title: Effects of Na+/H+ exchanger inhibitors on subcellular localisation of endocytic organelles and intracellular dynamics of protein transduction domains HIV-TAT peptide and octaarginine.
abstract: Protein transduction domains such as those derived from the HIV protein TAT have great potential as vectors for delivery of therapeutic entities such as genes and proteins into cells. Extensive studies have shown that a major fraction of the most studied variants enters cells via an endocytic mechanism. However, controversy surrounds the exact uptake mechanism and whether a specific pathway is utilised. Studies showing inhibition of uptake of protein transduction domains in the presence of ion-transport inhibitors such as amiloride and its more potent analogue 5-(N-ethyl-N-isopropyl) amiloride (EIPA) suggest a link between peptide internalisation and macropinocytosis. In this study, using immunolabelling of early and late components of the endocytic pathway, we show that treatment of cells with EIPA and to a lesser extent amiloride affects the morphology and subcellular location of early, late endosomes and lysosomes. Enlarged early and late endocytic structures were observed in EIPA-treated cells, and these organelles accumulated in a perinuclear region. Results from experiments investigating the effects of EIPA on distribution of fluorescent octaarginine were in agreement with the immunolocalisation studies. Treatment of the CD34(+) leukaemia cell line KG1a with EIPA in the presence of fluorescent conjugates of HIV-TAT peptide and octaarginine showed distinct vesicular staining in agreement with untreated cells but EIPA-treated cells were additionally characterized by increased localization of the peptides in the cytosol. At levels previously shown to inhibit uptake of HIV-TAT peptide and octaarginine in other cell lines, EIPA was without major effect on uptake of both peptides in KG1a cells.
authors.affiliation: Welsh School of Pharmacy, Redwood Building, Cardiff University, Cardiff, CF10 3XF, Wales, UK.
authors.forename: Marjan
authors.initials: M
authors.lastname: Fretz
authors.forename: Jing
authors.initials: J
authors.lastname: Jin
authors.forename: Robin
authors.initials: R
authors.lastname: Conibere
authors.forename: Neal A
authors.initials: NA
authors.lastname: Penning
authors.forename: Saly
authors.initials: S
authors.lastname: Al-Taei
authors.forename: Gert
authors.initials: G
authors.lastname: Storm
authors.forename: Shiroh
authors.initials: S
authors.lastname: Futaki
authors.forename: Toshihide
authors.initials: T
authors.lastname: Takeuchi
authors.forename: Ikuhiko
authors.initials: I
authors.lastname: Nakase
authors.forename: Arwyn T
authors.initials: AT
authors.lastname: Jones
mesh_terms: D000584:Amiloride / Q000031:analogs & derivatives* / Q000494:pharmacology / Q000633:toxicity; D002470:Cell Survival / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004705:Endocytosis / Q000187:drug effects*; D011992:Endosomes / Q000187:drug effects* / Q000378:metabolism; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D015696:Gene Products, tat / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D052119:Lysosomal-Associated Membrane Protein 2; D051907:Lysosomal Membrane Proteins / Q000378:metabolism; D008247:Lysosomes / Q000187:drug effects / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D009842:Oligopeptides / Q000378:metabolism*; D010873:Pinocytosis / Q000187:drug effects; D017923:Sodium-Hydrogen Exchangers / Q000037:antagonists & inhibitors* / Q000378:metabolism; D013997:Time Factors; D033921:Vesicular Transport Proteins / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; D015696:Gene Products, tat; C497811:LAMP2 protein, human; D052119:Lysosomal-Associated Membrane Protein 2; D051907:Lysosomal Membrane Proteins; D008565:Membrane Proteins; D009842:Oligopeptides; D017923:Sodium-Hydrogen Exchangers; D033921:Vesicular Transport Proteins; C093846:early endosome antigen 1; C448619:octaarginine; D000584:Amiloride; C039614:ethylisopropylamiloride