Apically and basolaterally internalized aminoglycosides colocalize in LLC- PK1 lysosomes and alter cell function

D. M. Ford, R. H. Dahl, C. A. Lamp, Bruce Molitoris

Research output: Contribution to journalArticle

23 Citations (Scopus)

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.

Original languageEnglish (US)
JournalAmerican Journal of Physiology - Cell Physiology
Volume266
Issue number1 35-1
StatePublished - 1994
Externally publishedYes

Fingerprint

Aminoglycosides
Lysosomes
Gentamicins
Membranes
LLC-PK1 Cells
Acetylglucosaminidase
Proteins
Tobramycin
Ferritins
Endocytosis
Epithelial Cells
Immunohistochemistry
Kidney
Processing

Keywords

  • endocytosis
  • gentamicin
  • immunocytochemistry
  • lysosomes
  • N- acetylglucosaminidase

ASJC Scopus subject areas

  • Cell Biology
  • Clinical Biochemistry
  • Physiology

Cite this

Apically and basolaterally internalized aminoglycosides colocalize in LLC- PK1 lysosomes and alter cell function. / Ford, D. M.; Dahl, R. H.; Lamp, C. A.; Molitoris, Bruce.

In: American Journal of Physiology - Cell Physiology, Vol. 266, No. 1 35-1, 1994.

Research output: Contribution to journalArticle

@article{30d251a9638747d7aa492cfb7bebcb0a,
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.",
keywords = "endocytosis, gentamicin, immunocytochemistry, lysosomes, N- acetylglucosaminidase",
author = "Ford, {D. M.} and Dahl, {R. H.} and Lamp, {C. A.} and Bruce Molitoris",
year = "1994",
language = "English (US)",
volume = "266",
journal = "American Journal of Physiology",
issn = "0193-1857",
publisher = "American Physiological Society",
number = "1 35-1",

}

TY - JOUR

T1 - Apically and basolaterally internalized aminoglycosides colocalize in LLC- PK1 lysosomes and alter cell function

AU - Ford, D. M.

AU - Dahl, R. H.

AU - Lamp, C. A.

AU - Molitoris, Bruce

PY - 1994

Y1 - 1994

N2 - 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.

AB - 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.

KW - endocytosis

KW - gentamicin

KW - immunocytochemistry

KW - lysosomes

KW - N- acetylglucosaminidase

UR - http://www.scopus.com/inward/record.url?scp=0028014526&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0028014526&partnerID=8YFLogxK

M3 - Article

C2 - 8304430

AN - SCOPUS:0028014526

VL - 266

JO - American Journal of Physiology

JF - American Journal of Physiology

SN - 0193-1857

IS - 1 35-1

ER -