Intracellular Ca2+ signals activate apoptosis in thymocytes: studies using the Ca(2+)-ATPase inhibitor thapsigargin.
The endoplasmic reticular Ca(2+)-ATPase inhibitor, thapsigargin, was used to study the role of an increase in cytosolic free calcium concentration ([Ca2+]i) as a signal for the activation of thymocyte apoptosis. Treatment of rat thymocytes with thapsigargin resulted in an early sustained increase in [Ca2+]i followed by extensive DNA fragmentation. Agarose gel electrophoresis revealed that the pattern of DNA fragments was typical of endonuclease-mediated internucleosomal cleavage. In addition, confocal microscopy studies showed the formation of apoptotic nuclei in thapsigargin-treated thymocytes. The concentrations of thapsigargin required to induce DNA fragmentation and [Ca2+]i increase in thymocytes were identical and so were the kinetics of thapsigargin-induced DNA fragmentation and formation of apoptotic nuclei. The lowest concentration of thapsigargin needed to activate apoptosis was 1 nM. Thapsigargin-induced [Ca2+]i increase and thymocyte apoptosis were inhibited in cells incubated in nominally Ca(2+)-free medium or pretreated with the intracellular Ca2+ chelator, bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/acetoxymethyl ester. Removal of extracellular free Ca2+ with 5 mM EGTA at different time points after thapsigargin addition revealed a time dependency of about 2 h for the sustained increase in [Ca2+]i to trigger apoptosis in thymocytes. Thus, we conclude that the signal provided by the thapsigargin-induced [Ca2+]i increase is sufficient to activate thymocyte apoptosis.
Jiang
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Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden.
Chow
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SC
Nicotera
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Orrenius
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D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors*; D002467:Cell Nucleus / Q000648:ultrastructure; D004305:Dose-Response Relationship, Drug; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000201:enzymology; D015759:Ionomycin / Q000494:pharmacology; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction; D013729:Terpenes / Q000494:pharmacology*; D019284:Thapsigargin; D013950:Thymus Gland / Q000166:cytology / Q000187:drug effects / Q000502:physiology*
D013729:Terpenes; D004533:Egtazic Acid; D015759:Ionomycin; D019284:Thapsigargin; D000252:Calcium-Transporting ATPases; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium