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

Ceramide can induce apoptosis through a caspase independent pathway. Bax ha s been described as able to kill cells in the absence of caspase activity, therefore we measured Bax in situ during ceramide-induced apoptosis using a nti-Bax antibodies and flow cytometry analysis. An early (< 30 min) increas e in Bax labeling was observed after the addition of several ceramide speci es to several hemopoietic-related cell types. On U937, this increase was no t due to antigens synthesis or processing, but rather an increased accessib ility or reactivity of Bax antigens for antibodies. This increased immuno-r eactivity of Bax was not inhibited by Z-VAD-fmk nor leupeptin, and preceded nuclear fragmentation by several hours. Such an increase in immuno-reactiv ity was also observed after Fas ligation, but it occurred later (>2 h) acco mpanying nuclear apoptosis, and was inhibited by Z-VAD-fmk. Bax immuno-reac tivity 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 Ba x immuno-reactivity and pHi increases were dependent on the mitochondrial p ermeability transition pore (PTP) status. It was concluded from these resul ts 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 respo nsible for further apoptosis in the C2-Cer pathway while it was a consequen ce of caspase activation in the Fas pathway.