Ford D M, Dahl R H, Lamp C A, Molitoris B A
Department of Pediatrics, University of Colorado Health Sciences Center, Children's Hospital, Denver 80218.
Am J Physiol. 1994 Jan;266(1 Pt 1):C52-7. doi: 10.1152/ajpcell.1994.266.1.C52.
Aminoglycosides bind to apical and basolateral (BL) membranes of renal epithelial cells. However, little is known regarding differential uptake and intracellular processing after internalization across these distinct surface membrane domains. To examine these processes independently, LLC-PK1 cells were grown on porous filters, which allow selective access to both domains. Apical and BL membrane uptakes of gentamicin (0.5 mM), quantified using [3H]gentamicin, were linear from 2 to 24 h (r = 0.99). The 4-h apical gentamicin uptake was 667 +/- 59 pmol/mg protein, the BL 748 +/- 26 pmol/mg protein, and concurrent apical and BL uptake 1,389 +/- 22 pmol/mg protein. Aminoglycoside uptake, documented using indirect immunogold techniques, occurred via the apical and BL endocytic systems and colocalized with cationic ferritin. Aminoglycosides internalized via the apical (gentamicin) and BL (tobramycin) membrane converged at the lysosomal level. Gentamicin incorporated via either domain significantly decreased lysosomal N-acetylglucosaminidase below control values (P < 0.05). We conclude that, after binding, aminoglycosides are internalized equally across apical and BL membranes of LLC-PK1 cells via receptor-mediated endocytosis, colocalize within the lysosomal compartment, and alter cellular function similarly.
氨基糖苷类药物可与肾上皮细胞的顶端膜和基底外侧（BL）膜结合。然而，关于其通过这些不同的表面膜结构域内化后的差异摄取和细胞内加工过程，我们了解甚少。为了独立研究这些过程，将LLC-PK1细胞培养在多孔滤器上，该滤器可选择性地接触这两个结构域。使用[3H]庆大霉素对庆大霉素（0.5 mM）的顶端和BL膜摄取进行定量，结果显示在2至24小时内呈线性（r = 0.99）。4小时时，顶端庆大霉素摄取量为667±59 pmol/mg蛋白质，BL摄取量为748±26 pmol/mg蛋白质，同时进行顶端和BL摄取时摄取量为1,389±22 pmol/mg蛋白质。使用间接免疫金技术记录的氨基糖苷类药物摄取是通过顶端和BL内吞系统发生的，并与阳离子铁蛋白共定位。通过顶端（庆大霉素）和BL（妥布霉素）膜内化的氨基糖苷类药物在溶酶体水平汇聚。通过任一结构域掺入的庆大霉素均使溶酶体N-乙酰葡糖胺酶显著低于对照值（P < 0.05）。我们得出结论，结合后，氨基糖苷类药物通过受体介导的内吞作用在LLC-PK1细胞的顶端和BL膜上以相同方式内化，在溶酶体区室中共定位，并同样改变细胞功能。
