Levade T, Vidal F, Vermeersch S, Andrieu N, Gatt S, Salvayre R
Laboratoire de Biochimie, CJF INSERM 9206, Institut Louis Bugnard, C.H.U. Rangueil, Toulouse, France.
Biochim Biophys Acta. 1995 Oct 5;1258(3):277-87. doi: 10.1016/0005-2760(95)00132-v.
The aim of the present study was to investigate the role of the entitled neutral, sphingomyelinase in the non-lysosomal pathway of sphingomyelin 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 of acid, lysosomal and neutral sphingomyelinases as determined in vitro on cell homogenates by their respective standard assays. For this purpose, neuroblastoma, skin fibroblasts, lymphoid and leukemic cell lines, some of them derived from patients with Niemann-Pick disease (deficient in the acid, lysosomal sphingomyelinase) were incubated with radioactive, [oleoyl-3H]sphingomyelin or fluorescent, pyrene-sulfonylaminoundecanoyl-sphingomyelin. Either compound was taken up by a pathway which was not receptor-mediated and hydrolyzed by all intact cells, including those derived from Niemann-Pick disease patients. Moreover, their degradation by the intact cells was not inhibited by treatment with chloroquine, indicating hydrolysis by a non-lysosomal sphingomyelinase. The intracellular sphingomyelin degradation rates showed no correlation with the activity of the 'classical' neutral sphingomyelinase as determined in vitro. In particular, fibroblasts derived from Niemann-Pick patients lacking the lysosomal sphingomyelinase, and having no detectable 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 dithiothreitol-dependent sphingomyelinase activities, that might contribute to the non-lysosomal pathway for sphingomyelin degradation to ceramide in intact cells.
本研究的目的是调查上述中性鞘磷脂酶在完整细胞鞘磷脂降解的非溶酶体途径中的作用（Spence等人，（1983年）《生物化学杂志》258卷，8595 - 8600页；Levade等人，（1991年）《生物化学杂志》266卷，13519 - 13529页）。使用在体外通过各自标准测定法测定细胞匀浆中酸性、溶酶体和中性鞘磷脂酶活性水平范围广泛的细胞系，研究完整活细胞对鞘磷脂的摄取和降解。为此，将神经母细胞瘤、皮肤成纤维细胞、淋巴细胞和白血病细胞系，其中一些来自尼曼 - 匹克病患者（缺乏酸性、溶酶体鞘磷脂酶），与放射性的[油酰 - 3H]鞘磷脂或荧光的芘 - 磺酰氨基十一烷酰 - 鞘磷脂一起孵育。这两种化合物均通过非受体介导的途径被摄取，并被所有完整细胞水解，包括来自尼曼 - 匹克病患者的细胞。此外，用氯喹处理不会抑制完整细胞对它们的降解，表明是由非溶酶体鞘磷脂酶进行水解。细胞内鞘磷脂降解速率与体外测定的“经典”中性鞘磷脂酶活性无关。特别是，来自缺乏溶酶体鞘磷脂酶且体外未检测到“经典”中性鞘磷脂酶活性的尼曼 - 匹克病患者的成纤维细胞，能够降解外源提供的鞘磷脂。实际上，体外研究表明这些细胞表现出中性、依赖镁和二硫苏糖醇的鞘磷脂酶活性，这可能有助于完整细胞中鞘磷脂降解为神经酰胺的非溶酶体途径。
