title: Apically and basolaterally internalized aminoglycosides colocalize in LLC-PK1 lysosomes and alter cell function.
abstract: 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.
authors.affiliation: Department of Pediatrics, University of Colorado Health Sciences Center, Children's Hospital, Denver 80218.
authors.forename: D M
authors.initials: DM
authors.lastname: Ford
authors.forename: R H
authors.initials: RH
authors.lastname: Dahl
authors.forename: C A
authors.initials: CA
authors.lastname: Lamp
authors.forename: B A
authors.initials: BA
authors.lastname: Molitoris
mesh_terms: D000617:Aminoglycosides / Q000378:metabolism*; D000818:Animals; D001485:Basement Membrane / Q000378:metabolism*; D001692:Biological Transport / Q000187:drug effects; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D005293:Ferritins / Q000493:pharmacokinetics; D005839:Gentamicins / Q000493:pharmacokinetics* / Q000494:pharmacology; D007150:Immunohistochemistry; D007425:Intracellular Membranes / Q000378:metabolism*; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000378:metabolism* / Q000502:physiology; D008247:Lysosomes / Q000378:metabolism*; D008353:Mannitol / Q000493:pharmacokinetics; D014018:Tissue Distribution; D014031:Tobramycin / Q000494:pharmacology
chemical_list: D000617:Aminoglycosides; D005839:Gentamicins; C010297:polycationic ferritin; D008353:Mannitol; D005293:Ferritins; D014031:Tobramycin