Immunocytochemical visualization of cathepsin D during oxidative stress
1998 (English)Licentiate thesis, comprehensive summary (Other academic)
Cathepsin D is the major aspartic protease in lysosomes, and it is found in almost all animal cells. In the present studies, a pre-embedding immunocytochemical method was used to visualize cathepsin D in cultured cells. The protein can be detected 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 in 4% paraformaldehyde (PFA) and 0.05% glutaraldehyde (GA) in 0.15 M sodium cacodylate buffer, followed by membrane permeabilization in sodium borohydride for 20 min at 4° c. Three cell types were used: human foreskin fibroblasts, mouse histiocytic lymphoma (1774) cells, and primary rat heart myocytes. Cathepsin D was found in lysosome-like structures in all three cell types.
Rat heart myocytes were treated with the redox cycling, xenobiotic compound naphthazarin (5,8-dihydroxy-1,4-naphthoquinone) to induce oxidative stress. Immunofluorescence staining and light rnicroscopy (LM) 30 min after exposure showed that cathepsin D fluorescence had changed from a mainly lysosomal pattern in control cells to a partly cytosolic location in cells exposed to oxidative stress. Electron microscopic morphometry 15, 30, and 45 min after exposure to naphthazarin confirmed that a statistically significant amount of cathepsin D was transferred from lysosome-like structures to the cytosol. Exposure to naphthazarin also caused a decrease in intracellular pH and ATP, whereas lipid peroxidation was not detected within the same time frame.
Following exposure to naphthazarin, cells were washed and normal culture conditions were reestablished for another 18 h. Thereafter many cells displayed apoptotic morphology (i.e. cellular shrinkage and chromatin condensation; analyzed by Giemsa staining). Also, 41% of the cells stained positive with the TUNEL technique, and DNA fragmentation was detected. Pretreatment of the cultures with α-tocopherol succinate largely prevented relocation of cathepsin D (shown by immunofluorescence) and also significantly decreased apoptosis.
Place, publisher, year, edition, pages
Linköping: Linköpings universitet , 1998. , 44 p.
Linköping Studies in Health Sciences. Thesis, ISSN 1100-6013 ; 39
Medical and Health Sciences
IdentifiersURN: urn:nbn:se:liu:diva-28268Local ID: 13016ISBN: 91-7219-318-2OAI: oai:DiVA.org:liu-28268DiVA: diva2:249072
Papers, included in licentiate theses, are not registered and included in the posts from 1999 and earlier.2009-10-092009-10-092013-07-08