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Relocalization of Cathepsin D and Cytochrome c Early in Apoptosis Revealed by Immunoelectron Microscopy
Linköping University, Department of Neuroscience and Locomotion, Pathology. Linköping University, Faculty of Health Sciences.
2001 (English)In: Laboratory Investigation, ISSN 0023-6837, E-ISSN 1530-0307, Vol. 81, no 2, 149-158 p.Article in journal (Refereed) Published
Abstract [en]

Cathepsin D was translocated from lysosomal structures to the cytosol in primary cultures of neonatal rat cardiomyocytes exposed to oxidative stress, and these cells underwent apoptotic death during subsequent incubation. Temporal aspects of cathepsin D relocalization, cytochrome c release, and decrease in mitochondrial transmembrane potential (Δψm) were studied in myocytes exposed to the redox-cycling xenobiotic naphthazarin (5,8-dihydroxy-1,4-naphthoquinone). Immunofluorescence labeling revealed that cathepsin D was translocated to the cytosol after 30 minutes of naphthazarin treatment, and cytochrome c was released from mitochondria to the cytosol after 2 hours. Western blotting and immunoelectron microscopy indicated a minor release of cytochrome c after only 30 minutes and 1 hour, respectively. Thereafter, a decrease in Δψm was detected using the Δψm-sensitive dye JC-1 and confocal microscopy, and ultrastructural analysis indicated apoptotic morphology. Pretreatment of the cultures with the cathepsin D inhibitor pepstatin A prevented release of cytochrome c from mitochondria and maintained the Δψm. Moreover, ultrastructural examination showed no apoptotic morphology. These findings suggest that lysosomal destabilization (detected as the release of cathepsin D) and release of cytochrome c from mitochondria take place early in apoptosis. Also, the former event probably occurs before the latter during apoptosis induced by oxidative stress because pretreatment with pepstatin A prevented release of cytochrome c and loss of Δψm in cardiomyocytes exposed to naphthazarin.

Place, publisher, year, edition, pages
2001. Vol. 81, no 2, 149-158 p.
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-27927Local ID: 12688OAI: oai:DiVA.org:liu-27927DiVA: diva2:248479
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2017-12-13Bibliographically 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, Karin

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