Oxidative stress causes relocation of the lysosomal enzyme cathepsin D with ensuing apoptosis in neonatal rat cardiomyocytes.
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.
Roberg
K
K
Division of Pathology II, Faculty of Health Sciences, Linköping University, Sweden.
Ollinger
K
K
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D017209:Apoptosis; D002402:Cathepsin D / Q000378:metabolism* / Q000648:ultrastructure; D002478:Cells, Cultured; D005260:Female; D019084:Fluorescent Antibody Technique, Indirect; D006321:Heart / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008247:Lysosomes / Q000201:enzymology* / Q000648:ultrastructure; D008297:Male; D016253:Microscopy, Immunoelectron; D009206:Myocardium / Q000166:cytology / Q000201:enzymology*; D009285:Naphthoquinones / Q000494:pharmacology; D018384:Oxidative Stress; D051381:Rats; D017207:Rats, Sprague-Dawley; D014810:Vitamin E / Q000494:pharmacology
D009285:Naphthoquinones; D014810:Vitamin E; C005503:naphthazarin; D000255:Adenosine Triphosphate; D007770:L-Lactate Dehydrogenase; D002402:Cathepsin D