Gentamicin traffics rapidly and directly to the Golgi complex in LLC-PK1 cells

To study the intracellular mechanisms of aminoglycoside toxicity, we used a 1:1 fluorescent conjugate of Texas Red and gentamicin (TRG) to quantify ea rly uptake dynamics in renal epithelial (LLC-PK1) cells. Utilizing a protoc ol that quenches TRG fluorescence from lysosomes, the bulk of intracellular accumulation, we determined a portion rapidly trafficked directly to the G olgi complex when identified by a FITC-conjugated lectin from Lens culinari s agglutinin (LCA). A kinetic study over 120 min on cells showing total and quenched TRG fluorescence was then carried out, and the fluorescence inten sity from the images was quantified. Trafficking of TRG to the Golgi comple x occurred within 15 min and accounted for similar to 20% of total cellular accumulation in the kinetic study. Colocalization studies using compartmen t-specific markers, 6-[ N-(7-nitrobenz- 2-oxa-1,3-diazol-4-yl)amino] hexano yl sphingosine (C6-NBD ceramide) and LCA, for the TGN trans-Golgi network, and the cis/medial-Golgi compartments, respectively, determined colocalizat ion 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.