title: Determination of specific receptor sites for platelet activating factor in bovine neutrophils.
abstract: OBJECTIVE
  To identify and characterize a platelet activating factor (PAF) receptor in bovine neutrophils by use of radioligand binding, reverse transcription-polymerase chain reaction (RT-PCR) assay, and western blot analysis.
  
  
  METHODS
  4 healthy adult cows.
  
  
  METHODS
  Bovine neutrophil membranes were isolated for association, dissociation, and saturation binding experiments with PAF labeled with hydrogen 3 (3H-PAF). The RT-PCR assay was performed with appropriate human primers, and western blot analysis was developed with a polyclonal antibody obtained from a peptide of bovine PAF receptor.
  
  
  RESULTS
  Analysis of kinetic binding data supported a single class of PAF receptor. Binding of 3H-PAF to membrane preparations was selectively displaced by PAF and a nonhydrolyzable analogue of guanine triphosphate (Gpp[NH]p) and by lyso-PAF (a biologically inactive analogue of PAF) to a lesser extent. Among other PAF receptor antagonists, 14-deoxyandrographolide and WEB 2086 were the most effective in inhibiting 3H-PAF binding sites in neutrophil membranes; 2 lignans, schisandrin-A and gamma-schisandrin were also effective, but 2 gingkolides (BN52020 and BN52021) only mildly inhibited 3H-PAF binding. Results of RT-PCR assay and western blot analysis of neutrophil crude membranes confirmed the presence of a PAF receptor.
  
  
  CONCLUSIONS
  Results indicated that bovine neutrophils express only 1 type of PAF receptor, and it is likely that this receptor is involved in inflammatory responses. The most effective PAF antagonists were 14-deoxyandrographolide and WEB 2086; these PAF antagonists may be potentially useful in the treatment of inflammatory processes in cattle.
authors.affiliation: Laboratory of Molecular Pharmacology, Institute of Pharmacology, Faculty of Veterinary Sciences, Universidad Austral de Chile, PO Box 567, Valdivia, Chile.
authors.forename: Rafael A
authors.initials: RA
authors.lastname: Burgos
authors.forename: María A
authors.initials: MA
authors.lastname: Hidalgo
authors.forename: Sabine M
authors.initials: SM
authors.lastname: Matthei
authors.forename: Ricardo
authors.initials: R
authors.lastname: Hermosilla
authors.forename: Hugo
authors.initials: H
authors.lastname: Folch
authors.forename: Juan L
authors.initials: JL
authors.lastname: Hancke
mesh_terms: D000818:Animals; D001381:Azepines / Q000378:metabolism; D001483:Base Sequence; D015153:Blotting, Western; D002417:Cattle; D034242:Cyclooctanes / Q000378:metabolism; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics; D004224:Diterpenes / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D046934:Ginkgolides; D018922:HL-60 Cells; D006801:Humans; D007700:Kinetics; D007783:Lactones / Q000378:metabolism; D017705:Lignans / Q000378:metabolism; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000378:metabolism*; D010972:Platelet Activating Factor / Q000378:metabolism*; D010980:Platelet Membrane Glycoproteins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D011083:Polycyclic Compounds / Q000378:metabolism; D011869:Radioligand Assay; D043562:Receptors, G-Protein-Coupled / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D014230:Triazoles / Q000378:metabolism; D014316:Tritium
chemical_list: C114791:14-deoxyandrographolide; D001381:Azepines; D034242:Cyclooctanes; D017931:DNA Primers; D018076:DNA, Complementary; D004224:Diterpenes; D046934:Ginkgolides; D007783:Lactones; D017705:Lignans; D010972:Platelet Activating Factor; D010980:Platelet Membrane Glycoproteins; D011083:Polycyclic Compounds; D043562:Receptors, G-Protein-Coupled; D014230:Triazoles; C039351:platelet activating factor receptor; C015499:schizandrin B; D014316:Tritium; C052518:WEB 2086; C034734:schizandrin A; C045856:ginkgolide B; C477042:ginkgolide A