Determination of specific receptor sites for platelet activating factor in bovine neutrophils.
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.
Burgos
Rafael A
RA
Laboratory of Molecular Pharmacology, Institute of Pharmacology, Faculty of Veterinary Sciences, Universidad Austral de Chile, PO Box 567, Valdivia, Chile.
Hidalgo
María A
MA
Matthei
Sabine M
SM
Hermosilla
Ricardo
R
Folch
Hugo
H
Hancke
Juan L
JL
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
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