title: Frog urinary bladder epithelial cells express TLR4 and respond to bacterial LPS by increase of iNOS expression and L-arginine uptake.
abstract: As in mammals, epithelium of the amphibian urinary bladder forms a barrier to pathogen entry and is a first line of defense against penetrating microorganisms. We investigated the effect of Escherichia coli LPS on generation of nitric oxide (NO), a critically important mediator during infectious processes, by primary cultured frog (Rana temporaria) urinary bladder epithelial cells (FUBEC). It was found that FUBEC constitutively express Toll-like receptor 4 (TLR4), a receptor of LPS, and respond to LPS (10 μg/ml) by stimulation of inducible nitric oxide synthase (iNOS) mRNA/protein expression and NOS activity measured by nitrite produced in the culture medium and by citrulline assay. We characterized uptake of l-arginine, a precursor in NO synthesis, by FUBEC and showed that it is mediated mainly by the y+ cationic amino acid transport system. LPS stimulated l-arginine uptake, and this effect was blocked by the iNOS inhibitor 1400W. Arginase II was found to be expressed in FUBEC. Inhibition of arginase activity by (S)-(boronoethyl)-l-cysteine increased generation of NO, suggesting contribution of arginase to NO production via competing with NOS for the substrate. LPS altered neither total arginase activity nor arginase II expression. Among epithelial cells, phagocytic macrophage-like cells were observed, but they did not contribute to LPS-induced NO production. These data demonstrate that amphibian urinary bladder epithelial cells recognize LPS and respond to it by increased generation of NO via stimulation of iNOS expression and l-arginine uptake, which appears to be essential for the regulation of the innate immune response and the inflammation in bladder epithelium.
authors.affiliation: I. M. Sechenov Institute of Evolutionary Physiology and Biochemistry of the Russian Academy of Sciences, St. Petersburg, Russia.
authors.forename: Svetlana
authors.initials: S
authors.lastname: Nikolaeva
authors.forename: Vera
authors.initials: V
authors.lastname: Bachteeva
authors.forename: Ekaterina
authors.initials: E
authors.lastname: Fock
authors.forename: Sabine
authors.initials: S
authors.lastname: Herterich
authors.forename: Elena
authors.initials: E
authors.lastname: Lavrova
authors.forename: Alexandra
authors.initials: A
authors.lastname: Borodkina
authors.forename: Stepan
authors.initials: S
authors.lastname: Gambaryan
authors.forename: Rimma
authors.initials: R
authors.lastname: Parnova
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000378:metabolism; D002478:Cells, Cultured; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects; D008070:Lipopolysaccharides / Q000633:toxicity*; D008297:Male; D008969:Molecular Sequence Data; D052247:Nitric Oxide Synthase Type II / Q000235:genetics / Q000378:metabolism; D011898:Ranidae / Q000502:physiology*; D051197:Toll-Like Receptor 4 / Q000235:genetics / Q000378:metabolism*; D001743:Urinary Bladder / Q000502:physiology*; D019459:Urothelium / Q000166:cytology*
chemical_list: D008070:Lipopolysaccharides; D051197:Toll-Like Receptor 4; D001120:Arginine; D052247:Nitric Oxide Synthase Type II