Ceramide 1-phosphate formation in neutrophils

In this study, we examined the metabolism of [H-3]N-hexanoylsphingosine [C-6-ceramide (Cer)] in neutrophils, erythrocytes, platelets and mononuclear cells. [H-3]C-6-Cer, exogenously added and incorporated into the inside of the cell, was found to be converted to other radioactive sphingolipids in these differentiated blood cells, except erythrocytes, which were unable to metabolize C6-Cer. Only in neutrophils was a specific radioactive band, comigrating with a standard C-6-Cer 1-phosphate (C-6-Cer-1-P) on thin layer chromatography, observed in a time-dependent manner. This metabolite was confirmed to be C6-Cer-1-P by its sensitivity to acid treatment and resistance to mild alkaline hydrolysis. Neutrophil [H-3]C-6-Cer conversion into [H-3]C-6-Cer-1-P, reflecting Cer kinase activity, was not affected by cell stimulation. Furthermore, extracellular release of [H-3]C-6-Cer-1-P was not observed either. Exogenous addition Of C-8-Cer-1-P failed to induce intracellular Ca2+ mobilization or affect the response induced by the formyl peptide. Furthermore, neutrophil morphology was not affected by C8-Cer-1-P. Although specific expression of Cer kinase suggests an important role for Cer-1-P in neutrophils, its functional role(s) remain to be clarified.