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A Pre-embedding Technique for Immunocytochemical Visualization of Cathepsin D in Cultured Cells Subjected to 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.ORCID iD: 0000-0003-4075-159X
1998 (English)In: Journal of Histochemistry and Cytochemistry, ISSN 0022-1554, E-ISSN 1551-5044, Vol. 46, no 3, 411-418 p.Article in journal (Refereed) Published
Abstract [en]

We describe a pre-embedding immunocytochemical method for visualization of the lysosomal enzyme cathepsin D in cultured cells. The protein was demonstrated at both light and electron microscopic levels by neutral-pH silver enhancement of ultrasmall (0.8-nm) gold particles conjugated to the antibodies. The best morphological preservation and the highest labeling density were achieved by initial fixation for 20 min at 4C in 4% paraformaldehyde (PFA) and 0.05% glutaraldehyde (GA) in 0.15 M sodium cacodylate buffer, followed by permeabilization in sodium borohydride. Three cell types were used: human foreskin fibroblasts, histocytic lymphoma (J-774) cells, and primary rat heart myocytes. In all three, cathepsin D was demonstrated in lysosome-like structures. The rat heart myocytes were also exposed to the redox cycling substance naphthazarin (5,8-dihydroxy-1,4-naph-thoquinone) to induce oxidative stress. This was done for such a short period of time that the cells initially did not show any signs of morphological damage and retained normal plasma membrane stability, although an early and clear redistribution of cathepsin D from membrane-bound structures to the cytosol was apparent. This redistribution was followed by cell degeneration and, eventually, by cell death.

Place, publisher, year, edition, pages
1998. Vol. 46, no 3, 411-418 p.
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-80993DOI: 10.1177/002215549804600316OAI: oai:DiVA.org:liu-80993DiVA: diva2:549669
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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