DEGRADATION OF FLUORESCENT AND RADIOLABELED SPHINGOMYELINS IN INTACT-CELLS BY A NONLYSOSOMAL PATHWAY

The aim of the present study was to investigate the role of the entitl ed neutral, sphingomyelinase in the non-lysosomal pathway of sphingomy elin degradation by intact cells (Spence et al. (1983) J. Biol. Chem. 258, 8595-8600; Levade et al, (1991) J. Biol Chem. 266, 13519-13529). The uptake and degradation of sphingomyelin by intact living cells was studied using cell lines exhibiting a wide range of activity levels o f acid, lysosomal and neutral sphingomyelinases as determined in vitro on cell homogenates by their respective standard assays. For this pur pose, neuroblastoma, skin fibroblasts, lymphoid and leukemic cell line s, some of them derived from patients with Niemann-Pick disease (defic ient in the acid, lysosomal sphingomyelinase) were incubated with radi oactive, [oleoyl-H-3]sphingomyelin or fluorescent, pyrene-sulfonylamin oundecanoyl-sphingomyelin. Either compound was taken up by a pathway w hich was not receptor-mediated and hydrolyzed by all intact cells, inc luding those derived from Niemann-Pick disease patients. Moreover, the ir degradation by the intact cells was not inhibited by treatment with chloroquine, indicating hydrolysis by a non-lysosomal sphingomyelinas e. The intracellular sphingomyelin degradation rates showed no correla tion with the activity of the 'classical' neutral sphingomyelinase as determined in vitro. In particular, fibroblasts derived from Niemann-P ick patients lacking the lysosomal sphingomyelinase, and having no det ectable in vitro activity of the 'classical' neutral sphingomyelinase, were able to degrade the exogenously supplied sphingomyelins. Indeed, in vitro these cells were shown to exhibit neutral, magnesium- and di thiothreitol-dependent sphingomyelinase activities, that, might contri bute to the non-lysosomal pathway for sphingomyelin degradation to cer amide in intact cells.