Understanding the mechanism of apoptosis is necessary to improve the use of stem cells for the treatment of neurodegenerative disorders. In HN9 embryonic hippocampal cells, serum deprivation induces apoptosis preceded by sphingomyelinase (SMase) activation and raise of ceramide levels. It is not known so far whether the SMase degrades all the species of sphingomyelins (SMs) in the same way and if it is the only enzyme that uses SM to produce ceramide. In order to begin to address this aspect still unclear about the relationship between SM and HN9.10 apoptosis, we carried out the analysis of the main species of SM and phosphatidylcholine (PC) with the idea that SM could be partly used as a source of phosphorylcholine to synthesize PC. To better investigate this possibility we measured the enzymatic activity of the reverse-SM synthase. In addition, since the ceramides involved in starvation-induced cell death are unknown, we have studied the content of ceramide and sphingosine-1-phosphate species as metabolites of SM produced by both SMase and reverse-SMsynthase. Cells have been incubated with 10% (control) or 0.2% fetal calf serum for 48 h (deprived). Lipid species has been separated and measured by ultraperformance liquid chromatography tandem mass spectrometry. Reverse-SMsynthase activity was assayed by using 14C labelled SM. The results show that serum deprivation reduces total SM and in particular 18:1/16:0 SM whereas it increases total PC and in particular 18:1/18:0 PC. There is a 2-fold increase of ceramides 18:1/ 16:0 and 18:1/ 24:0 and of sphingosine-1-phoshate 18:1. Following serum deprivation, the activity of Reverse-SM synthase remains constant for 8 h, increases afterwards up to 3.82 –fold compared to control at 48h. In conclusion, SMase activation might lead to decrease of SM 18:1/16:0, increase in ceramide 18:1/16:0 which can originate sphingosine-1-phoshate 18:1. In addition SM could be a source for PC synthesis with fatty acid rearrangement. Individual ceramide species regulated by specific pathways in distinct subcellular compartments might execute distinct cellular functions
SERUM DEPRIVATION-DEPENDENT MODULATION OF SPHINGOMYELIN SPECIES IN HN9.10 EMBRYONIC HIPPOCAMPAL CELLS.
GARCIA GIL, MARIA de las MERCEDES;
2013-01-01
Abstract
Understanding the mechanism of apoptosis is necessary to improve the use of stem cells for the treatment of neurodegenerative disorders. In HN9 embryonic hippocampal cells, serum deprivation induces apoptosis preceded by sphingomyelinase (SMase) activation and raise of ceramide levels. It is not known so far whether the SMase degrades all the species of sphingomyelins (SMs) in the same way and if it is the only enzyme that uses SM to produce ceramide. In order to begin to address this aspect still unclear about the relationship between SM and HN9.10 apoptosis, we carried out the analysis of the main species of SM and phosphatidylcholine (PC) with the idea that SM could be partly used as a source of phosphorylcholine to synthesize PC. To better investigate this possibility we measured the enzymatic activity of the reverse-SM synthase. In addition, since the ceramides involved in starvation-induced cell death are unknown, we have studied the content of ceramide and sphingosine-1-phosphate species as metabolites of SM produced by both SMase and reverse-SMsynthase. Cells have been incubated with 10% (control) or 0.2% fetal calf serum for 48 h (deprived). Lipid species has been separated and measured by ultraperformance liquid chromatography tandem mass spectrometry. Reverse-SMsynthase activity was assayed by using 14C labelled SM. The results show that serum deprivation reduces total SM and in particular 18:1/16:0 SM whereas it increases total PC and in particular 18:1/18:0 PC. There is a 2-fold increase of ceramides 18:1/ 16:0 and 18:1/ 24:0 and of sphingosine-1-phoshate 18:1. Following serum deprivation, the activity of Reverse-SM synthase remains constant for 8 h, increases afterwards up to 3.82 –fold compared to control at 48h. In conclusion, SMase activation might lead to decrease of SM 18:1/16:0, increase in ceramide 18:1/16:0 which can originate sphingosine-1-phoshate 18:1. In addition SM could be a source for PC synthesis with fatty acid rearrangement. Individual ceramide species regulated by specific pathways in distinct subcellular compartments might execute distinct cellular functionsI documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
