title: Poly-L-arginine enhances paracellular permeability via serine/threonine phosphorylation of ZO-1 and tyrosine dephosphorylation of occludin in rabbit nasal epithelium.
abstract: OBJECTIVE
  The purpose of the present study is to explore whether a poly-L-arginine (poly-L-Arg)-induced increase in tight junctions (TJ) permeability of fluorescein isothiocyanate-labeled dextran (MW 4.4 kDa, FD-4) is associated with the Ca2+-dependent signaling and occurs following the phosphorylation/dephosphorylation of TJ proteins.
  
  
  METHODS
  Excised rabbit nasal epithelium was mounted in an Ussing-type chamber for measurement of FD-4 transport and membrane conductance (Gt) in the presence of various inhibitors that are involved in the Ca2+-dependent pathway and the phosphorylation/dephosphorylation of TJ proteins. The resultant distribution of TJ proteins was observed using confocal laser scanning microscopy (CLSM) in an immunostaining.
  
  
  RESULTS
  The increase in TJ permeability of FD4 induced by 0.2 mg/ml poly-L-Arg was not altered by treatment with inhibitors (of possible Ca2+ mobilization pathways followed by exposure of poly-L-Arg, suggesting that the promoting effect of poly-L-Arg is independent of Ca2+-related signaling. On the other hand, the protein kinase C (PKC) and tyrosine phosphatase inhibitors suppress the increase in TJ permeability by poly-L-Arg, indicating that serine/threonine phosphorylation by way of Ca2+-independent PKC and tyrosine dephosphorylation of junction proteins may have occurred. Furthermore, immunofluorescent monitoring of ZO-1, a TJ associated protein, and occludin, an integral membrane protein localizing at TJ, after preincubation with PKC and tyrosine phosphatase inhibitors followed by poly-L-Arg treatment has shown that the internalization of ZO-1 and occludin occurred by way of serine/threonine phosphorylation by PKC activation and by way of tyrosine dephosphorylation, respectively, providing TJ disassembly.
  
  
  CONCLUSIONS
  We conclude that poly-L-Arg enhances the paracellular permeability of FD-4 (i.e., macromolecules), at least, by way of both serine/threonine phosphorylation of ZO-1 and tyrosine dephosphorylation of occludin in rabbit nasal epithelium.
authors.affiliation: Faculty of Pharmaceutical Sciences, Josai University, 1-1 Keyakidai, Sakado, Saitama 350-0295, Japan.
authors.forename: Kazuo
authors.initials: K
authors.lastname: Ohtake
authors.forename: Takuya
authors.initials: T
authors.lastname: Maeno
authors.forename: Hideo
authors.initials: H
authors.lastname: Ueda
authors.forename: Masahiko
authors.initials: M
authors.lastname: Ogihara
authors.forename: Hideshi
authors.initials: H
authors.lastname: Natsume
authors.forename: Yasunori
authors.initials: Y
authors.lastname: Morimoto
mesh_terms: D000818:Animals; D001120:Arginine / Q000737:chemistry; D002463:Cell Membrane Permeability; D066298:In Vitro Techniques; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism*; D009297:Nasal Mucosa / Q000166:cytology / Q000378:metabolism*; D062793:Occludin; D010455:Peptides / Q000737:chemistry*; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011817:Rabbits; D012694:Serine / Q000378:metabolism*; D013912:Threonine / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism*; D062826:Zonula Occludens-1 Protein
chemical_list: D008565:Membrane Proteins; D062793:Occludin; D010455:Peptides; D010750:Phosphoproteins; D062826:Zonula Occludens-1 Protein; C015462:polyarginine; D013912:Threonine; D014443:Tyrosine; D012694:Serine; D001120:Arginine