Stable 5,6-Epoxyeicosatrienoic Acid Analog Relaxes Coronary Arteries Through Potassium Channel Activation

Abstract

Abstract-5,6-Epoxyeicosatrienoic acid (5,6-EET) is a cytochrome P450 epoxygenase metabolite of arachidonic acid that causes vasorelaxation. However, investigations of its role in biological systems have been limited by its chemical instability. We developed a stable agonist of 5,6-EET, 5-(pentadeca-3(Z),6(Z),9(Z)-trienyloxy)pentanoic acid (PTPA), in which the 5,6-epoxide was replaced with a 5-ether. PTPA obviates chemical and enzymatic hydrolysis. In bovine coronary artery rings precontracted with U46619, PTPA (1 nmol/L to 10 mol/L) induced concentration-dependent relaxations, with maximal relaxation of 86Ϯ5% and EC 50 of 1 mol/L. The relaxations were inhibited by the cyclooxygenase inhibitor indomethacin (10 mol/L; max relaxation 43Ϯ9%); the ATP-sensitive K ϩ channel inhibitor glybenclamide (10 mol/L; max relaxation 49Ϯ6%); and the large conductance calcium-activated K ϩ channel inhibitor iberiotoxin (100 nmol/L; max relaxation 38Ϯ6%) and abolished by the combination of iberiotoxin with indomethacin or glybenclamide or increasing extracellular K ϩ to 20 mmol/L. Whole-cell outward K ϩ current was increased nearly 6-fold by PTPA (10 mol/L), which was also blocked by iberiotoxin. Additionally, we synthesized 5-(pentadeca-6(Z),9(Z)-dienyloxy)pentanoic acid and 5-(pentadeca-3(Z),9(Z)-dienyloxy)pentanoic acid (PDPA), PTPA analogs that lack the 8,9 or 11,12 double bonds of arachidonic acid and therefore are not substrates for cyclooxygenase. The PDPAs caused concentration-dependent relaxations (max relaxations 46Ϯ13% and 52Ϯ7%, respectively; EC 50 1mol/L), which were not altered by glybenclamide but blocked by iberiotoxin. These studies suggested that PTPA induces relaxation through 2 mechanisms: (1) cyclooxygenase-dependent metabolism to 5-ether-containing prostaglandins that activate ATP-sensitive K ϩ channels and (2) Key Words: arachidonic acids Ⅲ cyclooxygenase Ⅲ endothelium-derived factors C ytochrome P450 epoxygenases metabolize arachidonic acid to 4 regioisomeric epoxyeicosatrienoic acids (EETs): 14,15-EET, 11,12-EET, 8,9-EET, and 5,6-EET. 1 The EETs have a range of biological activities including vasodilation, 2,3 release of catecholamines 4 and hypothalamic hormones, 5 and alteration of sodium transport. 6 In some instances, the 4 EET regioisomers are equipotent in biological activity, 2 and in other instances, a particular regioisomer is active, whereas other isomers are inactive or less active. 7 EETs are metabolized by soluble epoxide hydrolase to dihydroxyeicosatrienoic acids (DHETs) 8 by ␤-oxidation to short-chain epoxides 9 or by incorporation into membrane phospholipids. 10 These pathways represent mechanisms of cellular inactivation and EET recycling. With the exception of 5,6-EET, the EETs are chemically stable in alkaline aqueous solution. 5,6-EET is hydrolyzed to 5,6-DHET and its ␦-lactone under neutral and acidic conditions. 11,12 In some studies, the methyl ester of 5,6-EET is used because of its improved chemical stability. The chemical instability has limited investigation of this EET regioiso...

14,15-Dihydroxy-eicosa-5(Z)-enoic Acid Selectively Inhibits 14,15-Epoxyeicosatrienoic Acid-Induced Relaxations in Bovine Coronary Arteries

“…Unlike other EET regioisomers, 5,6-EET is a substrate for cyclooxygenase that metabolizes it to vasodilatory 5,6-epoxy-prostaglandins (Oliw, 1984;Carroll et al, 1993). This effect was substantiated in the present study when pretreatment of arteries with indomethacin (10 M), a cyclooxygenase inhibitor, caused marked reduction of 5,6-EET-Me-induced relaxation (data not shown) (Yang et al, 2005). 13-HTDA pretreatment (10 M) caused significant inhibition of 8,9-EET-, 11,12-EET-, and 14,15-EET-induced relaxations.…”

“…BCAs were prepared as described under Vascular Reactivity Studies (Rosolowsky and Campbell, 1993;Yang et al, 2005). 14,15-EE5ZE or 13-HTDA (1 M) was incubated for 10 or 30 min at 37°C in HEPES buffer (10 mM HEPES, 150 mM NaCl, 5 mM KCl, 2 mM CaCl 2 , 1 mM MgCl 2 , and 6 mM glucose, pH 7.4) in the presence and absence of coronary artery rings.…”

“…The samples were dried under a stream of nitrogen and analyzed by liquid chromatographyelectrospray ionization-mass spectrometry (LC/ESI-MS; 1100 LC/ MSD, SL model; Agilent, Palo Alto, CA) as described previously (Nithipatikom et al, 2001;Yang et al, 2005). 14,15-EE5ZE, 14,15-DHE5ZE, and 13-HTDA were measured in the selected ion monitoring mode by detecting the M-H ions (molecular mass minus a hydro-gen ion) of m/z 323, m/z 341, and m/z 325, respectively.…”

“…Previous studies have revealed that 5,6-EET is unstable at physiological buffer and is hydrolyzed to 5,6-DHET. The methyl ester analog of 5,6-EET (5,6-EET-Me) is more stable, so it was used as an alternative 5,6-EET agonist (VanderNoot and VanRollins, 2002;Yang et al, 2005). Unlike other EET regioisomers, 5,6-EET is a substrate for cyclooxygenase that metabolizes it to vasodilatory 5,6-epoxy-prostaglandins (Oliw, 1984;Carroll et al, 1993).…”

Endothelium-Derived Epoxyeicosatrienoic Acids and Vascular Function

“…18 This difference accounts for the finding that relaxation of the bovine coronary artery induced by a novel stable agonist of 5,6-EET, 5-(pentadeca-3(Z),6(Z),9(Z)-trienyloxy) pentanoic acid was inhibited by the cyclooxygenase inhibitor indomethacin. 19 The recent development of sEH inhibitors has helped to determine the physiological consequences of enhanced CYP activity and EET formation. Inhibitors of this enzyme enhance iberiotoxin-sensitive, EETinduced vasodilator responses in isolated human coronary arterioles, 20 highlighting the potential relevance of this pathway in humans.…”