title: Evidence for a plasma membrane-mediated permeability barrier to Tat basic domain in well-differentiated epithelial cells: lack of correlation with heparan sulfate.
abstract: Membrane permeation peptides, such as Tat basic domain, have emerged as useful membrane transduction agents with potential utility in therapeutic delivery and diagnostic imaging. While generally thought to universally permeate all cells by a nonselective process, the mechanism of membrane transduction remains poorly characterized. To examine vectorial transport properties of Tat basic domain in well-differentiated epithelial cells possessing tight junctions, L and D stereoisomers of Tat(48-57) peptide conjugates labeled with (99m)Tc were quantitatively analyzed in confluent monolayers of MDCK renal epithelial and CaCo-2 colonic carcinoma cells grown in transwell configurations. In both cell lines, vectorial transepithelial apparent permeability coefficients (P(app)) for L- and D-[(99m)Tc]Tat-peptides ranged from 30 to 70 nm/s, comparable to values for the macromolecular impermeant marker inulin in both apical-to-basolateral and basolateral-to-apical directions, but 100-fold less than the P(app) values for propranolol, a highly permeable control compound. Upon direct instillation of [(99m)Tc]Tat-peptide into the urinary bladder of living rats in vivo, no transepithelial permeation into other tissues was identified. Furthermore, MDCK and CaCo-2 cells showed a complete lack of intracellular accumulation of fluorescein conjugated Tat-peptide. However, translocation into cells was induced by treatment with plasma membrane permeabilizing agents such as digitonin and acetone/methanol, while cholesterol depletion with beta-methyl-cyclodextrin and metabolic inhibition with CCCP or 4 degrees C showed no effect. By contrast, in Hela and KB 3-1 cells, epithelial lines that do not form tight junctions in monolayer culture, baseline cytoplasmic and nucleolar accumulation was readily observed. Because all four cell lines expressed heparan sulfate proteoglycans, putative receptors for Tat basic peptides, we found no correlation between heparan sulfate and the permeation barrier observed in MDCK and CaCo-2 cells. The unanticipated presence of a permeation barrier to Tat-peptides in well-differentiated epithelial cells suggests the existence of cell-specific mechanisms for mediated translocation of these permeation peptides.
authors.affiliation: Molecular Imaging Center, Mallinckrodt Institute of Radiology, and Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
authors.forename: Stefania
authors.initials: S
authors.lastname: Violini
authors.forename: Vijay
authors.initials: V
authors.lastname: Sharma
authors.forename: Julie L
authors.initials: JL
authors.lastname: Prior
authors.forename: Mary
authors.initials: M
authors.lastname: Dyszlewski
authors.forename: David
authors.initials: D
authors.lastname: Piwnica-Worms
mesh_terms: D000283:Administration, Intravesical; D000818:Animals; D018938:Caco-2 Cells; D002460:Cell Line; D002463:Cell Membrane Permeability / Q000502:physiology*; D004285:Dogs; D004847:Epithelial Cells / Q000378:metabolism / Q000502:physiology*; D005260:Female; D015696:Gene Products, tat / Q000008:administration & dosage / Q000737:chemistry / Q000502:physiology*; D015497:HIV-1 / Q000737:chemistry / Q000502:physiology*; D006367:HeLa Cells; D006497:Heparan Sulfate / Q000096:biosynthesis / Q000502:physiology*; D006801:Humans; D007624:KB Cells; D008856:Microscopy, Fluorescence; D009842:Oligopeptides / Q000008:administration & dosage / Q000737:chemistry / Q000502:physiology; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D019459:Urothelium / Q000378:metabolism / Q000502:physiology; D054322:tat Gene Products, Human Immunodeficiency Virus
chemical_list: D015696:Gene Products, tat; D006497:Heparan Sulfate; D009842:Oligopeptides; D054322:tat Gene Products, Human Immunodeficiency Virus