Roarke M C, Wagner D D, Marder V J, Sporn L A
Department of Medicine, University of Rochester School of Medicine and Dentistry, NY.
Eur J Cell Biol. 1989 Apr;48(2):337-43.
The effect of reduced temperature on the post-translational processing and stimulated release of von Willebrand factor (vWf) from human umbilical vein endothelial cells was studied. Following pulse-labeling, cells were incubated for 4 h at 18 degrees C or 20 degrees C. Post-translational processing was reversibly arrested at the dimer stage, dimers were composed of Endo H-sensitive precursor subunits, and no vWf was detected in the culture medium. This block was reversible, since warming cells to 37 degrees C relieved it and resulted in the appearance of fully processed vWf in the cells and the culture medium. The same results were obtained when cells were incubated with carbonyl cyanide m-chlorophenol hydrazone or dinitrophenol which inhibit mitochondrial oxidative phosphorylation, known to block exit of secretory proteins from the endoplasmic reticulum (ER). This indicated that ER exit is not required for the complete dimerization of vWf. Reduced temperature (18 degrees C and 20 degrees C) also reversibly and nearly completely inhibited the secretagogue-induced release of vWf from Weibel-Palade bodies without affecting the microtubular cytoskeleton. We add reduced temperature to the list of useful tools for the study of the vWf secretory pathway in endothelial cells.
研究了降低温度对人脐静脉内皮细胞中血管性血友病因子（vWf）的翻译后加工及刺激释放的影响。脉冲标记后，将细胞在18℃或20℃下孵育4小时。翻译后加工在二聚体阶段可逆地停滞，二聚体由内切糖苷酶H敏感的前体亚基组成，并且在培养基中未检测到vWf。这种阻滞是可逆的，因为将细胞升温至37℃可解除阻滞，并导致细胞和培养基中出现完全加工的vWf。当细胞与羰基氰化物间氯苯腙或二硝基苯酚一起孵育时也得到相同结果，这两种物质抑制线粒体氧化磷酸化，已知其可阻断分泌蛋白从内质网（ER）的输出。这表明vWf的完全二聚化不需要从ER输出。降低温度（18℃和20℃）还可逆且几乎完全抑制了促分泌剂诱导的vWf从魏贝尔-帕拉德小体的释放，而不影响微管细胞骨架。我们将降低温度添加到研究内皮细胞中vWf分泌途径的有用工具列表中。
