Figure 7

Effects of oxPL on aSMase activity and aSMase-mediated apoptosis. Panel A: RAW264.7 cells were incubated with DMEM containing 25 μM of lipid or 1% v/v EtOH (control) for 5 or 15 minutes. Cells were harvested and lysed and sphingomyelinase activity was determined as described under materials and methods. Results are shown as relative values (folds of controls). The absolute enzyme activities of the control cells in the individual experimental series ranged from 4 to 8 pmol/min/μg protein. POVPC and E-POVPC increase aSMase activity within 5 minutes. PGPC and E-PGPE had a slightly inhibitory effect on enzyme activity as compared to the control. Panel B: RAW264.7 cells were incubated with 50 μM oxPLs in media under low serum conditions (0,1% FCS) for 4 h and analyzed by flow cytometry. The fraction of apoptotic cells was determined from Annexin V staining of externalized phosphatidylserine. Control cells were incubated with 1% v/v EtOH with or without NB19 in the incubation media. Inhibition of aSMase with NB19 (10 nmol/ml) leads to significant decrease in apoptosis induced by POVPC and PGPC. NB19 alone had no significant effect on cell viability. Results are means of 4 replicates out of 3 experiments. Data are expressed as means +/− SD relative to the control. Significance was determined by Student’s t-test (two tailed, unpaired). * p ≤ 0,05, **p ≤ 0,01.