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1 Department of Medicine and Division of Nephrology, Indiana University School of Medicine, Indianapolis, IN, USA; Indiana Center for Biological Microscopy, Indianapolis, IN, USA; Roudebush VAMC, Indianapolis, IN, USA
* To whom correspondence should be addressed. E-mail: bmolitor{at}iupui.edu.
Previous mechanisms describing how aminoglycosides exert their cellular toxicity including lysosomal accumulation, rupture and release, cannot account for the rapidity and extent of the observed subcellular and organ effects. Using immuno-amplification techniques and colocalization with epitopes of the Endoplasmic Reticulum (ER), we report rapid retrograde transport of gentamicin to the ER. Additionally, exposure times of 2 and 4 hours in LLC-PK1 cells produced cytosolic release and nuclear association. Cellular internalization and trafficking of aminoglycoside structural analogs, amine containing cationic fluorescent dextrans of 3,000 MW, corroborated these findings. However, identical anionic fluorescent dextrans, or larger cationic dextrans, of 10,000 MW, failed to traverse from the ER into the cytosol or localize within the nucleus. These studies suggest a pathway exists that transports internalized aminoglycosides, and other small MW cationic compounds, in a retrograde manner through the Golgi Complex and to the ER. From there, these compounds move into the cytosol for delivery throughout the cell. To quantify the potential toxic effects of cytosolic aminoglycoside release, experiments examining mitochondrial membrane potential in the continued presence of extracellular gentamicin were undertaken and demonstrated a significant reduction after 4 and 8 hours. These observations provide a mechanism for the rapidly induced known cellular alterations including aberrant vesicle fusion, mitochondrial toxicity/ free radical generation and decreased protein synthesis either by reduced transcription or translation following aminoglycoside exposure.
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