Early mobilization of Ca2+ is not required for glucocorticoid-induced apoptosis in thymocytes.
Contradictory results have been reported on the question of the role of Ca2+ in glucocorticoid-induced apoptosis in thymocytes. To resolve this problem, we investigated the effect of dexamethasone, a synthetic glucocorticoid, on intracellular Ca2+ concentration ([Ca2+]i), by microscopic fluorometry that enables us to monitor real-time [Ca2+]i of cells loaded with fura-2, a fluorescent Ca2+ indicator, on a single cell basis. The results indicated that dexamethasone does not induce an increase in [Ca2+]i above control level both in murine and rat thymocytes at least for 1 h after the start of the culture. We also investigated whether the depletion of extracellular Ca2+ with EGTA or buffering intracellular Ca2+ with quin-2/AM inhibited glucocorticoid-induced apoptosis as reported on rat thymocytes. Dexamethasone-induced apoptosis in both murine and rat thymocytes, however, was not inhibited by EGTA. High concentrations (25 microM and over) of quin-2/AM inhibited DNA fragmentation, but failed to inhibit cytolysis. Calmodulin inhibitors, trifluoperazine and calmidazolium, also inhibited DNA fragmentation as reported, although they markedly enhanced cytolysis. Therefore, glucocorticoid-induced death is not inhibited by quin-2/AM or calmodulin inhibitors. Furthermore, we have previously found that a proper combination of the calcium ionophore, ionomycin, and the protein kinase activator, PMA, inhibits corticosterone-induced apoptosis. These results suggest that an early increase in [Ca2+]i is neither induced by glucocorticoids nor responsible for glucocorticoid-induced apoptosis in thymocytes.
Iseki
R
R
Department of Cellular Recognition Research, Mitsubishi Kasei Institute of Life Sciences, Tokyo, Japan.
Kudo
Y
Y
Iwata
M
M
D000634:Aminoquinolines / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000037:antagonists & inhibitors; D004247:DNA / Q000378:metabolism; D003907:Dexamethasone / Q000494:pharmacology*; D007093:Imidazoles / Q000494:pharmacology; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D051381:Rats; D017207:Rats, Sprague-Dawley; D017260:Receptor-CD3 Complex, Antigen, T-Cell / Q000502:physiology; D013601:T-Lymphocytes / Q000187:drug effects* / Q000378:metabolism
D000634:Aminoquinolines; D002147:Calmodulin; D007093:Imidazoles; D017260:Receptor-CD3 Complex, Antigen, T-Cell; C031938:calmidazolium; D003907:Dexamethasone; D004247:DNA; C035739:Quin2; D002118:Calcium