Kendler A, Dawson G
Department of Pediatrics, Joseph P. Kennedy Jr. Mental Retardation Research Center, University of Chicago, Illinois.
J Neurosci Res. 1992 Feb;31(2):205-11. doi: 10.1002/jnr.490310202.
Previous studies have shown that gradual progressive hypoxia specifically inhibits the synthesis of the major myelin lipid galactosylceramide (GalCer) in cultured neonatal rat oligodendrocytes (OLG) (Kendler and Dawson, J Biol Chem 265:12259-12266, 1990). The inhibition of de novo synthesized GalCer (measured by [3H]palmitate incorporation) was accompanied by an increase in the [3H]labeled pool of nonhydroxy fatty acid ceramide, the precursor of GalCer. The decreased galactosylation of NFACer was not due to an inhibition of UDP-Gal:ceramide:galactosyltransferase activity or to a depletion in available UDP-Gal. Analysis of subcellular fractionations of OLG membranes on Percoll gradients indicated that NFA ceramide was accumulating in the endoplasmic reticulum (ER) during hypoxia, suggesting that the transport of NFACer from its site of synthesis (ER) to its site of galactosylation, presumably the Golgi, was blocked by hypoxia. This accumulation of ceramide was replicated by lowering ATP levels to 80-90% of control by treating OLG with 12 nM oligomycin, and was reversed by reoxygenation of the cells. Conversion of [3H]palmitate-labeled NFACer to GalCer in semi-intact OLG required both exogenous UDP-Gal and ATP, further suggesting that the transport of NFACer from the ER to its site of synthesis (cis-Golgi) is an energy-dependent step that is highly susceptible to relatively minor ATP depletion associated with early hypoxic injury. Our results further suggest that ceramide appears to be a good marker for ER and GalCer is a good marker for the cis-Golgi.
先前的研究表明，逐渐进行性缺氧会特异性抑制培养的新生大鼠少突胶质细胞（OLG）中主要髓磷脂脂质半乳糖基神经酰胺（GalCer）的合成（肯德勒和道森，《生物化学杂志》265:12259 - 12266，1990年）。从头合成的GalCer受到抑制（通过[3H]棕榈酸掺入量来衡量）的同时，GalCer的前体非羟基脂肪酸神经酰胺的[3H]标记池增加。非羟基脂肪酸神经酰胺（NFACer）半乳糖基化的减少并非由于UDP - Gal：神经酰胺：半乳糖基转移酶活性受到抑制，也不是由于可用UDP - Gal的耗竭。对OLG膜在Percoll梯度上进行亚细胞分级分离分析表明，缺氧期间NFACer在内质网（ER）中积累，这表明NFACer从其合成位点（ER）运输到其半乳糖基化位点（大概是高尔基体）被缺氧阻断。通过用12 nM寡霉素处理OLG将ATP水平降至对照的80 - 90%，可复制这种神经酰胺的积累，并且细胞再给氧后这种积累会逆转。在半完整的OLG中，[3H]棕榈酸标记的NFACer转化为GalCer既需要外源性UDP - Gal也需要ATP，这进一步表明NFACer从ER运输到其合成位点（顺式高尔基体）是一个能量依赖步骤，对与早期缺氧损伤相关的相对较小的ATP消耗高度敏感。我们的结果进一步表明，神经酰胺似乎是内质网的良好标志物，而GalCer是顺式高尔基体的良好标志物。
