title: Early transitory rise in intracellular pH leads to Bax conformation change during ceramide-induced apoptosis.
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 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.
authors: Laboratoire d'Hématologie, H pital Haut-Léveque, Pessac, France.
authors: M A
authors: MA
authors: Belaud-Rotureau
authors: N
authors: N
authors: Leducq
authors: F
authors: F
authors: Macouillard Poulletier de Gannes
authors: P
authors: P
authors: Diolez
authors: L
authors: L
authors: Lacoste
authors: F
authors: F
authors: Lacombe
authors: P
authors: P
authors: Bernard
authors: F
authors: F
authors: Belloc
mesh_terms: D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D015153:Blotting, Western; D002478:Cells, Cultured / Q000187:drug effects* / Q000378:metabolism; D002518:Ceramides / Q000378:metabolism / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology; D006801:Humans; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D007424:Intracellular Fluid / Q000187:drug effects* / Q000378:metabolism; D007425:Intracellular Membranes / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D017434:Protein Structure, Tertiary / Q000187:drug effects / Q000502:physiology; D011518:Proto-Oncogene Proteins / Q000187:drug effects* / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein
chemical_list: C494826:BAX protein, human; D002518:Ceramides; D004791:Enzyme Inhibitors; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein