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Early transitory rise in intracellular pH leads to Bax conformation change during ceramide-induced apoptosis

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Abstract

Ceramide can induce apoptosis through a caspase independent pathway. Bax has been described as able to kill cells in the absence of caspase activity, therefore we measured Bax in situ during ceramide-induced apoptosis using anti-Bax antibodies and flow cytometry analysis. An early (<30 min) increase in Bax labeling was observed after the addition of several ceramide species to several hemopoietic-related cell types. On U937, this increase was not due to antigens synthesis or processing, but rather an increased accessibility or reactivity of Bax antigens for antibodies. This increased immuno-reactivity of Bax was not inhibited by Z-VAD-fmk nor leupeptin, and preceded nuclear fragmentation by several hours. Such an increase in immuno-reactivity was also observed after Fas ligation, but it occurred later (>2 h) accompanying nuclear apoptosis, and was inhibited by Z-VAD-fmk. Bax immuno-reactivity was found to be related to intracellular pH (pHi), and C2-Ceramide (C2-Cer) induced a very early (<10 min) transitory increase in pHi. Both Bax immuno-reactivity and pHi increases were dependent on the mitochondrial permeability transition pore (PTP) status. It was concluded from these results that C2-Cer induced a transitory increase in pHi in relation to the PTP. This rise in pHi led to conformational changes in Bax which could be responsible for further apoptosis in the C2-Cer pathway while it was a consequence of caspase activation in the Fas pathway.

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Authors and Affiliations

  1. Laboratoire d'Hématologie, Hôpital Haut-Léveque, Pessac, France;

    M.A. Belaud-Rotureau, L. Lacoste, F. Lacombe, P. Bernard & F. Belloc

  2. Laboratoire Universitaire d'Hématologie, Université Victor Segalen, Bordeaux, France

    M.A. Belaud-Rotureau, P. Bernard & F. Belloc

  3. CNRS-UMR 5536, Université Victor Segalen, Bordeaux, France

    N. Leducq, F. Macouillard Poulletier de Gannes & P. Diolez

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  1. M.A. Belaud-Rotureau
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  2. N. Leducq
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Belaud-Rotureau, M., Leducq, N., de Gannes, F.M.P. et al. Early transitory rise in intracellular pH leads to Bax conformation change during ceramide-induced apoptosis. Apoptosis 5, 551–560 (2000). https://doi.org/10.1023/A:1009693630664

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  • DOI: https://doi.org/10.1023/A:1009693630664

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