Cytoplasmic and intra-nuclear binding of gentamicin does not require endocytosis.
Understanding the cellular mechanism(s) by which the oto- and nephrotoxic aminoglycoside antibiotics penetrate cells, and the precise intracellular distribution of these molecules, will enable identification of aminoglycoside-sensitive targets, and potential uptake blockers. Clones of two kidney cell lines, OK and MDCK, were treated with the aminoglycoside gentamicin linked to the fluorophore Texas Red (GTTR). As in earlier reports, endosomal accumulation was observed in live cells, or cells fixed with formaldehyde only. However, delipidation of fixed cells revealed GTTR fluorescence in cytoplasmic and nuclear compartments. Immunolabeling of both GTTR and unconjugated gentamicin corresponded to the cytoplasmic distribution of GTTR fluorescence. Intra-nuclear GTTR binding co-localized with labeled RNA in the nucleoli and trans-nuclear tubules. Cytoplasmic and nuclear distribution of GTTR was quenched by phosphatidylinositol-bisphosphate (PIP2), a known ligand for gentamicin. Cytoplasmic and nuclear GTTR binding increased over time (at 37 degrees C, or on ice to inhibit endocytosis), and was serially competed off by increasing concentrations of unconjugated gentamicin, i.e., GTTR binding is saturable. In contrast, little or no reduction of endocytotic GTTR uptake was observed when cells were co-incubated with up to 4 mg/mL unconjugated gentamicin. Thus, cytoplasmic and nuclear GTTR uptake is time-dependent, weakly temperature-dependent and saturable, suggesting that it occurs via an endosome-independent mechanism, implicating ion channels, transporters or pores in the plasma membrane as bioregulatory routes for gentamicin entry into cells.
Myrdal
Sigrid E
SE
Oregon Hearing Research Center, Oregon Health & Science University, 3181 SW Sam Jackson Park Road, Portland, OR 97239, USA.
Johnson
Katherine C
KC
Steyger
Peter S
PS
D000818:Animals; D000900:Anti-Bacterial Agents / Q000009:adverse effects / Q000378:metabolism*; D001665:Binding Sites / Q000187:drug effects; D002460:Cell Line; D004285:Dogs; D004305:Dose-Response Relationship, Drug; D004705:Endocytosis / Q000502:physiology*; D005456:Fluorescent Dyes; D005839:Gentamicins / Q000009:adverse effects / Q000378:metabolism*; D007150:Immunohistochemistry; D007686:Kidney Tubules, Distal / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D018613:Microscopy, Confocal; D009474:Neurons / Q000187:drug effects; D009893:Opossums; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism / Q000494:pharmacology; D014966:Xanthenes
D000900:Anti-Bacterial Agents; D005456:Fluorescent Dyes; D005839:Gentamicins; D019269:Phosphatidylinositol 4,5-Diphosphate; D014966:Xanthenes; C034657:Texas red