title: Adenosine triphosphate induced P2Y2 receptor activation induces proinflammatory cytokine release in uroepithelial cells.
abstract: OBJECTIVE
  We characterized and identified the uroepithelial P2 receptor responsible for adenosine triphosphate mediated release of the cytokines interleukin-8 and 6.
  
  
  METHODS
  The human renal epithelial cell line A498 (ATCC™) was cultured and stimulated with different purinergic agonists with or without prior inhibition with different antagonists or signaling pathway inhibitors. Supernatant was analyzed for interleukin-8 and 6 by enzyme-linked immunosorbent assay. P2 receptor mRNA expression was assessed by real-time reverse transcriptase-polymerase chain reaction. The candidate receptor was knocked down with siRNA technology. Interleukin-8 and 6 responses were measured after purinergic stimulation of knocked down cells.
  
  
  RESULTS
  ATP and ATP-γ-S (Roche Diagnostics, Mannheim, Germany) were equipotent as inducers of interleukin-8 and 6 release. Agonist profile experiments using different P2 receptor agonists indicated that P2Y(2) was the main contributor to this release, although P2Y(11) and P2X(7) activation could not be excluded. Signaling pathway experiments showed that interleukin-8 release involved phospholipase C and inositol trisphosphate mediated signaling, indicating a P2Y receptor subtype. Antagonist experiments indicated P2Y(2) as the responsible receptor. Gene expression analysis of P2 receptors showed that strong expression of P2Y(2) receptor and subsequent knockdown of P2Y(2) receptor mRNA for 72 and 96 hours abrogated interleukin-8 and 6 release after purinergic stimulation with adenosine triphosphate-γ-S.
  
  
  CONCLUSIONS
  Interleukin-8 and 6 release after purinergic stimulation in uroepithelial A498 cells is mediated through P2Y(2) receptor activation.
authors.affiliation: School of Health and Medical Sciences, Örebro University, Örebro and School of Natural Sciences, Kalmar, Sweden. Robert.Kruse@oru.se
authors.forename: Robert
authors.initials: R
authors.lastname: Kruse
authors.forename: Susanne
authors.initials: S
authors.lastname: Säve
authors.forename: Katarina
authors.initials: K
authors.lastname: Persson
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology*; D020013:Calcium Signaling / Q000187:drug effects; D002478:Cells, Cultured; D016207:Cytokines / Q000187:drug effects / Q000378:metabolism; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D015850:Interleukin-6 / Q000096:biosynthesis*; D016209:Interleukin-8 / Q000096:biosynthesis*; D034741:RNA, Small Interfering / Q000378:metabolism; D058473:Receptors, Purinergic P2Y2 / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D015398:Signal Transduction; D014552:Urinary Tract Infections / Q000503:physiopathology; D019459:Urothelium / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism
chemical_list: D016207:Cytokines; D015850:Interleukin-6; D016209:Interleukin-8; D034741:RNA, Small Interfering; D058473:Receptors, Purinergic P2Y2; D000255:Adenosine Triphosphate