Gentamicin traffics retrograde through the secretory pathway and is released in the cytosol via the endoplasmic reticulum

Ruben M. Sandoval, Bruce Molitoris

Research output: Contribution to journalArticle

72 Citations (Scopus)

Abstract

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 immunoamplification 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 h in LLC-PK 1 cells produced cytosolic release and nuclear association. Cellular internalization and trafficking of aminoglycoside structural analogs, amine-containing cationic fluorescent dextrans of 3,000 molecular weight, corroborated these findings. However, identical anionic fluorescent dextrans, or larger cationic dextrans, of 10,000 molecular weight, failed to traverse from the ER into the cytosol or localize within the nucleus. These studies suggest that a pathway exists that transports internalized aminoglycosides, and other small-molecular-weight 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 h. 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 after aminoglycoside exposure.

Original languageEnglish
JournalAmerican Journal of Physiology - Renal Physiology
Volume286
Issue number4 55-4
StatePublished - Apr 2004

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Secretory Pathway
Aminoglycosides
Gentamicins
Endoplasmic Reticulum
Cytosol
Dextrans
Molecular Weight
Mitochondrial Dynamics
Mitochondrial Membrane Potential
Poisons
Golgi Apparatus
Free Radicals
Amines
Epitopes
Rupture
Proteins

Keywords

  • Endoplasmic reticulum
  • Texas red
  • Tyramide signal amplification

ASJC Scopus subject areas

  • Physiology

Cite this

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abstract = "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 immunoamplification 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 h in LLC-PK 1 cells produced cytosolic release and nuclear association. Cellular internalization and trafficking of aminoglycoside structural analogs, amine-containing cationic fluorescent dextrans of 3,000 molecular weight, corroborated these findings. However, identical anionic fluorescent dextrans, or larger cationic dextrans, of 10,000 molecular weight, failed to traverse from the ER into the cytosol or localize within the nucleus. These studies suggest that a pathway exists that transports internalized aminoglycosides, and other small-molecular-weight 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 h. 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 after aminoglycoside exposure.",
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N2 - 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 immunoamplification 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 h in LLC-PK 1 cells produced cytosolic release and nuclear association. Cellular internalization and trafficking of aminoglycoside structural analogs, amine-containing cationic fluorescent dextrans of 3,000 molecular weight, corroborated these findings. However, identical anionic fluorescent dextrans, or larger cationic dextrans, of 10,000 molecular weight, failed to traverse from the ER into the cytosol or localize within the nucleus. These studies suggest that a pathway exists that transports internalized aminoglycosides, and other small-molecular-weight 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 h. 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 after aminoglycoside exposure.

AB - 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 immunoamplification 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 h in LLC-PK 1 cells produced cytosolic release and nuclear association. Cellular internalization and trafficking of aminoglycoside structural analogs, amine-containing cationic fluorescent dextrans of 3,000 molecular weight, corroborated these findings. However, identical anionic fluorescent dextrans, or larger cationic dextrans, of 10,000 molecular weight, failed to traverse from the ER into the cytosol or localize within the nucleus. These studies suggest that a pathway exists that transports internalized aminoglycosides, and other small-molecular-weight 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 h. 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 after aminoglycoside exposure.

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