Oda T, Chen C H, Wu H C
Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814-4799.
J Biol Chem. 1995 Feb 24;270(8):4088-92. doi: 10.1074/jbc.270.8.4088.
We have found that C6 ceramide, a cell-permeable ceramide analog, partially restored the brefeldin A (BFA) sensitivity in a BFA-resistant mutant of Vero cells (BER-40) and in the naturally BFA-resistant Madin-Darby canine kidney (MDCK) cells. Incubation of BER-40 and MDCK cells with low concentrations of C6 ceramide resulted in (i) a pronounced increase in BFA cytotoxicity as measured by the inhibition of [3H]thymidine incorporation and the inhibition of colony formation by BFA, (ii) a significant protection by BFA against ricin cytotoxicity, and (iii) an inhibition of bulk protein secretion by BFA in BER-40 and MDCK cells. Related sphingolipids including sphingosine, sphingomyelin, and lactosylceramide and other unrelated lipid second messengers such as arachidonic acid and 1,2-diacylglycerol did not elicit the protection of BER-40 and MDCK cells against ricin cytotoxicity by BFA. C6 ceramide was the most effective among the ceramides with different acyl chain lengths. Interestingly, dihydro-C6 ceramide, which lacks the trans double bond in the sphingoid base, had no effect. On the other hand, C6 ceramide did not enhance BFA sensitivity in BFA-sensitive Vero cells. The LD50 of C6 ceramide were similar in Vero and BER-40 cells. Fluorescence microscopic studies revealed that C6 ceramide induced the redistribution of beta-COP from the Golgi membranes to a more dispersed localization in both BFA-sensitive and BFA-resistant cell lines, mimicking the effect of BFA. Suboptimal concentration of C6 ceramide also restored the effect of BFA on the beta-COP distribution in BER-40 and MDCK cells. These results indicate that C6 ceramide restores the BFA sensitivity in BFA-resistant BER-40 and MDCK cells.
我们发现，C6神经酰胺（一种可穿透细胞的神经酰胺类似物）可部分恢复Vero细胞的BFA抗性突变体（BER - 40）以及天然具有BFA抗性的Madin - Darby犬肾（MDCK）细胞对布雷菲德菌素A（BFA）的敏感性。用低浓度的C6神经酰胺孵育BER - 40和MDCK细胞导致：（i）通过抑制[3H]胸苷掺入和BFA对集落形成的抑制作用来衡量，BFA细胞毒性显著增加；（ii）BFA对蓖麻毒素细胞毒性有显著保护作用；（iii）BFA抑制BER - 40和MDCK细胞中的大量蛋白质分泌。包括鞘氨醇、鞘磷脂和乳糖神经酰胺在内的相关鞘脂以及其他不相关的脂质第二信使如花生四烯酸和1,2 - 二酰甘油，均未引发BFA对BER - 40和MDCK细胞蓖麻毒素细胞毒性的保护作用。在具有不同酰基链长度的神经酰胺中，C6神经酰胺最为有效。有趣的是，在鞘氨醇碱基中缺乏反式双键的二氢C6神经酰胺没有效果。另一方面，C6神经酰胺并未增强对BFA敏感的Vero细胞对BFA的敏感性。C6神经酰胺在Vero细胞和BER - 40细胞中的半数致死剂量相似。荧光显微镜研究表明，C6神经酰胺诱导β - COP从高尔基体膜重新分布到两种对BFA敏感和抗性的细胞系中更分散的定位，模拟了BFA的作用。次优浓度的C6神经酰胺也恢复了BFA对BER - 40和MDCK细胞中β - COP分布的影响。这些结果表明，C6神经酰胺恢复了对BFA抗性的BER - 40和MDCK细胞对BFA的敏感性。
