Effect of pertussis toxin and B-oligomer on platelet-activating factor-induced generation of inositol phosphates in porcine alveolar macrophages.
OBJECTIVE
To test the hypothesis that platelet-activating factor (PAF) induces inositol phosphate turnover through a receptor-linked, pertussis toxin-sensitive guanine nucleotide-binding (G) protein-dependent pathway in porcine alveolar macrophages.


METHODS
Randomized complete block design was used with 2 or 3 replicates/block.


METHODS
Porcine alveolar macrophages were obtained by lavage of excised lungs from Yorkshire-type pigs (mean +/- SEM, 21 +/- 2 kg).


METHODS
Phospholipase C activation was assessed, using anion exchange chromatography to measure accumulation of inositol phosphates in [3H]myo-inositol-labeled alveolar macrophages. Macrophages were incubated with saline solution, pertussis toxin (4.75 nM), or B-oligomer (4.75 nM) for 2 hours. Cells then were washed and incubated for 5 minutes with PAF (0, 0.1, 1.0, or 10 microM; n = 15). Results were expressed as total inositol phosphates (inositol monophosphate, bisphosphate, trisphosphate, and tetrakisphosphate).


RESULTS
Concentrations of total inositol phosphates were significantly (P < 0.05) increased to 162 +/- 7, 172 +/- 4, and 194 +/- 9% of control in response to 0.1, 1.0, and 10 microM PAF, respectively. Pertussis toxin attenuated the PAF-induced increase in total inositol phosphates by approximately 50% (P < 0.05). The B-oligomer of pertussis toxin failed to modify PAF-induced increases in total inositol phosphates. The specific PAF receptor antagonist WEB 2086 markedly attenuated PAF-induced. (10 microM) increase in inositol phosphates.


CONCLUSIONS
We conclude that PAF stimulates accumulation of inositol phosphates through a specific receptor and that a pertussis toxin-sensitive G protein is involved in the signal transduction process leading to activation of phospholipase C in porcine alveolar macrophages.
Dodam
J R
JR
Department of Veterinary Medicine and Surgery, College of Veterinary Medicine, University of Missouri, Columbia 65211, USA.
Adler
K B
KB
Olson
N C
NC
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000077410:Aluminum Chloride; D017607:Aluminum Compounds / Q000494:pharmacology; D000704:Analysis of Variance; D000818:Animals; D001381:Azepines / Q000494:pharmacology; D018893:Bronchoalveolar Lavage; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D002712:Chlorides / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism*; D007294:Inositol / Q000378:metabolism; D007295:Inositol Phosphates / Q000378:metabolism*; D007770:L-Lactate Dehydrogenase; D016676:Macrophages, Alveolar / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D009243:NAD / Q000378:metabolism; D037342:Pertussis Toxin; D010972:Platelet Activating Factor / Q000494:pharmacology*; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology; D011897:Random Allocation; D012969:Sodium Fluoride / Q000494:pharmacology; D013552:Swine; D014230:Triazoles / Q000494:pharmacology; D010738:Type C Phospholipases / Q000378:metabolism; D010566:Virulence Factors, Bordetella / Q000494:pharmacology*
D017607:Aluminum Compounds; D001381:Azepines; D002712:Chlorides; D007295:Inositol Phosphates; D010972:Platelet Activating Factor; D010975:Platelet Aggregation Inhibitors; D014230:Triazoles; D010566:Virulence Factors, Bordetella; D009243:NAD; C052518:WEB 2086; D000246:Adenosine Diphosphate Ribose; D000077410:Aluminum Chloride; D007294:Inositol; D012969:Sodium Fluoride; D007770:L-Lactate Dehydrogenase; D037342:Pertussis Toxin; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins