Yamamoto A, Tagawa Y, Yoshimori T, Moriyama Y, Masaki R, Tashiro Y
Department of Physiology and Liver Research Center (Cell Biology), Kansai Medical University, Osaka, Japan.
Cell Struct Funct. 1998 Feb;23(1):33-42. doi: 10.1247/csf.23.33.
We studied the effects of bafilomycin A1, a potent and specific inhibitor of vacuolar H+ ATPase (V-ATPase), on the process of autophagy in rat hepatoma cell line, H-4-II-E cells. To induce autophagy, cells were transferred from Dulbecco's modified Eagle medium containing 12% fetal calf serum into Hanks' balanced salt solution. When bafilomycin A1 was added to Hanks' balanced salt solution, endogenous protein degradation was strongly inhibited and numerous autophagosomes accumulated in H-4-II-E cells, whereas autolysosomes decreased in number. Acid phosphatase activity was not detected in the autophagosomes which accumulated in the presence of bafilomycin A1, suggesting that fusion between autophagosomes and lysosomes was disturbed by this drug. Inhibition of the fusion was reversible, and the autophagosomes changed into autolysosomes after the removal of the inhibitor. Bafilomycin A1 also prevented the appearance of endocytosed HRP in autophagic vacuoles. These results suggested that acidification of the lumenal space of autophagosomes or lysosomes by V-ATPase is important for the fusion between autophagosomes and lysosomes.
我们研究了液泡H⁺-ATP酶（V-ATP酶）的强效特异性抑制剂巴弗洛霉素A1对大鼠肝癌细胞系H-4-II-E细胞自噬过程的影响。为诱导自噬，将细胞从含12%胎牛血清的杜氏改良 Eagle培养基转移至汉克斯平衡盐溶液中。当向汉克斯平衡盐溶液中添加巴弗洛霉素A1时，内源性蛋白质降解受到强烈抑制，H-4-II-E细胞中积累了大量自噬体，而自溶酶体数量减少。在巴弗洛霉素A1存在的情况下积累的自噬体中未检测到酸性磷酸酶活性，这表明该药物干扰了自噬体与溶酶体之间的融合。融合抑制是可逆的，去除抑制剂后自噬体转变为自溶酶体。巴弗洛霉素A1还阻止了自噬泡中内吞的辣根过氧化物酶的出现。这些结果表明，V-ATP酶使自噬体或溶酶体腔酸化对于自噬体与溶酶体之间的融合很重要。
