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. 1997 Jan 1;321(Pt 1):217–225. doi: 10.1042/bj3210217

A novel method for the study of autophagy: destruction of hepatocytic lysosomes, but not autophagosomes, by the photosensitizing porphyrin tetra(4-sulphonatophenyl)porphine.

P E Strømhaug 1, T O Berg 1, K Berg 1, P O Seglen 1
PMCID: PMC1218057  PMID: 9003422

Abstract

A photoactivatable porphyrin, tetra(4-sulphonatophenyl)porphine (TPPS4), was shown to accumulate in rat hepatocytes as a linear function of dose after intravenous injection, and to localize predominantly in hepatocytic lysosomes. A major fraction of the lysosomal enzymes acid phosphatase and N-acetyl-beta-D-glucosaminidase was inactivated by TPPS4 after 20 h of contact with the drug in vivo in the absence of photoactivation. On exposure of isolated hepatocytes to light, photoactivated TPPS4 caused additional inactivation of the lysosomal enzymes as well as inactivation of intralysosomal lactate dehydrogenase (LDH), a cytosolic enzyme that accumulated in lysosomes as a result of autophagy during a 2 h incubation of hepatocytes at 37 degrees C in the dark (in the presence of the proteinase inhibitor leupeptin to prevent degradation of intralysosomal LDH). Photoactivation of TPPS4 also induced lysosomal rupture, with a loss of lysosomal enzymes, autophagocytosed LDH, endocytosed 125I-tyramine-cellobiose-asialo-orosomucoid and TPPS4 from the lysosomes. However, LDH-containing autophagosomes, accumulated in the presence of vinblastine (a microtubule inhibitor used to prevent the fusion of lysosomes with autophagosomes or endosomes), were not affected by TPPS4. TPPS4 may thus be useful as a selective lysosomal (or endosomal) perturbant in the study of autophagic-endocytic-lysosomal interactions.

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Selected References

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  1. Berg K., Madslien K., Bommer J. C., Oftebro R., Winkelman J. W., Moan J. Light induced relocalization of sulfonated meso-tetraphenylporphines in NHIK 3025 cells and effects of dose fractionation. Photochem Photobiol. 1991 Feb;53(2):203–210. doi: 10.1111/j.1751-1097.1991.tb03924.x. [DOI] [PubMed] [Google Scholar]
  2. Berg K., Moan J. Lysosomes as photochemical targets. Int J Cancer. 1994 Dec 15;59(6):814–822. doi: 10.1002/ijc.2910590618. [DOI] [PubMed] [Google Scholar]
  3. Berg K., Moan J. The influence of the cysteine protease inhibitor L-trans-epoxysuccinyl-leucyl amido(4-guanidio)butane (E64) on photobiological effects of tetra(4-sulfonatophenyl)porphine. Cancer Lett. 1995 Jan 27;88(2):227–236. doi: 10.1016/0304-3835(94)03620-x. [DOI] [PubMed] [Google Scholar]
  4. Berg K., Prydz K., Moan J. Photochemical treatment with the lysosomally localized dye tetra(4-sulfonatophenyl)porphine results in lysosomal release of the dye but not of beta-N-acetyl-D-glucosaminidase activity. Biochim Biophys Acta. 1993 Nov 28;1158(3):300–306. doi: 10.1016/0304-4165(93)90029-8. [DOI] [PubMed] [Google Scholar]
  5. Berg T. O., Strømhaug E., Løvdal T., Seglen O., Berg T. Use of glycyl-L-phenylalanine 2-naphthylamide, a lysosome-disrupting cathepsin C substrate, to distinguish between lysosomes and prelysosomal endocytic vacuoles. Biochem J. 1994 May 15;300(Pt 1):229–236. doi: 10.1042/bj3000229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Berg T. O., Strømhaug P. E., Berg T., Seglen P. O. Separation of lysosomes and autophagosomes by means of glycyl-phenylalanine-naphthylamide, a lysosome-disrupting cathepsin-C substrate. Eur J Biochem. 1994 Apr 1;221(1):595–602. doi: 10.1111/j.1432-1033.1994.tb18771.x. [DOI] [PubMed] [Google Scholar]
  7. Berg T., Kindberg G. M., Ford T., Blomhoff R. Intracellular transport of asialoglycoproteins in rat hepatocytes. Evidence for two subpopulations of lysosomes. Exp Cell Res. 1985 Dec;161(2):285–296. doi: 10.1016/0014-4827(85)90086-2. [DOI] [PubMed] [Google Scholar]
  8. Fengsrud M., Roos N., Berg T., Liou W., Slot J. W., Seglen P. O. Ultrastructural and immunocytochemical characterization of autophagic vacuoles in isolated hepatocytes: effects of vinblastine and asparagine on vacuole distributions. Exp Cell Res. 1995 Dec;221(2):504–519. doi: 10.1006/excr.1995.1402. [DOI] [PubMed] [Google Scholar]
  9. Freitas I., Novarina A. Dark effects of hematoporphyrin derivative on lactate dehydrogenase activity and distribution in HeLa cells: cytochemical evaluation. Photochem Photobiol. 1987 Nov;46(5):699–706. doi: 10.1111/j.1751-1097.1987.tb04835.x. [DOI] [PubMed] [Google Scholar]
  10. Furuno K., Ishikawa T., Kato K. Isolation and characterization of autolysosomes which appeared in rat liver after leupeptin treatment. J Biochem. 1982 Jun;91(6):1943–1950. doi: 10.1093/oxfordjournals.jbchem.a133888. [DOI] [PubMed] [Google Scholar]
  11. Gordon P. B., Høyvik H., Seglen P. O. Prelysosomal and lysosomal connections between autophagy and endocytosis. Biochem J. 1992 Apr 15;283(Pt 2):361–369. doi: 10.1042/bj2830361. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gordon P. B., Høyvik H., Seglen P. O. Sugar uptake by fluid-phase pinocytosis and diffusion in isolated rat hepatocytes. Biochem J. 1987 May 1;243(3):655–660. doi: 10.1042/bj2430655. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gordon P. B., Seglen P. O. Autophagic sequestration of [14C]sucrose, introduced into rat hepatocytes by reversible electro-permeabilization. Exp Cell Res. 1982 Nov;142(1):1–14. doi: 10.1016/0014-4827(82)90402-5. [DOI] [PubMed] [Google Scholar]
  14. Gordon P. B., Seglen P. O. Prelysosomal convergence of autophagic and endocytic pathways. Biochem Biophys Res Commun. 1988 Feb 29;151(1):40–47. doi: 10.1016/0006-291x(88)90556-6. [DOI] [PubMed] [Google Scholar]
  15. Gordon P. B., Seglen P. O. Use of electrical methods in the study of hepatocytic autophagy. Biomed Biochim Acta. 1986;45(11-12):1635–1645. [PubMed] [Google Scholar]
  16. Holen I., Strømhaug P. E., Gordon P. B., Fengsrud M., Berg T. O., Seglen P. O. Inhibition of autophagy and multiple steps in asialoglycoprotein endocytosis by inhibitors of tyrosine protein kinases (tyrphostins). J Biol Chem. 1995 May 26;270(21):12823–12831. doi: 10.1074/jbc.270.21.12823. [DOI] [PubMed] [Google Scholar]
  17. Høyvik H., Gordon P. B., Berg T. O., Strømhaug P. E., Seglen P. O. Inhibition of autophagic-lysosomal delivery and autophagic lactolysis by asparagine. J Cell Biol. 1991 Jun;113(6):1305–1312. doi: 10.1083/jcb.113.6.1305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kishore B. K., Fuming L. u., Maldague P., Tulkens P. M., Courtoy P. J. Mechanism of the thesaurismosis and altered lysosomal dynamics induced by poly-D-glutamic acid in kidney proximal tubular cells. Lab Invest. 1996 Jun;74(6):1025–1037. [PubMed] [Google Scholar]
  19. Kominami E., Hashida S., Khairallah E. A., Katunuma N. Sequestration of cytoplasmic enzymes in an autophagic vacuole-lysosomal system induced by injection of leupeptin. J Biol Chem. 1983 May 25;258(10):6093–6100. [PubMed] [Google Scholar]
  20. Kongshaug M., Moan J., Brown S. B. The distribution of porphyrins with different tumour localising ability among human plasma proteins. Br J Cancer. 1989 Feb;59(2):184–188. doi: 10.1038/bjc.1989.38. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kopitz J., Kisen G. O., Gordon P. B., Bohley P., Seglen P. O. Nonselective autophagy of cytosolic enzymes by isolated rat hepatocytes. J Cell Biol. 1990 Sep;111(3):941–953. doi: 10.1083/jcb.111.3.941. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kovács A. L., Reith A., Seglen P. O. Accumulation of autophagosomes after inhibition of hepatocytic protein degradation by vinblastine, leupeptin or a lysosomotropic amine. Exp Cell Res. 1982 Jan;137(1):191–201. doi: 10.1016/0014-4827(82)90020-9. [DOI] [PubMed] [Google Scholar]
  23. Marzella L., Ahlberg J., Glaumann H. Isolation of autophagic vacuoles from rat liver: morphological and biochemical characterization. J Cell Biol. 1982 Apr;93(1):144–154. doi: 10.1083/jcb.93.1.144. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Moan J., Berg K. Photochemotherapy of cancer: experimental research. Photochem Photobiol. 1992 Jun;55(6):931–948. doi: 10.1111/j.1751-1097.1992.tb08541.x. [DOI] [PubMed] [Google Scholar]
  25. Moan J., Berg K. The photodegradation of porphyrins in cells can be used to estimate the lifetime of singlet oxygen. Photochem Photobiol. 1991 Apr;53(4):549–553. doi: 10.1111/j.1751-1097.1991.tb03669.x. [DOI] [PubMed] [Google Scholar]
  26. Mossin L., Blankson H., Huitfeldt H., Seglen P. O. Ploidy-dependent growth and binucleation in cultured rat hepatocytes. Exp Cell Res. 1994 Oct;214(2):551–560. doi: 10.1006/excr.1994.1293. [DOI] [PubMed] [Google Scholar]
  27. Qian P., Evensen J. F., Rimington C., Moan J. A comparison of different photosensitizing dyes with respect to uptake C3H-tumors and tissues of mice. Cancer Lett. 1987 Jul;36(1):1–10. doi: 10.1016/0304-3835(87)90096-6. [DOI] [PubMed] [Google Scholar]
  28. Seglen P. O., Bohley P. Autophagy and other vacuolar protein degradation mechanisms. Experientia. 1992 Feb 15;48(2):158–172. doi: 10.1007/BF01923509. [DOI] [PubMed] [Google Scholar]
  29. Seglen P. O., Gordon P. B. 3-Methyladenine: specific inhibitor of autophagic/lysosomal protein degradation in isolated rat hepatocytes. Proc Natl Acad Sci U S A. 1982 Mar;79(6):1889–1892. doi: 10.1073/pnas.79.6.1889. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Seglen P. O., Gordon P. B. Amino acid control of autophagic sequestration and protein degradation in isolated rat hepatocytes. J Cell Biol. 1984 Aug;99(2):435–444. doi: 10.1083/jcb.99.2.435. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Seglen P. O. Preparation of isolated rat liver cells. Methods Cell Biol. 1976;13:29–83. doi: 10.1016/s0091-679x(08)61797-5. [DOI] [PubMed] [Google Scholar]
  32. Seglen P. O., Solheim A. E. Conversion of dense lysosomes into light lysosomes during hepatocytic autophagy. Exp Cell Res. 1985 Apr;157(2):550–555. doi: 10.1016/0014-4827(85)90141-7. [DOI] [PubMed] [Google Scholar]
  33. Seglen P. O., Solheim A. E. Valine uptake and incorporation into protein in isolated rat hepatocytes. Nature of the precursor pool for protein synthesis. Eur J Biochem. 1978 Apr;85(1):15–25. doi: 10.1111/j.1432-1033.1978.tb12208.x. [DOI] [PubMed] [Google Scholar]
  34. WINKELMAN J. The distribution of tetraphenylporphinesulfonate in the tumor-bearing rat. Cancer Res. 1962 Jun;22:589–596. [PubMed] [Google Scholar]
  35. Wessels J. M., Strauss W., Seidlitz H. K., Rück A., Schneckenburger H. Intracellular localization of meso-tetraphenylporphine tetrasulphonate probed by time-resolved and microscopic fluorescence spectroscopy. J Photochem Photobiol B. 1992 Feb 28;12(3):275–284. doi: 10.1016/1011-1344(92)85029-t. [DOI] [PubMed] [Google Scholar]
  36. Woodside K. H., Mortimore G. E. Suppression of protein turnover by amino acids in the perfused rat liver. J Biol Chem. 1972 Oct 25;247(20):6474–6481. [PubMed] [Google Scholar]

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