Biosynthesis of platelet-activating factor (PAF) in human polymorphonuclear leucocytes. The role of lyso-PAF disposal and free arachidonic acid.
Theophylline and 1-methyl-3-isobutylxanthine (MIX), compounds that block eicosanoid formation and modulate phospholipase A2 activity, inhibited in a dose-dependent manner the formation of both leukotriene B4 (LTB4) and platelet-activating factor (PAF) by human polymorphonuclear leucocytes (PMN) in response to ionophore A23187. Theophylline and MIX lacked any inhibitory effect on acetyl-CoA: lyso-PAF acetyltransferase activity, which is the rate-limiting step for PAF biosynthesis in PMN. The effect of theophylline and MIX on PAF formation could be reversed by incubating the cells in the presence of 1-10 microM exogenous lyso-PAF. Incubation of PMN homogenates in the presence of unsaturated non-esterified fatty acids resulted in dose-dependent inhibition of the acetyltransferase. This effect was linked to the presence of a free carboxyl group, since both arachidonic acid methyl ester and palmitoyl-arachidonoyl phosphatidylcholine lacked inhibitory activity. This inhibitory effect was also dependent on the number of double bonds, since arachidonic acid (C20:4) and eicosapentaenoic acid (C20:5) displayed maximal effect. Kinetic analysis showed that the effect of arachidonic acid was consistent with competitive inhibition, with a Ki value of about 19 microM. Oxidative metabolites of arachidonic acid showed a lesser inhibitory effect with the following order of potency: arachidonic acid greater than 15-HETE (15-hydroxy-6,8,11,14-eicosatetraenoic acid) greater than LTB4 greater than 5-HETE (5-hydroxy-6,8,11,14-eicosatetraenoic acid) greater than lipoxin A4. Examination of enzymes involved in CoA-dependent acylation revealed a low activity of both arachidonoyl-CoA synthetase and arachidonoyl-CoA: lyso-PAF arachidonoyltransferase. These data indicate a strong influence on PAF biosynthesis of the products of the phospholipase A2 reaction, with lyso-PAF disposal being a critical event for PAF formation, and unsaturated fatty acids acting as feed-back inhibitors. The conversion of arachidonic acid via oxidative metabolism into less active inhibitors of acetyl-CoA:lyso-PAF acetyltransferase seems to be an additional mechanism of modulation of this enzyme activity, linked to the function of lipoxygenases. Finally, the enzyme activities involved in arachidonoyl-CoA-dependent acylation of lyso-PAF show a low efficiency in capturing arachidonic acid.
Garcia
M C
MC
Instituto de Investigaciones Medicas, Fundacion Jimenez Diaz, CSIC, Madrid, Spain.
Fernandez-Gallardo
S
S
Gijon
M A
MA
Garcia
C
C
Nieto
M L
ML
Sanchez Crespo
M
M
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000123:Acetyltransferases / Q000037:antagonists & inhibitors / Q000378:metabolism; D000217:Acyltransferases / Q000378:metabolism; D016718:Arachidonic Acid; D001095:Arachidonic Acids / Q000378:metabolism* / Q000494:pharmacology; D000001:Calcimycin / Q000494:pharmacology; D003066:Coenzyme A Ligases / Q000378:metabolism; D005227:Fatty Acids / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D007975:Leukotriene B4 / Q000096:biosynthesis; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism*; D010972:Platelet Activating Factor / Q000031:analogs & derivatives* / Q000096:biosynthesis* / Q000378:metabolism / Q000494:pharmacology; D013806:Theophylline / Q000494:pharmacology
D001095:Arachidonic Acids; D005227:Fatty Acids; C029271:O-deacetyl platelet activating factor; D010972:Platelet Activating Factor; D007975:Leukotriene B4; D016718:Arachidonic Acid; D000001:Calcimycin; D013806:Theophylline; D000217:Acyltransferases; D000123:Acetyltransferases; C018764:2-acylglycerophosphate acyltransferase; C046035:1-alkylglycerophosphocholine acetyltransferase; D003066:Coenzyme A Ligases; C033896:arachidonate - CoA ligase; D015056:1-Methyl-3-isobutylxanthine