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. 1992 Oct 1;119(1):99–110. doi: 10.1083/jcb.119.1.99

Second messengers regulate endosomal acidification in Swiss 3T3 fibroblasts

PMCID: PMC2289635  PMID: 1382079

Abstract

Acidification of the endosomal pathway is important for ligand and receptor sorting, toxin activation, and protein degradation by lysosomal acid hydrolases. Fluorescent probes and imaging methods were developed to measure pH to better than 0.2 U accuracy in individual endocytic vesicles in Swiss 3T3 fibroblasts. Endosomes were pulse labeled with transferrin (Tf), alpha 2-macroglobulin (alpha 2M), or dextran, each conjugated with tetramethylrhodamine and carboxyfluorescein (for pH 5-8) or dichlorocarboxyfluorescein (for pH 4- 6); pH in individual labeled vesicles was measured by ratio imaging using a cooled CCD camera and novel image analysis software. Tf-labeled endosomes acidified to pH 6.2 +/- 0.1 with a t1/2 of 4 min at 37 degrees C, and remained small and near the cell periphery. Dextran- and alpha 2M-labeled endosomes acidified to pH 4.7 +/- 0.2, becoming larger and moving toward the nucleus over 30 min; approximately 15% of alpha 2M-labeled endosomes were strongly acidic (pH less than 5.5) at only 1 min after labeling. Replacement of external Cl by NO3 or isethionate strongly and reversibly inhibited acidification. Addition of ouabain (1 mM) at the time of labeling strongly enhanced acidification in the first 5 min; Tf-labeled endosomes acidified to pH 5.3 without a change in morphology. Activation of phospholipase C by vasopressin (50 nM) enhanced acidification of early endosomes; activation of protein kinase C by PMA (100 nM) enhanced acidification strongly, whereas elevation of intracellular Ca by A23187 (1 microM) had no effect on acidification. Activation of protein kinase A by CPT-cAMP (0.5 mM) or forskolin (50 microM) inhibited acidification. Lysosomal pH was not affected by ouabain or the protein kinase activators. These results establish a methodology for quantitative measurement of pH in individual endocytic vesicles, and demonstrate that acidification of endosomes labeled with Tf and alpha 2M (receptor-mediated endocytosis) and dextran (fluid- phase endocytosis) is sensitive to intracellular anion composition, Na/K pump inhibition, and multiple intracellular second messengers.

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Selected References

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  1. Backer J. M., King G. L. Regulation of receptor-mediated endocytosis by phorbol esters. Biochem Pharmacol. 1991 May 1;41(9):1267–1277. doi: 10.1016/0006-2952(91)90097-o. [DOI] [PubMed] [Google Scholar]
  2. Bae H. R., Verkman A. S. Protein kinase A regulates chloride conductance in endocytic vesicles from proximal tubule. Nature. 1990 Dec 13;348(6302):637–639. doi: 10.1038/348637a0. [DOI] [PubMed] [Google Scholar]
  3. Barasch J., Gershon M. D., Nunez E. A., Tamir H., al-Awqati Q. Thyrotropin induces the acidification of the secretory granules of parafollicular cells by increasing the chloride conductance of the granular membrane. J Cell Biol. 1988 Dec;107(6 Pt 1):2137–2147. doi: 10.1083/jcb.107.6.2137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Barasch J., Kiss B., Prince A., Saiman L., Gruenert D., al-Awqati Q. Defective acidification of intracellular organelles in cystic fibrosis. Nature. 1991 Jul 4;352(6330):70–73. doi: 10.1038/352070a0. [DOI] [PubMed] [Google Scholar]
  5. Benveniste M., Schlessinger J., Kam Z. Characterization of internalization and endosome formation of epidermal growth factor in transfected NIH-3T3 cells by computerized image-intensified three-dimensional fluorescence microscopy. J Cell Biol. 1989 Nov;109(5):2105–2115. doi: 10.1083/jcb.109.5.2105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Blair H. C., Teitelbaum S. L., Tan H. L., Koziol C. M., Schlesinger P. H. Passive chloride permeability charge coupled to H(+)-ATPase of avian osteoclast ruffled membrane. Am J Physiol. 1991 Jun;260(6 Pt 1):C1315–C1324. doi: 10.1152/ajpcell.1991.260.6.C1315. [DOI] [PubMed] [Google Scholar]
  7. Cain C. C., Murphy R. F. A chloroquine-resistant Swiss 3T3 cell line with a defect in late endocytic acidification. J Cell Biol. 1988 Feb;106(2):269–277. doi: 10.1083/jcb.106.2.269. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cain C. C., Sipe D. M., Murphy R. F. Regulation of endocytic pH by the Na+,K+-ATPase in living cells. Proc Natl Acad Sci U S A. 1989 Jan;86(2):544–548. doi: 10.1073/pnas.86.2.544. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chao A. C., Dix J. A., Sellers M. C., Verkman A. S. Fluorescence measurement of chloride transport in monolayer cultured cells. Mechanisms of chloride transport in fibroblasts. Biophys J. 1989 Dec;56(6):1071–1081. doi: 10.1016/S0006-3495(89)82755-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cuppoletti J., Aures-Fischer D., Sachs G. The lysosomal H+ pump: 8-azido-ATP inhibition and the role of chloride in H+ transport. Biochim Biophys Acta. 1987 May 29;899(2):276–284. doi: 10.1016/0005-2736(87)90409-3. [DOI] [PubMed] [Google Scholar]
  11. Dautry-Varsat A., Ciechanover A., Lodish H. F. pH and the recycling of transferrin during receptor-mediated endocytosis. Proc Natl Acad Sci U S A. 1983 Apr;80(8):2258–2262. doi: 10.1073/pnas.80.8.2258. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Dunn K. W., McGraw T. E., Maxfield F. R. Iterative fractionation of recycling receptors from lysosomally destined ligands in an early sorting endosome. J Cell Biol. 1989 Dec;109(6 Pt 2):3303–3314. doi: 10.1083/jcb.109.6.3303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Forster S., Lloyd J. B. Solute translocation across the mammalian lysosome membrane. Biochim Biophys Acta. 1988 Oct 11;947(3):465–491. doi: 10.1016/0304-4157(88)90004-4. [DOI] [PubMed] [Google Scholar]
  14. Fuchs R., Mâle P., Mellman I. Acidification and ion permeabilities of highly purified rat liver endosomes. J Biol Chem. 1989 Feb 5;264(4):2212–2220. [PubMed] [Google Scholar]
  15. Fuchs R., Schmid S., Mellman I. A possible role for Na+,K+-ATPase in regulating ATP-dependent endosome acidification. Proc Natl Acad Sci U S A. 1989 Jan;86(2):539–543. doi: 10.1073/pnas.86.2.539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gruenberg J., Howell K. E. Membrane traffic in endocytosis: insights from cell-free assays. Annu Rev Cell Biol. 1989;5:453–481. doi: 10.1146/annurev.cb.05.110189.002321. [DOI] [PubMed] [Google Scholar]
  17. Hilden S. A., Ghoshroy K. B., Madias N. E. Na(+)-H+ exchange, but not Na(+)-K(+)-ATPase, is present in endosome-enriched microsomes from rabbit renal cortex. Am J Physiol. 1990 May;258(5 Pt 2):F1311–F1319. doi: 10.1152/ajprenal.1990.258.5.F1311. [DOI] [PubMed] [Google Scholar]
  18. Hopkins C. R., Gibson A., Shipman M., Miller K. Movement of internalized ligand-receptor complexes along a continuous endosomal reticulum. Nature. 1990 Jul 26;346(6282):335–339. doi: 10.1038/346335a0. [DOI] [PubMed] [Google Scholar]
  19. Inoué S. Imaging of unresolved objects, superresolution, and precision of distance measurement with video microscopy. Methods Cell Biol. 1989;30:85–112. doi: 10.1016/s0091-679x(08)60976-0. [DOI] [PubMed] [Google Scholar]
  20. Issandou M., Rozengurt E. Bradykinin transiently activates protein kinase C in Swiss 3T3 cells. Distinction from activation by bombesin and vasopressin. J Biol Chem. 1990 Jul 15;265(20):11890–11896. [PubMed] [Google Scholar]
  21. Lencer W. I., Verkman A. S., Arnaout M. A., Ausiello D. A., Brown D. Endocytic vesicles from renal papilla which retrieve the vasopressin-sensitive water channel do not contain a functional H+ ATPase. J Cell Biol. 1990 Aug;111(2):379–389. doi: 10.1083/jcb.111.2.379. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lukacs G. L., Rotstein O. D., Grinstein S. Determinants of the phagosomal pH in macrophages. In situ assessment of vacuolar H(+)-ATPase activity, counterion conductance, and H+ "leak". J Biol Chem. 1991 Dec 25;266(36):24540–24548. [PubMed] [Google Scholar]
  23. 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]
  24. Moriyama Y., Nelson N. The purified ATPase from chromaffin granule membranes is an anion-dependent proton pump. J Biol Chem. 1987 Jul 5;262(19):9175–9180. [PubMed] [Google Scholar]
  25. Mulberg A. E., Tulk B. M., Forgac M. Modulation of coated vesicle chloride channel activity and acidification by reversible protein kinase A-dependent phosphorylation. J Biol Chem. 1991 Nov 5;266(31):20590–20593. [PubMed] [Google Scholar]
  26. Reenstra W. W., Sabolic I., Bae H. R., Verkman A. S. Protein kinase A dependent membrane protein phosphorylation and chloride conductance in endosomal vesicles from kidney cortex. Biochemistry. 1992 Jan 14;31(1):175–181. doi: 10.1021/bi00116a026. [DOI] [PubMed] [Google Scholar]
  27. Shi L. B., Fushimi K., Bae H. R., Verkman A. S. Heterogeneity in ATP-dependent acidification in endocytic vesicles from kidney proximal tubule. Measurement of pH in individual endocytic vesicles in a cell-free system. Biophys J. 1991 Jun;59(6):1208–1217. doi: 10.1016/S0006-3495(91)82336-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Shi L. B., Wang Y. X., Verkman A. S. Regulation of the formation and water permeability of endosomes from toad bladder granular cells. J Gen Physiol. 1990 Oct;96(4):789–808. doi: 10.1085/jgp.96.4.789. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Sipe D. M., Jesurum A., Murphy R. F. Absence of Na+,K(+)-ATPase regulation of endosomal acidification in K562 erythroleukemia cells. Analysis via inhibition of transferrin recycling by low temperatures. J Biol Chem. 1991 Feb 25;266(6):3469–3474. [PubMed] [Google Scholar]
  30. Sipe D. M., Murphy R. F. High-resolution kinetics of transferrin acidification in BALB/c 3T3 cells: exposure to pH 6 followed by temperature-sensitive alkalinization during recycling. Proc Natl Acad Sci U S A. 1987 Oct;84(20):7119–7123. doi: 10.1073/pnas.84.20.7119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Van Dyke R. W. Anion inhibition of the proton pump in rat liver multivesicular bodies. J Biol Chem. 1986 Dec 5;261(34):15941–15948. [PubMed] [Google Scholar]
  32. Van Dyke R. W. Proton pump-generated electrochemical gradients in rat liver multivesicular bodies. Quantitation and effects of chloride. J Biol Chem. 1988 Feb 25;263(6):2603–2611. [PubMed] [Google Scholar]
  33. Wang Y. X., Shi L. B., Verkman A. S. Functional water channels and proton pumps are in separate populations of endocytic vesicles in toad bladder granular cells. Biochemistry. 1991 Mar 19;30(11):2888–2894. doi: 10.1021/bi00225a023. [DOI] [PubMed] [Google Scholar]
  34. Yamashiro D. J., Borden L. A., Maxfield F. R. Kinetics of alpha 2-macroglobulin endocytosis and degradation in mutant and wild-type Chinese hamster ovary cells. J Cell Physiol. 1989 May;139(2):377–382. doi: 10.1002/jcp.1041390221. [DOI] [PubMed] [Google Scholar]
  35. Yamashiro D. J., Maxfield F. R. Acidification of endocytic compartments and the intracellular pathways of ligands and receptors. J Cell Biochem. 1984;26(4):231–246. doi: 10.1002/jcb.240260404. [DOI] [PubMed] [Google Scholar]
  36. Yamashiro D. J., Maxfield F. R. Acidification of morphologically distinct endosomes in mutant and wild-type Chinese hamster ovary cells. J Cell Biol. 1987 Dec;105(6 Pt 1):2723–2733. doi: 10.1083/jcb.105.6.2723. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Yamashiro D. J., Maxfield F. R. Kinetics of endosome acidification in mutant and wild-type Chinese hamster ovary cells. J Cell Biol. 1987 Dec;105(6 Pt 1):2713–2721. doi: 10.1083/jcb.105.6.2713. [DOI] [PMC free article] [PubMed] [Google Scholar]

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