Inhibiting long-chain fatty acyl CoA synthetase does not increase agonist-induced release of arachidonate metabolites from human endothelial cells.
BACKGROUND
Triacsin C, a fatty acid analog, inhibits endothelial nitric oxide synthetase (eNOS) palmitoylation, increases nitric oxide synthesis and enhances methacholine-induced relaxation of vascular rings. The experiments presented here tested the hypothesis that triacsin C increases the synthesis of PGI(2) and/or endothelial-derived hyperpolarizing factor.


METHODS
Long-chain fatty acyl CoA synthetase activity (LCFACoAS), agonist-induced prostacyclin synthesis and agonist-induced release of radioactivity in endothelial cells labeled with [(3)H]arachidonic acid were measured in the presence and absence of triacsin C.


RESULTS
Inhibition by triacsin C of palmitoyl CoA formation was significantly greater than inhibition of arachidonoyl CoA formation in solubilized endothelial cell preparations. While 24-hour triacsin C treatment significantly reduced basal 6-keto synthesis, it had no effect on agonist-stimulated synthesis. The release of arachidonic acid metabolites was examined in [(3)H]arachidonate-labeled cells. Triacsin C treatment had no effect on basal or vasopressin-, angiotensin-II-, bradykinin- or ionomycin-induced release of radioactivity, but significantly reduced release in response to isoproterenol or phenylephrine. Expression of neither immunoreactive eNOS nor immunoreactive inducible nitric oxide synthetase (iNOS) was changed by triacsin C treatment, but the fraction of immunoreactive eNOS in the cytoplasm of treated cells was significantly greater as compared to vehicle control cells. Phorbol myristoyl acetate or fenofibrate significantly increased in vitro LCFACoAS activity, and significantly decreased the nitrite/eNOS ratio.


CONCLUSIONS
These data indicate that, while triacsin C can inhibit arachidonoyl CoA synthetase in endothelial cells, it does not increase the availability of endogenous substrate for basal or agonist-induced PGI(2) synthesis, nor does it enhance release of arachidonic acid or its metabolites.
Weis
Margaret T
MT
Department of Pharmaceutical Sciences, Texas Tech University Health Science Center, Amarillo, 79106, USA. margaret.weis@tuhsc.edu
Brady
Monica
M
Moore
Mike
M
Crumley
Jason
J
Stallone
John N
JN
D001011:Aorta / Q000201:enzymology; D016718:Arachidonic Acid / Q000378:metabolism*; D002478:Cells, Cultured; D003066:Coenzyme A Ligases / Q000037:antagonists & inhibitors*; D003331:Coronary Vessels / Q000201:enzymology; D042783:Endothelial Cells / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D011464:Epoprostenol / Q000096:biosynthesis; D006801:Humans; D009569:Nitric Oxide / Q000096:biosynthesis; D019001:Nitric Oxide Synthase / Q000378:metabolism; D014226:Triazenes / Q000494:pharmacology*; D000098859:Long-Chain-Fatty-Acid-CoA Ligase
D004791:Enzyme Inhibitors; D014226:Triazenes; D016718:Arachidonic Acid; D009569:Nitric Oxide; C034613:triacsin C; D011464:Epoprostenol; D019001:Nitric Oxide Synthase; D003066:Coenzyme A Ligases; D000098859:Long-Chain-Fatty-Acid-CoA Ligase