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Determination of specific receptor sites for platelet activating factor in bovine neutrophils

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  • 1 Laboratory of Molecular Pharmacology, Institute of Pharmacology, Faculty of Veterinary Sciences, Universidad Austral de Chile, PO Box 567, Valdivia, Chile
  • | 2 Laboratory of Molecular Pharmacology, Institute of Pharmacology, Faculty of Veterinary Sciences, Universidad Austral de Chile, PO Box 567, Valdivia, Chile.
  • | 3 Laboratory of Molecular Pharmacology, Institute of Pharmacology, Faculty of Veterinary Sciences, Universidad Austral de Chile, PO Box 567, Valdivia, Chile.
  • | 4 Institute of Pharmacology, Charité-Universitätsmedizin Berlin, Berlin, Germany, Forschungsinstitut für Molekulare Pharmakologie, Thielallee 69-73 14195, Berlin, Germany.
  • | 5 Institute of Immunology, Faculty of Medicine, Universidad Austral de Chile, PO Box 567, Valdivia, Chile.
  • | 6 Laboratory of Molecular Pharmacology, Institute of Pharmacology, Faculty of Veterinary Sciences, Universidad Austral de Chile, PO Box 567, Valdivia, Chile.

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.

Animals—4 healthy adult cows.

Procedure—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 γ-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 and Clinical Relevance—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. ( Am J Vet Res 2004;65:628–636)

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.

Animals—4 healthy adult cows.

Procedure—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 γ-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 and Clinical Relevance—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. ( Am J Vet Res 2004;65:628–636)