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Lysosomal release of Cathepsin D precedes relocation of Cytochrome C and loss of mitochondrial transmembrane potential during apoptosis induced by oxidative stress
Linköping University, Department of Neuroscience and Locomotion, Pathology. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Neuroscience and Locomotion, Pathology. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Neuroscience and Locomotion, Pathology. Linköping University, Faculty of Health Sciences.ORCID iD: 0000-0003-4075-159X
1999 (English)In: Free Radical Biology & Medicine, ISSN 0891-5849, E-ISSN 1873-4596, Vol. 27, no 11-12, 1228-1237 p.Article in journal (Refereed) Published
Abstract [en]

Apoptosis was induced in human foreskin fibroblasts by the redox-cycling quinone naphthazarin (5,8-dihydroxy-1,4-naphthoquinone). Most of the cells displayed ultrastructure typical of apoptosis after 8 h of exposure to naphthazarin. Apoptosis was inhibited in fibroblasts pretreated with the cathepsin D inhibitor pepstatin A. Immunofluorescence analysis of the intracellular distribution of cathepsin D revealed a distinct granular pattern in control cells, whereas cells treated with naphthazarin for 30 min exhibited more diffuse staining that corresponded to release of the enzyme from lysosomes to the cytosol. After 2 h, release of cytochrome c from mitochondria to the cytosol was indicated by immunofluorescence. The membrane-potential–sensitive probe JC-1 and flow cytometry did not detect a permanent decrease in mitochondrial transmembrane potential (ΔΨm) until after 5 h of naphthazarin treatment. Our findings show that, during naphthazarin-induced apoptosis, lysosomal destabilization (measured as release of cathepsin D) precedes release of cytochrome c, loss of ΔΨm, and morphologic alterations. Moreover, apoptosis could be inhibited by pretreatment with pepstatin A.

Place, publisher, year, edition, pages
1999. Vol. 27, no 11-12, 1228-1237 p.
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-80995DOI: 10.1016/S0891-5849(99)00146-XOAI: oai:DiVA.org:liu-80995DiVA: diva2:549678
Available from: 2012-09-05 Created: 2012-09-05 Last updated: 2017-12-07Bibliographically approved
In thesis
1. The role of cathepsin D in apoptosis induced by oxidative stress
Open this publication in new window or tab >>The role of cathepsin D in apoptosis induced by oxidative stress
2001 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The lysosomal protease cathepsin D is translocated from lysosomes to the cytosol during apoptosis induced by oxidative stress. In the present studies, the redox-cycling, xenobiotic compound naphthazarin (5,8-dihydroxy-1,4-naphthoquinone) was used to create oxidative stress in rat cardiomyocytes and human foreskin fibroblasts. In naphthazarin exposed cells, lysosomal release of cathepsin D preceded liberation of cytochrome c from mitochondria and a decrease in the mitochondrial transmembrane potential (ΔΨm).

A pre-embedding immunocytochemical method was used for ultrastructural examination of cathepsin D and cytochrome c in cultured cells. Electron microscopic morphometry confirmed that a statistically significant amount of cathepsin D was transferred from lysosome-like structures to the cytosol before any biochemical or morphological signs of apoptosis were detected. Pretreatment of the cells with atocopherol succinate largely prevented translocation of cathepsin D and also significantly decreased apoptosis. Electron microscopy also revealed that, during exposure to naphthazarin, a minor release of cytochrome c has occured after one hour and a more extensive release after two hours, and these results were verified by Western blotting. After the translocation of cathepsin D and cytochrome c, a decrease in ΔΨm was detected using the ΔΨm-sensitive probe JC-1 and confocal microscopy or measured by flow cytornetry. Pretreatment with the cathepsin D inhibitor pepstatin A prevented release of cytochrome c from mitochondria, maintained the ΔΨm and inhibited apoptosis.

In conclusion, these findings show that translocation of cathepsin D precedes important incidents in mitochondria, such as release of cytochrome c and loss of ΔΨm during apoptosis induced by oxidative stress. Moreover, inhibition of cathepsin D prevented the apoptosis and the mitochondrial changes, which indicates that cathepsin D is an inducer of apoptosis upstream of cytochrome c release.

Place, publisher, year, edition, pages
Linköping: Linköpings universitet, 2001. 87 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 688
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-28070 (URN)12834 (Local ID)91-7219-979-2 (ISBN)12834 (Archive number)12834 (OAI)
Public defence
2001-09-28, Berzeliussalen, Hälsouniversitetet, Linköping, 13:00 (Swedish)
Opponent
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2013-07-08Bibliographically approved

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Roberg, KarinJohansson, UnoÖllinger, Karin

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