title: Reorganization of ZO-1 by sodium-dependent glucose transporter activation after heat stress in LLC-PK1 cells.
abstract: Heat stress (HS) induces activation of high-affinity sodium-dependent glucose transporter (SGLT1) in porcine renal LLC-PK(1) cells. In this study, we investigated the roles of SGLT1 activation in reorganization of zonula occludens-1 (ZO-1), a cytosolic tight junction (TJ) protein, after HS. HS (42 degrees C, 3 h) caused decrease in transepithelial electrical resistance (TER). Subsequent incubation at 37 degrees C for 12 h increased TER above pre-HS level. The treatment of phloridzin, a potent SGLT1 inhibitor, or the replacement of glucose with a nonmetabolizable glucose analog blocked the recovery of TER and increased the transepithelial flux of FITC-dextran (4,000 Da). Immunofluorescent staining of ZO-1 showed that HS diffused ZO-1 from cell contact to cytosolic sites. Furthermore, the fraction of ZO-1 was distributed from the Triton X-100 insoluble to the Triton X-100 soluble pool. After incubation at 37 degrees C for 12 h, cell contact and ZO-1 extractability with Triton X-100 returned to pre-HS conditions, but the recovery was completely prevented by phloridzin. Tyrosine kinases activity was increased by HS that was inhibited by phloridzin. Genistein and CGP77675, tyrosine kinases inhibitors, blocked the recovery of TER and increased the transepithelial flux of FITC-dextran. Furthermore, these inhibitors prevented the recovery of cell contact and ZO-1 extractability with Triton X-100 as same as phloridzin. These findings suggested that the activation of SGLT1 reorganized ZO-1 mediated by elevation of tyrosine kinases activity after heat injury.
authors.affiliation: Department of Environmental Biochemistry and Toxicology, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka, Japan. ikari@u-shizuoka-ken.ac.jp
authors.forename: Akira
authors.initials: A
authors.lastname: Ikari
authors.forename: Mika
authors.initials: M
authors.lastname: Nakano
authors.forename: Yasunobu
authors.initials: Y
authors.lastname: Suketa
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Harada
authors.forename: Kuniaki
authors.initials: K
authors.lastname: Takagi
mesh_terms: D000818:Animals; D002450:Cell Communication / Q000187:drug effects / Q000502:physiology; D002463:Cell Membrane Permeability / Q000502:physiology; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D003911:Dextrans / Q000493:pharmacokinetics; D004058:Diffusion / Q000187:drug effects; D017097:Electric Impedance; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000378:metabolism*; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives* / Q000493:pharmacokinetics; D018869:Heat-Shock Response / Q000502:physiology*; D018374:LLC-PK1 Cells; D008562:Membrane Glycoproteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008564:Membrane Potentials / Q000502:physiology; D008565:Membrane Proteins / Q000378:metabolism*; D009004:Monosaccharide Transport Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000187:drug effects / Q000378:metabolism; D020127:Recovery of Function / Q000502:physiology; D051269:Sodium-Glucose Transporter 1; D013552:Swine; D019108:Tight Junctions / Q000378:metabolism*; D015854:Up-Regulation / Q000187:drug effects / Q000502:physiology; D062826:Zonula Occludens-1 Protein
chemical_list: D003911:Dextrans; D004791:Enzyme Inhibitors; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; D010750:Phosphoproteins; D051269:Sodium-Glucose Transporter 1; D062826:Zonula Occludens-1 Protein; C015219:fluorescein isothiocyanate dextran; D011505:Protein-Tyrosine Kinases; D016650:Fluorescein-5-isothiocyanate