Biosynthesis of paf-acether. Activators of protein kinase C stimulate cultured mast cell acetyltransferase without stimulating paf-acether synthesis.
Antigen stimulation of cultured bone-marrow-derived mast cells sensitized with specific monoclonal IgE induced cell degranulation and paf-acether (paf; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) biosynthesis via the deacylation/acetylation (remodelling) pathway. Phorbol myristate acetate (PMA; 20-100 ng/ml) triggered only acetyltransferase activation, without concomitant lyso-paf (1-O-alkyl-sn-glycero-3-phosphocholine) and paf formation. A low concentration of PMA (5 ng/ml) potentiated antigen-induced degranulation, acetyltransferase activation and paf formation by about 30% but did not change the level of lyso-paf formation. Stimulation of mast cells with antigen increased intracellular Ca2+ from 61 to 269 nM, whereas no modification of Ca2+ influx was observed when cells were pretreated with PMA (5 ng/ml) before antigen challenge. Gas chromatography coupled to electron capture detection revealed that the composition of paf formed by cells stimulated by antigen alone was similar to that of paf formed by PMA-primed antigen-stimulated cells; 84 +/- 8% and 79 +/- 2% (means +/- S.E.M., n = 3) of molecules respectively bore the C16:0 alkyl chain moiety, with the remainder bearing essentially C18:0 molecules. Overnight treatment of mast cells with PMA (200 ng/ml) caused disappearance of protein kinase C (PKC) from both cytosol and membranes. When such cells were stimulated further with antigen, they failed to degranulate, and acetyltransferase activation, paf production and lyso-paf production were decreased by 33 +/- 11%, 57 +/- 4% and 96 +/- 3% respectively (n = 3 or 5). The PKC inhibitors chlorpromazine and staurosporine inhibited to a significant extent both cell degranulation and all steps leading to paf biosynthesis. Our data suggest that PKC-dependent mechanisms are operational during cell degranulation and contribute only in part to paf biosynthesis. The PKC-dependent signal directly generated by PMA or diacylglycerol is not sufficient to trigger the full cell response, which is obtained only through receptor-operated antigen challenge.
Joly
F
F
INSERM U 200, Université Paris-Sud, Clamart, France.
Vigrain
I
I
Bossant
M J
MJ
Bessou
G
G
Benveniste
J
J
Ninio
E
E
D000123:Acetyltransferases / Q000378:metabolism*; D000470:Alkaloids / Q000494:pharmacology; D000818:Animals; D001854:Bone Marrow Cells; D002118:Calcium / Q000378:metabolism; D002746:Chlorpromazine / Q000494:pharmacology; D002849:Chromatography, Gas; D003594:Cytoplasmic Granules / Q000502:physiology; D004789:Enzyme Activation / Q000187:drug effects; D007073:Immunoglobulin E / Q000494:pharmacology; D008407:Mast Cells / Q000201:enzymology* / Q000648:ultrastructure; D051379:Mice; D008807:Mice, Inbred BALB C; D010972:Platelet Activating Factor / Q000096:biosynthesis*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D001619:beta-N-Acetylhexosaminidases / Q000378:metabolism
D000470:Alkaloids; D010972:Platelet Activating Factor; D007073:Immunoglobulin E; D000123:Acetyltransferases; D011493:Protein Kinase C; D001619:beta-N-Acetylhexosaminidases; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium; D002746:Chlorpromazine