Primary stimuli of icosanoid release inhibit arachidonoyl-CoA synthetase and lysophospholipid acyltransferase. Mechanism of action of hydrogen peroxide and methyl mercury in platelets.
Icosanoid formation in platelets depends on the concentration of free arachidonate that is mainly liberated from membrane phospholipids by phospholipase A2. The concentration of free arachidonate is also controlled by the activities of the reacylating enzymes arachidonoyl-CoA synthetase and lysophospholipid acyltransferase. In human platelet microsomes we determined the high enzyme activities of 5.9 nmol.min-1.(10(9) platelets)-1 for the arachidonoyl-CoA synthetase and 37 nmol.min-1.(10(9) platelets)-1 for the lysophospholipid acyltransferase. The activities of these reacylating enzymes were strongly reduced by hydrogen peroxide (H2O2) and methyl mercury that are primary stimuli of arachidonate release in intact platelets. H2O2 inhibited the arachidonoyl-CoA synthetase with an IC50 of 3.3 mmol/l without affecting the lysophospholipid acyltransferase. Sulfhydryl group protection by 3-mercapto-1,2-propanediol did not overcome the inhibition but glutathione prevented the inhibition of the arachidonoyl-CoA synthetase by H2O2. This suggests that glutathione by virtue of the glutathione peroxidase reduces H2O2 rather than that it protects free sulfhydryl groups of the arachidonoyl-CoA synthetase. Methyl mercury left the arachidonoyl-CoA synthetase activity unaffected but inhibited the lysophospholipid acyltransferase activity with an IC50 of 3.4 mumol/l. The inhibition is probably evoked by the blockade of sulfhydryl groups of the lysophospholipid acyltransferase because it disappeared when 3-mercapto-1,2-propanediol was added at a concentration higher than that of methyl mercury. Thrombin as a physiological full agonist, Ca2+ less than or equal to 1 mmol/l, the calcium ionophore A23187 and phorbol 12-myristate 13-acetate (TPA) and 1-oleoyl-2-acetylglycerol as model stimuli of protein kinase C neither influenced arachidonoyl-CoA synthetase nor lysophospholipid acyltransferase. It is concluded that the inhibitory effect of H2O2 and methyl mercury on the arachidonate-reacylating enzymes arachidonoyl-CoA synthetase or lysophospholipid acyltransferase, respectively, are responsible for their capacity to stimulate icosanoid release in intact cells. Thrombin and its intracellular messengers Ca2+ and diacylglycerol do not directly affect arachidonoyl-CoA synthetase and lysophospholipid acyltransferase.
Hornberger
W
W
Institute for Clinical Chemistry, Klinikum Mannheim, University of Heidelberg, Federal Republic of Germany.
Patscheke
H
H
D000217:Acyltransferases / Q000037:antagonists & inhibitors / Q000097:blood*; D001792:Blood Platelets / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology; D003066:Coenzyme A Ligases / Q000037:antagonists & inhibitors / Q000097:blood*; D004075:Diglycerides / Q000494:pharmacology; D015777:Eicosanoids / Q000097:blood*; D005978:Glutathione / Q000494:pharmacology; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D007700:Kinetics; D008245:Lysophospholipase / Q000037:antagonists & inhibitors / Q000097:blood*; D008767:Methylmercury Compounds / Q000494:pharmacology*; D008861:Microsomes / Q000187:drug effects / Q000378:metabolism; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors / Q000097:blood*; D010740:Phospholipases / Q000097:blood*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013917:Thrombin / Q000494:pharmacology
D004075:Diglycerides; D015777:Eicosanoids; D008767:Methylmercury Compounds; D009097:Multienzyme Complexes; D000001:Calcimycin; C038237:1-oleoyl-2-acetylglycerol; D006861:Hydrogen Peroxide; D000217:Acyltransferases; D010740:Phospholipases; D008245:Lysophospholipase; C021782:lysophospholipase-transacylase; D013917:Thrombin; D003066:Coenzyme A Ligases; C033896:arachidonate - CoA ligase; D005978:Glutathione; D013755:Tetradecanoylphorbol Acetate; C004925:methylmercuric chloride; D002118:Calcium