(1-2):12–19. doi: 10.1139/o94-003. [DOI] [PubMed] [Google Scholar]
  15. Lake J. R., Licko V., Van Dyke R. W., Scharschmidt B. F. Biliary secretion of fluid-phase markers by the isolated perfused rat liver. Role of transcellular vesicular transport. J Clin Invest. 1985 Aug;76(2):676–684. doi: 10.1172/JCI112021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lang F., Stehle T., Häussinger D. Water, K+, H+, lactate and glucose fluxes during cell volume regulation in perfused rat liver. Pflugers Arch. 1989 Jan;413(3):209–216. doi: 10.1007/BF00583532. [DOI] [PubMed] [Google Scholar]
  17. Lencer W. I., Weyer P., Verkman A. S., Ausiello D. A., Brown D. FITC-dextran as a probe for endosome function and localization in kidney. Am J Physiol. 1990 Feb;258(2 Pt 1):C309–C317. doi: 10.1152/ajpcell.1990.258.2.C309. [DOI] [PubMed] [Google Scholar]
  18. Meijer A. J., Gimpel J. A., Deleeuw G. A., Tager J. M., Williamson J. R. Role of anion translocation across the mitochondrial membrane in the regulation of urea synthesis from ammonia by isolated rat hepatocytes. J Biol Chem. 1975 Oct 10;250(19):7728–7738. [PubMed] [Google Scholar]
  19. Mellman I., Fuchs R., Helenius A. Acidification of the endocytic and exocytic pathways. Annu Rev Biochem. 1986;55:663–700. doi: 10.1146/annurev.bi.55.070186.003311. [DOI] [PubMed] [Google Scholar]
  20. Murphy R. F. Analysis and isolation of endocytic vesicles by flow cytometry and sorting: demonstration of three kinetically distinct compartments involved in fluid-phase endocytosis. Proc Natl Acad Sci U S A. 1985 Dec;82(24):8523–8526. doi: 10.1073/pnas.82.24.8523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Ohkuma S., Moriyama Y., Takano T. Identification and characterization of a proton pump on lysosomes by fluorescein-isothiocyanate-dextran fluorescence. Proc Natl Acad Sci U S A. 1982 May;79(9):2758–2762. doi: 10.1073/pnas.79.9.2758. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Ohkuma S., Poole B. Fluorescence probe measurement of the intralysosomal pH in living cells and the perturbation of pH by various agents. Proc Natl Acad Sci U S A. 1978 Jul;75(7):3327–3331. doi: 10.1073/pnas.75.7.3327. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Palmgren M. G. Acridine orange as a probe for measuring pH gradients across membranes: mechanism and limitations. Anal Biochem. 1991 Feb 1;192(2):316–321. doi: 10.1016/0003-2697(91)90542-2. [DOI] [PubMed] [Google Scholar]
  24. Saha N., Schreiber R., vom Dahl S., Lang F., Gerok W., Häussinger D. Endogenous hydroperoxide formation, cell volume and cellular K+ balance in perfused rat liver. Biochem J. 1993 Dec 15;296(Pt 3):701–707. doi: 10.1042/bj2960701. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Seglen P. O., Grinde B., Solheim A. E. Inhibition of the lysosomal pathway of protein degradation in isolated rat hepatocytes by ammonia, methylamine, chloroquine and leupeptin. Eur J Biochem. 1979 Apr 2;95(2):215–225. doi: 10.1111/j.1432-1033.1979.tb12956.x. [DOI] [PubMed] [Google Scholar]
  26. Star R. A., Zhang B. X., Loessberg P. A., Muallem S. Regulatory volume decrease in the presence of HCO3- by single osteosarcoma cells UMR-106-01. J Biol Chem. 1992 Sep 5;267(25):17665–17669. [PubMed] [Google Scholar]
  27. Stoll B., Gerok W., Lang F., Häussinger D. Liver cell volume and protein synthesis. Biochem J. 1992 Oct 1;287(Pt 1):217–222. doi: 10.1042/bj2870217. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Thomas J. A., Buchsbaum R. N., Zimniak A., Racker E. Intracellular pH measurements in Ehrlich ascites tumor cells utilizing spectroscopic probes generated in situ. Biochemistry. 1979 May 29;18(11):2210–2218. doi: 10.1021/bi00578a012. [DOI] [PubMed] [Google Scholar]
  29. Völkl H., Busch G. L., Häussinger D., Lang F. Alkalinization of acidic cellular compartments following cell swelling. FEBS Lett. 1994 Jan 24;338(1):27–30. doi: 10.1016/0014-5793(94)80110-x. [DOI] [PubMed] [Google Scholar]
  30. Völkl H., Friedrich F., Häussinger D., Lang F. Effect of cell volume on Acridine Orange fluorescence in hepatocytes. Biochem J. 1993 Oct 1;295(Pt 1):11–14. doi: 10.1042/bj2950011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Wettstein M., vom Dahl S., Lang F., Gerok W., Häussinger D. Cell volume regulatory responses of isolated perfused rat liver. The effect of amino acids. Biol Chem Hoppe Seyler. 1990 Jun;371(6):493–501. doi: 10.1515/bchm3.1990.371.1.493. [DOI] [PubMed] [Google Scholar]
  32. Wilson L., Friedkin M. The biochemical events of mitosis. I. Synthesis and properties of colchicine labeled with tritium in its acetyl moiety. Biochemistry. 1966 Jul;5(7):2463–2468. doi: 10.1021/bi00871a042. [DOI] [PubMed] [Google Scholar]
  33. Yoshimori T., Yamamoto A., Moriyama Y., Futai M., Tashiro Y. Bafilomycin A1, a specific inhibitor of vacuolar-type H(+)-ATPase, inhibits acidification and protein degradation in lysosomes of cultured cells. J Biol Chem. 1991 Sep 15;266(26):17707–17712. [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES