caa a22 4500 397557868 CHVBK 20180308164808.0 cr unu---uuuuu 161202e199605 xx s 000 0 eng 10.1093/oxfordjournals.jbchem.a021326 doi (NATIONALLICENCE)oxford-10.1093/oxfordjournals.jbchem.a021326 Wortmannin and Li+ Specifically Inhibit Clathrin-Independent Endocytic Internalization of Bulk Fluid [Elektronische Daten] [Satoshi B. Sato, Takahisa Taguchi, Shohei Yamashina, Sakuji Toyama] Incubation of a human fibrosarcoma cell line HT-1080 in Li+-containing medium inhibited internalization of a fluid marker, horseradish peroxidase (HRP), by more than 80percnt;. The ion inhibited the activity enhanced by Ca2+ or phorbol 12-myristate 13-acetate. We also found that wortmannin (WT), a potent inhibitor of phosphoinositide (PI) 3-kinase (PI 3-k), inhibited the non-stimulated and the two stimulated types of endocytosis to the same extent as Li+. In contrast, neither WT nor Li+ influenced the early internalization of transferrin (Tfn), EGF or platelet-derived growth factor. Neither targeting to early endosomes nor recycling of the once-internalized Tfn was influenced. When the cytoplasmic pH was lowered by chasing cells that had been preincubated with 25 mM NH4C1 in an amiloride-containing Na+-free medium, more than 90% of internalization of Tfn in HT-1080 cells was inhibited, while that of HBP was reduced by only 34%. In contrast, WT reduced the uptake of HBP by KB cells by 34%, while 60% of the activity was inhibited by the treatment for cytoplasmic acidification. Comparison of other cells i.e., A-549 and a human diploid cell line Miyajima, indicated that cells showing higher sensitivity to WT were less sensitive to low cytoplasmic pH. These results suggest that, in all the cells studied, bulk fluid is internalized either via a clathrin-independent/PI 3-k-dependent route or via a clathrin-dependent/PI 3-k-independent one, though the ratio varied among them. We also found that internalization of a mAb directed toward the 116 (100)-kDa subunit of vacuolar ATPase [0SW2; Sato and Toyama (1994) J. Cell BioL 127, 39-53] in the fluid phase was inhibited by WT, but the antibody was still internalized in a surface-bound form. Regardless of the treatment with WT, most of the antibody was transported to endosomes that were associated with Tfn receptor. These results suggest that both internalization routes are targeted to the same early endosomal compartments. © by the Journal of Biochemistry Regular Papers nationallicence endocytosis nationallicence growth factor internalization nationallicence PI-3k nationallicence Pi-turnover nationallicence transferrin nationallicence Sato Satoshi B. Cell and Information, PRESTO, Research Development Corporation of Japan and Department of Biophysics, Faculty of Science, Kyoto University Kyoto 606-01 aut Taguchi Takahisa Department of Organic Materials, Osaka National Research Institute Ikeda, Osaka 563 aut Yamashina Shohei Department of Anatomy, Kitasato University School of Medicine KUasato, Sagamihara, Kanagawa 228 aut Toyama Sakuji Institute for Virus Research, Kyoto University Kyoto 606-01 aut The Journal of Biochemistry Oxford University Press 119/5(1996-05), 887-897 0021-924X 119:5<887 1996 119 jbchem https://doi.org/10.1093/oxfordjournals.jbchem.a021326 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1093/oxfordjournals.jbchem.a021326 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Sato Satoshi B. Cell and Information, PRESTO, Research Development Corporation of Japan and Department of Biophysics, Faculty of Science, Kyoto University Kyoto 606-01 aut NATIONALLICENCE 700 1- Taguchi Takahisa Department of Organic Materials, Osaka National Research Institute Ikeda, Osaka 563 aut NATIONALLICENCE 700 1- Yamashina Shohei Department of Anatomy, Kitasato University School of Medicine KUasato, Sagamihara, Kanagawa 228 aut NATIONALLICENCE 700 1- Toyama Sakuji Institute for Virus Research, Kyoto University Kyoto 606-01 aut NATIONALLICENCE 773 0- The Journal of Biochemistry Oxford University Press 119/5(1996-05), 887-897 0021-924X 119:5<887 1996 119 jbchem Metadata rights reserved CC BY-NC-4.0 http://creativecommons.org/licenses/by-nc/4.0 nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-oxford