title: Urothelial cell platelet-activating factor production mediated by calcium-independent phospholipase A2γ.
abstract: OBJECTIVE
  To determine the effect of phospholipase A(2) (PLA(2)) inhibitors on urothelial cell platelet-activating factor (PAF) production in response to tryptase stimulation.
  
  
  METHODS
  Urothelial cells isolated from normal human ureters were immortalized with the human papillomavirus type 16E6E7 cell line (TEU-2 cells). PLA(2) activity in TEU-2 cells was measured using (16:0, [(3)H]18:1) plasmenylcholine and phosphatidylcholine substrates in the presence and absence of calcium. [(3)H]PAF production was measured in TEU-2 cells prelabeled with [(3)H] acetic acid. PAF-acetylhydrolase activity was measured by determining the amount of [(3)H] acetate hydrolyzed from [(3)H]PAF incubated with TEU-2 cellular protein. Adherence of human polymorphonuclear leukocyte (PMN) to TEU-2 cells was assessed by measuring myeloperoxidase activity in adherent PMNs after incubation with TEU-2 cells.
  
  
  RESULTS
  Most PLA(2) activity measured in TEU-2 cells was determined to be membrane-associated, calcium-independent PLA(2) and selective for plasmenylcholine substrate. Stimulation of TEU-2 cells with tryptase results in increased production of PAF and increased PMN adherence that were inhibited completely by pretreatment with the membrane-associated, calcium-independent PLA(2)γ-selective inhibitor (R)-bromoenol lactone. Pretreatment with the cytosolic PLA(2) inhibitor methyl arachidonyl fluorophosphonate resulted in potentiation of tryptase-stimulated PAF production and PMN adherence to TEU-2 cells that is a result of PAF-acetylhydrolase inhibition.
  
  
  CONCLUSIONS
  Tryptase stimulation of TEU-2 cells results in activation of membrane-associated, calcium-independent PLA(2)γ, leading to an increase in PAF production and increased PMN adherence. Inhibition of TEU-2 cell PAF-acetylhydrolase activity with methyl arachidonyl fluorophosphonate potentiated tryptase-stimulated PAF production and PMN adherence.
authors.affiliation: Department of Pathology, Saint Louis University School of Medicine, St. Louis, Missouri, USA.
authors.forename: Prerna
authors.initials: P
authors.lastname: Rastogi
authors.forename: Alice
authors.initials: A
authors.lastname: Rickard
authors.forename: David J
authors.initials: DJ
authors.lastname: Klumpp
authors.forename: Jane
authors.initials: J
authors.lastname: McHowat
mesh_terms: D002118:Calcium / Q000502:physiology; D002478:Cells, Cultured; D054513:Group IV Phospholipases A2 / Q000502:physiology*; D006801:Humans; D010972:Platelet Activating Factor / Q000096:biosynthesis*; D014513:Ureter / Q000166:cytology / Q000378:metabolism*; D019459:Urothelium / Q000166:cytology / Q000378:metabolism*
chemical_list: D010972:Platelet Activating Factor; D054513:Group IV Phospholipases A2; D002118:Calcium