Inhibition of cyclooxygenases 1 and 2 by the phospholipase-blocker, arachidonyl trifluoromethyl ketone.
OBJECTIVE
Arachidonyl trifluoromethyl ketone (ATK) is widely used as an inhibitor of cytosolic group IV phospholipase A(2) (cPLA(2)) and calcium-independent group VI phospholipase A(2) (iPLA(2)). ATK thus reduces arachidonic acid (AA) substrate for cyclooxygenase (COX; also known as prostaglandin H synthase) and attenuates prostaglandin (PG) synthesis. It has been shown previously, that ATK blocks thromboxane B(2) production induced by exogenous AA in human platelets. It remains, however, unknown whether ATK also directly modulates the activity of cyclooxygenase (COX).


METHODS
Time courses for inhibition of COX by ATK was obtained using osteoblast-like MC3T3-E1 cells, with exogenous AA as substrate and the pure enzymes COX-1 and COX-2. PGE(2) was measured by GC-MS.


RESULTS
ATK was a potent inhibitor of COX-1 and COX-2 with IC(50) values of 0.5 and 0.1 microM in MC3T3-E1 cells and of 1.7 and 2.6 microM using the pure enzymes. Inhibition was reversible, with slow- and tight-binding characteristics. The arachidonyl carbon chain was essential, as the saturated palmitoyl analogue had no effect.


CONCLUSIONS
Attenuation of PG synthesis by ATK is taken to be the consequence of PLA(2) inhibition and the findings of many studies are interpreted on that basis. If there are, however, alternative routes for AA liberation (such as phospholipase C/diacyl glycerol lipase or phospholipase D), this interpretation can lead to false conclusions. As ATK is a widely used and important pharmacological tool in eicosanoid research, knowledge of its interactions with other major enzymes of the cascade is of considerable importance.
Leis
H J
HJ
Medicinal University of Graz, University Children's Hospital, Research Unit of Osteology and Analytical Mass Spectrometry, Auenbruggerplatz 30, Graz, Austria. hans.leis@meduni-graz.at
Windischhofer
W
W
D000818:Animals; D001095:Arachidonic Acids / Q000494:pharmacology*; D015153:Blotting, Western; D002460:Cell Line; D051545:Cyclooxygenase 1 / Q000096:biosynthesis / Q000378:metabolism*; D051546:Cyclooxygenase 2 / Q000096:biosynthesis / Q000378:metabolism*; D016861:Cyclooxygenase Inhibitors / Q000494:pharmacology*; D008401:Gas Chromatography-Mass Spectrometry; D051379:Mice; D010006:Osteoblasts / Q000187:drug effects / Q000201:enzymology; D010740:Phospholipases / Q000037:antagonists & inhibitors*; D054512:Phospholipases A2, Calcium-Independent / Q000037:antagonists & inhibitors; D054510:Phospholipases A2, Cytosolic / Q000037:antagonists & inhibitors; D013379:Substrate Specificity
D001095:Arachidonic Acids; D016861:Cyclooxygenase Inhibitors; C081565:arachidonyltrifluoromethane; D051545:Cyclooxygenase 1; D051546:Cyclooxygenase 2; D010740:Phospholipases; D054512:Phospholipases A2, Calcium-Independent; D054510:Phospholipases A2, Cytosolic