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

• Published in: Apoptosis (London) Vol. 5; no. 6; pp. 551 - 560
• Main Authors: Belaud-Rotureau, M A, Leducq, N, Macouillard Poulletier de Gannes, F, Diolez, P, Lacoste, L, Lacombe, F, Bernard, P, Belloc, F
• Format: Journal Article
• Language: English
• Published: Netherlands Springer Nature B.V 01-12-2000
• Subjects: Apoptosis; Apoptosis - drug effects; Apoptosis - physiology; bcl-2-Associated X Protein; Blotting, Western; Cells; Cells, Cultured - drug effects; Cells, Cultured - metabolism; Cellular biology; Ceramides - metabolism; Ceramides - pharmacology; Enzyme Inhibitors - pharmacology; Gene Expression Regulation - drug effects; Gene Expression Regulation - physiology; Humans; Hydrogen-Ion Concentration - drug effects; Intracellular Fluid - drug effects; Intracellular Fluid - metabolism; Intracellular Membranes - drug effects; Intracellular Membranes - metabolism; Intracellular Membranes - ultrastructure; Mitochondria - drug effects; Mitochondria - metabolism; Mitochondria - ultrastructure; Protein Structure, Tertiary - drug effects; Protein Structure, Tertiary - physiology; Proto-Oncogene Proteins - drug effects; Proto-Oncogene Proteins - metabolism; Proto-Oncogene Proteins c-bcl-2
• ISSN: 1360-8185; 1573-675X
• DOI: 10.1023/A:1009693630664

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 immunoreactivity 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.