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

Rafael A. BurgosLaboratory of Molecular Pharmacology, Institute of Pharmacology, Faculty of Veterinary Sciences, Universidad Austral de Chile, PO Box 567, Valdivia, Chile

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María A. HidalgoLaboratory of Molecular Pharmacology, Institute of Pharmacology, Faculty of Veterinary Sciences, Universidad Austral de Chile, PO Box 567, Valdivia, Chile.

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Sabine M. MattheiLaboratory of Molecular Pharmacology, Institute of Pharmacology, Faculty of Veterinary Sciences, Universidad Austral de Chile, PO Box 567, Valdivia, Chile.

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Ricardo HermosillaInstitute of Pharmacology, Charité-Universitätsmedizin Berlin, Berlin, Germany, Forschungsinstitut für Molekulare Pharmakologie, Thielallee 69-73 14195, Berlin, Germany.

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Hugo FolchInstitute of Immunology, Faculty of Medicine, Universidad Austral de Chile, PO Box 567, Valdivia, Chile.

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Juan L. HanckeLaboratory of Molecular Pharmacology, Institute of Pharmacology, Faculty of Veterinary Sciences, Universidad Austral de Chile, PO Box 567, Valdivia, Chile.

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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)