title: Bradykinin does not induce gap formation between human endothelial cells.
abstract: Generally, a formation of paracellular gaps is considered to be the main pathway for fluid passage across endothelia. A model substance for studies in vitro is the vasodilatory peptide bradykinin, which has important functions in inflammation and vascular fluid balance. The mechanisms by which it increases endothelial permeability are not as yet clearly defined. Paracellular gap formation was approached using atomic force microscopy (AFM) on human umbilical vein endothelial cells grown on permeable filter supports. To further distinguish between para- vs transcellular fluid passage, a standard permeability assay was modified by a rapid cooling protocol to specifically inhibit vesicular transport pathways. Cell layers stimulated with bradykinin (1 microM) did not show significant alterations at the cellular junctions. However, gap formation was easily detectable by AFM after addition of the Ca(2+)-ionophore ionomycin (1 microM), which was taken as a positive control for cellular contraction. At 37 degrees C, bradykinin enhanced fluorescein isothiocyanate-dextran permeability by 48 +/- 11%. This was blocked by rapid cooling of the sample, indicating a vesicular mechanism of fluid transport. Contrastingly, ionomycin-induced permeability (259 +/- 43%) persisted after cooling (230 +/- 44%), thereby confirming paracellular gap formation. Accordingly, endocytotic vesicle formation, as detected by fluorescence microscopy, was upregulated by 68 +/- 15% through bradykinin action, while ionomycin did not show a significant effect (7 +/- 26%). The combined results of both permeability and morphometric studies lead to the conclusion that bradykinin promotes transcellular fluid passage rather than increasing paracellular diffusion.
authors.affiliation: Institute of Physiology II, University of Münster, Robert-Koch-Str. 27b, 48149 Münster, Germany.
authors.forename: Pia
authors.initials: P
authors.lastname: Jungmann
authors.forename: Marianne
authors.initials: M
authors.lastname: Wilhelmi
authors.forename: Hans
authors.initials: H
authors.lastname: Oberleithner
authors.forename: Christoph
authors.initials: C
authors.lastname: Riethmüller
mesh_terms: D001920:Bradykinin / Q000494:pharmacology*; D002021:Buffers; D002118:Calcium / Q000502:physiology; D002463:Cell Membrane Permeability / Q000187:drug effects; D002478:Cells, Cultured; D003080:Cold Temperature; D004705:Endocytosis / Q000187:drug effects; D042783:Endothelial Cells / Q000187:drug effects*; D016650:Fluorescein-5-isothiocyanate; D017629:Gap Junctions / Q000187:drug effects*; D006801:Humans; D018625:Microscopy, Atomic Force; D008856:Microscopy, Fluorescence; D014470:Umbilical Cord / Q000166:cytology; D014617:Vacuoles / Q000187:drug effects
chemical_list: D002021:Buffers; D016650:Fluorescein-5-isothiocyanate; D001920:Bradykinin; D002118:Calcium