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Oxidative stress causes relocation of the lysosomal enzyme cathepsin D with ensuing apoptosis in neonatal rat cardiomyocytes
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
1998 (English)In: American Journal of Pathology, ISSN 0002-9440, E-ISSN 1525-2191, Vol. 152, no 5, 1151-1156 p.Article in journal (Refereed) Published
Abstract [en]

Exposing neonatal rat heart myocytes to the redox cycling quinone naphthazarin (5,8-dihydroxy-1,4-naphthoquinone) for 15 to 45 minutes led to a time-dependent release of cathepsin D from many secondary lysosomes to the cytosol, as analyzed by morphometry. Cathepsin D was detected electron microscopically using a pre-embedding immunostaining technique that utilizes antibodies conjugated to ultra-small (0.8-nm) gold particles and subsequent silver enhancement. The exposure to naphthazarin also caused a decrease in both the pH and the ATP level of the cells within the same time frame. Lipid peroxidation was, however, not detected. Pretreatment of the cultures with alpha-tocopherol succinate prevented cathepsin D relocation, as shown by immunofluorescence. After exposure to naphthazarin, cells were washed, and normal culture conditions were re-established for 18 hours. Many cells then showed apoptotic morphology (ie, cellular shrinkage and chromatin condensation) as analyzed by Giemsa staining. Also, 41% of the cells stained positive with the TUNEL technique, and DNA fragmentation was detected by separation of intact and fragmented DNA. Apoptosis was significantly decreased in cultures pretreated with alpha-tocopherol succinate.

Place, publisher, year, edition, pages
1998. Vol. 152, no 5, 1151-1156 p.
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-80994PubMedID: 9588882OAI: oai:DiVA.org:liu-80994DiVA: diva2:549675
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, KarinÖllinger, Karin

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