Gentamicin traffics rapidly and directly to the Golgi complex in LLC-PK(1) cells

To study the intracellular mechanisms of aminoglycoside toxicity, we used a 1:1 fluorescent conjugate of Texas Red and gentamicin (TRG) to quantify early uptake dynamics in renal epithelial (LLC-PK(1)) cells. Utilizing a protocol that quenches TRG fluorescence from lysosomes, the bulk of intracellul...

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Bibliographic Details
Published in:American journal of physiology. Renal physiology 2000-11, Vol.279 (5), p.F884
Main Authors: Sandoval, R M, Dunn, K W, Molitoris, B A
Format: Article
Language:English
Subjects:
Animals
Anti-Bacterial Agents - chemistry
Anti-Bacterial Agents - pharmacokinetics
Biological Transport
Epithelial Cells - cytology
Epithelial Cells - metabolism
Fluorescent Dyes - chemistry
Gentamicins - chemistry
Gentamicins - pharmacokinetics
Golgi Apparatus - metabolism
Golgi Apparatus - ultrastructure
Kidney - cytology
Kidney - metabolism
LLC-PK1 Cells
Swine
Xanthenes - chemistry
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Summary:To study the intracellular mechanisms of aminoglycoside toxicity, we used a 1:1 fluorescent conjugate of Texas Red and gentamicin (TRG) to quantify early uptake dynamics in renal epithelial (LLC-PK(1)) cells. Utilizing a protocol that quenches TRG fluorescence from lysosomes, the bulk of intracellular accumulation, we determined a portion rapidly trafficked directly to the Golgi complex when identified by a FITC-conjugated lectin from Lens culinaris agglutinin (LCA). A kinetic study over 120 min on cells showing total and quenched TRG fluorescence was then carried out, and the fluorescence intensity from the images was quantified. Trafficking of TRG to the Golgi complex occurred within 15 min and accounted for approximately 20% of total cellular accumulation in the kinetic study. Colocalization studies using compartment-specific markers, 6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]hexanoyl sphingosine (C6-NBD ceramide) and LCA, for the TGN trans-Golgi network, and the cis/medial-Golgi compartments, respectively, determined colocalization occurred with both Golgi compartments. These data support the existence of a pathway that directly and rapidly shuttles a portion of internalized gentamicin to the Golgi complex. We believe this pathway may be responsible for the early negative effects seen on protein synthesis in renal proximal epithelia after aminoglycoside administration.
ISSN:1931-857X