Published online before print February 7, 2005, doi: 10.1161/01.HYP.0000153790.12735.f9
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(Hypertension. 2005;45:681.)
© 2005 American Heart Association, Inc.
Original Articles |
Stable 5,6-Epoxyeicosatrienoic Acid Analog Relaxes Coronary Arteries Through Potassium Channel Activation
From the Department of Pharmacology and Toxicology (W.Y., K.M.G., K.N., W.B.C.), Medical College of Wisconsin, Milwaukee; and Department of Biochemistry (L.M.R., B.S., K.K.S., J.R.F.), University of Texas Southwestern Medical Center, Dallas.
Correspondence to William B. Campbell, PhD, Department of Pharmacology and Toxicology, Medical College of Wisconsin, 8701 Watertown Plank Rd, Milwaukee, WI 53226. E-mail wbcamp{at}mcw.edu
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Abstract |
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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 µmol/L) induced concentration-dependent relaxations, with maximal relaxation of 86±5% and EC50 of 1 µmol/L. The relaxations were inhibited by the cyclooxygenase inhibitor indomethacin (10 µmol/L; max relaxation 43±9%); the ATP-sensitive K+ channel inhibitor glybenclamide (10 µmol/L; max relaxation 49±6%); and the large conductance calcium-activated K+ channel inhibitor iberiotoxin (100 nmol/L; max relaxation 38±6%) and abolished by the combination of iberiotoxin with indomethacin or glybenclamide or increasing extracellular K+ to 20 mmol/L. Whole-cell outward K+ current was increased nearly 6-fold by PTPA (10 µmol/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 46±13% and 52±7%, respectively; EC50 1µmol/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+ channels and (2) activation of smooth muscle large conductance calcium-activated K+ channels. PDPAs only activate large conductance calcium-activated K+ channels.
Key Words: arachidonic acids • cyclooxygenase • endothelium-derived factors
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Introduction |
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Cytochrome 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 catecholamines4 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 ß-oxidation to short-chain epoxides9 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 regioisomer and therefore has restricted our understanding of the role of 5,6-EET in biological systems. In addition, because the 8,9-double bonds, 11,12-double-bonds, and 14,15-double bonds are intact in 5,6-EET, it is a substrate for cyclooxygenase (COX) and is metabolized to biologically active 5,6-epoxy-prostaglandins (PGs).13–15 Because of the biological importance of 5,6-EET and limitations in chemical stability, we developed a stable 5,6-EET agonist, 5-(pentadeca-3(Z),6(Z),9(Z)-trienyloxy)pentanoic acid (PTPA), in which the 5,6-epoxide was replaced with an ether group. PTPA resists chemical and enzymatic hydrolysis and has similar vascular activity as 5,6-EET. PTPA and 5,6-EET relax bovine coronary arteries with equal potency. Relaxations induced by PTPA are mediated by increased opening of large conductance calcium-activated K+ (BKCa) channels and metabolism by COX to metabolites that open ATP-sensitive K+ (KATP) channels. These studies suggest that PTPA is a stable 5,6-EET agonist and a useful tool for investigating the activity of 5,6-EET in biological systems.
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Materials and Methods |
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Vascular Reactivity of Bovine Coronary Arteries
Bovine (Bos taurus) hearts were purchased from a local slaughterhouse, and the left anterior descending coronary artery was dissected and cleaned of connective tissue. Arteries of 2-mm diameter were cut into rings (3-mm width) and suspended on a pair of stainless hooks in a 6-mL water-jacketed organ chamber in Kreb’s buffer consisting of (in mmol/L) 119 NaCl, 4.8 KCl, 24 NaHCO3, 0.2 KH2PO4, 0.2 MgSO4, 11 glucose, 0.02 EDTA, and 3.2 CaCl2. The buffer was equilibrated with 95% O2-5%CO2 and maintained at 37°C. Tensions were recorded as described previously.2,16 The thromboxane-mimetic U46619 (10 to 20 nmol/L) was administered to contract the vessels to 50% to 75% of KCl-induced contraction. Cumulative additions of 5,6-EET or 5,6-EET analogs were added to the chamber. Concentrations were added after stable relaxations were reached, which averaged 4 to 10 minutes. In some studies, the arteries were incubated with indomethacin (10 mmol/L), iberiotoxin (IBTX; 100 nmol/L), glybenclamide (10 mmol/L), or vehicle for 10 minutes before U46619 contraction. In the high extracellular K+ studies, K+ was increased to 20 mmol/L by substitution of Na+. In a subset of experiments, endothelium was removed from the artery rings by gentle rubbing with a wooden stick. Results are expressed as percentage relaxation with 100% relaxation representing basal tension.
Whole-Cell Patch Clamp
Coronary smooth muscle cells (SMCs) were enzymatically dispersed using published vascular SMC methods.7 Whole-cell recordings of K+ currents were obtained in freshly isolated SMCs using an Axopatch 200B amplifier (Axon Instruments), pClamp 8 software (Axon Instruments), and standard methods and solutions as described previously.17 Briefly, macroscopic K+ currents were generated by progressive stepwise 10-mV depolarizing steps (500-ms duration, 5-s intervals) from a constant holding potential of –60 mV. Currents were sampled at 3 kHz and filtered at 1 kHz. After stable control (vehicle) currents were recorded, currents were obtained after application of PTPA (10 µmol/L) and PTPA plus IBTX (100 nmol/L). Membrane cell capacitance was estimated by integrating the capacitive current generated by a 10-mV hyperpolarizing pulse after electronic cancellation of pipette-patch capacitance.
Stability of 5,6-EET and PTPA
5,6-EET and PTPA (10 ng/mL each) were incubated in distilled water at 37°C. At 0, 10, 30, 60, and 120 minutes, an aliquot (5 µL) was analyzed for 5,6-EET and PTPA by liquid chromatography/mass spectrometry (LC/MS) using a modification of a method described previously.18 The LC/MS analysis used an Agilent 1100 mass selective detection mass spectrometer with electrospray ionization (ESI). The eicosanoids were separated on a Phemomenex Prodigy C-18 (5 µm; 1.0x50 mm) column. Solvent A was water and solvent B was acetonitrile, with both solvents containing 0.005% glacial acetic acid. 5,6-EET eluted at 3.99 minutes and PTPA at 5.56 minutes using a 4-minute linear gradient from 55% to 65% B in A at a flow rate of 0.1 mL/min. Detection was in the negative ion mode for the M-H ions 319 and 321 mass/charge for 5,6-EET and PTPA, respectively. Results are expressed as percentage of control with the integrated area under the curve at time 0 equal to 100%.
Chemicals
PTPA was synthesized as described in the supplemental material (available online at http://www.hypertensionaha.org). IBTX, indomethacin, and glybenclamide were obtained from Sigma. U46619 was obtained form Cayman Chemical. Indomethacin was dissolved in 95% ethanol, and all other drugs were dissolved in distilled water.
Statistics
Data are expressed as means±SEM. Statistical evaluation of the data were performed by 1-way ANOVA followed by the Student-Newman–Keuls multiple comparison test when significant differences were present. P<0.05 was considered statistically significant.
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Results |
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The structures and chemical abbreviations for 5,6-EET and the 5,6-EET analogs tested for vascular activity are shown in the figures. The analogs differed from 5,6-EET (Figure 1) by (1) substitution of the C-1 carboxyl with a methylester, 5,6-EET-Me (Figure 2); (2) substitution of the 5,6 epoxide with an ether, PTPA (Figure 1); (3) substitution of the 5,6 epoxide with an ether and elimination of the
8 double bond, P-6,9-DPA (see Figure 5); and (4) substitution of the 5,6 epoxide with an ether and elimination of the 11 double bond, P-3,9-DPA (see Figure 5).
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significantly different from PDPA; P<0.05.5,6-EET and PTPA were incubated at 37°C in water for various times (Figure 1). When the eicosanoids were measured by LC/MS, the concentration of 5,6-EET decreased with a half life <10 minutes. In contrast, the concentration of PTPA was unchanged. This demonstrates that PTPA is a chemically stable analog of 5,6-EET.
In bovine coronary arteries precontracted with U46619, 5,6-EET-Me caused concentration-dependent relaxation, with an EC50 of 1 µmol/L and a maximum relaxation of 79±9% at 10 µmol/L (Figure 2A). Pretreatment with indomethacin (10 µmol/L) markedly attenuated 5,6-EET-Me–induced relaxations (maximal relaxation 27±11%). Similar to 5,6-EET-Me, PTPA relaxed U46619-precontracted arteries with maximal relaxation of 86±5% at 10 µmol/L and EC50 of 1 µmol/L (Figure 2B). Pretreatment with indomethacin significantly attenuated the PTPA-mediated relaxation (maximal relaxation 43±9%). Because COX metabolism is endothelium dependent, we examined PTPA relaxations in endothelium-denuded vessels. Removal of endothelium reduced PTPA-mediated vasodilation (maximum relaxation 46±9%; Figure 2B). Addition of indomethacin to denuded vessels did not further alter the relaxation response (Figure 2B). These results suggest that PTPA retained full agonist activity and potency compared with 5,6-EET-Me, and similar to 5,6-EET and 5,6-EET-Me, PTPA relaxations are mediated, at least in part by endothelial COX metabolism.
We next examined the effect of K+ channel inhibition on PTPA-mediated relaxations. Increasing extracellular K+ from 4 to 20 mmol/L eliminated relaxations to PTPA (maximum relaxation 2±2%; Figure 3A). The BKCa channel blocker IBTX (100 nmol/L) also inhibited the PTPA-induced relaxation by 50% (maximum relaxation 38±6%; Figure 3A). The combination of IBTX plus indomethacin abolished PTPA-induced relaxation (maximum relaxation 11±2%; Figure 3A). Similarly, pretreatment with the KATP channel blocker glybenclamide (10 µmol/L) inhibited relaxations to PTPA by 45% (maximal relaxation 49±6%; Figure 3B), and the combination of glybenclamide plus IBTX blocked PTPA-induced relaxations (maximum relaxation 8±4%; Figure 3B). Figure 4 shows tracings of macroscopic whole-cell K+ currents that were generated by 10-mV depolarizing steps from –60 to +60 mV in an isolated bovine coronary SMC. Application of PTPA (10 µmol/L) increased outward current of this cell >6-fold from vehicle control (245±12 to 1513±24 pA). Further addition of IBTX (100 nmol/L) decreased the outward K+ current to 96±6 pA. Capacitance of this cell was 12.2 pF. Thus, direct application of PTPA to smooth muscle activates IBTX-sensitive BKCa currents. Together, these results demonstrate that PTPA-induced relaxation is mediated by activation of KATP and BKCa channels.
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significantly different from PTPA plus glybenclamide; P<0.05.
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To further evaluate the contribution of COX metabolism and KATP channels, we examined the vasoactivities of 2 PTPA analogs, P-6,9-DPA and P-3,9-DPA, which lack the 8 and 11 double bonds, respectively. COX metabolism of arachidonic acid and its congeners requires the 8 and 11 double bonds; therefore, these PTPA analogs do not serve as COX substrates. P-3,9-DPA and P-6,9-DPA relaxed precontracted coronary arteries in a concentration-dependent manner but were less potent than PTPA (Figure 5A and 5B). Maximal relaxations were 52±6.5% and 53±10% for P-3,9-DPA and P-6,9-DPA, respectively, which are comparable to the PTPA-induced relaxation in the presence of indomethacin. IBTX blocked relaxations to P-3,9-DPA and P-6,9-DPA (maximal relaxations were 9.0±4% and 10±3%, respectively; Figure 5A and 5B), whereas treatment with indomethacin or glybenclamide, or removal of endothelium did not alter relaxations to P-6,9-DPA (Figure 5B and 5C). These results demonstrated that 5-(pentadeca-3(Z),9(Z)-dienyloxy)pentanoic acid (PDPA)-induced relaxations are exclusively mediated by BKCa channel activation.
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Discussion |
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Previous studies by our laboratory and many others have shown that 5,6-EET causes relaxation in multiple vascular beds.2,3,16,19–21 However, 5,6-EET is unstable in physiological buffer, with a short half-life
8 minutes.12 It spontaneously hydrolyzes into 5,6-DHET and its -lactone.11,12 Epoxide hydrolysis is catalyzed by acid or epoxide hydrolase.8 The C-1 carboxyl group of 5,6-EET is in close proximity to the 5,6-epoxide group and acts as an acid catalyst for hydration. Esterification of the carboxyl group greatly stabilized the compound, and 5,6-EET-Me is a widely used agonist of 5,6-EET. Here, by substituting the 5,6-epoxide with an ether group, we developed a structural analogue of 5,6-EET (ie, PTPA) that does not contain a labile epoxide. Our studies showed that PTPA causes concentration-dependent relaxation of coronary arteries and retained full agonist potency and activity compared with 5,6-EET-Me and 5,6-EET. We reported previously that 5,6-EET induces concentration-dependent relaxation of bovine coronary arteries with an EC50 of 1 µmol/L and maximal effect of 80% to 90%.2,16,22 PTPA relaxed bovine coronary arteries, with a similar EC50 of 1 µmol/L and maximal effect of 86%±5.
In the coronary circulation, EETs function as endothelium-derived hyperpolarizing factors.2,23–26 EETs are released by endothelial cells and activate vascular smooth muscle BKCa channels.2 PTPA-induced relaxation was partly blocked by IBTX, a specific inhibitor of BKCa channels. Direct application of PTPA increased SMC outward K+ current, and the current is abolished by IBTX. These results suggest that PTPA activates IBTX-sensitive K+ channels, hence it causes hyperpolarization and relaxation of vascular smooth muscle. In addition to the IBTX-sensitive component of PTPA-induced relaxation, there is a portion of the relaxation that remains unaffected by IBTX. Unlike other EETs regioisomers, 5,6-EET is a substrate for COX, which transforms it into 5,6-epoxy-PGs.13–15 With intact 8 and 11 double bonds, PTPA should also be a substrate for COX. Indomethacin and endothelium removal inhibited PTPA-mediated relaxation by 54% and 50%, respectively, whereas addition of indomethacin to denuded vessels did not further attenuate PTPA-mediated vasodilation. Additionally, blockade of KATP channel activity with glybenclamide similarly inhibited the PTPA-mediated relaxation, and the IBTX-resistant component of PTPA-induced relaxation was blocked by indomethacin or glybenclamide. Similar to the PGs, some COX metabolites of 5,6-EET have vasoactivity. 5,6-epoxy-PGE1 was equipotent to PGE2 as a vasodilator in the isolated perfused rabbit kidney.15 PGI2, the major endothelial COX metabolite of arachidonic acid, activates KATP channels.27,28 Hence, PTPA may undergo COX metabolism to vasoactive prostanoids that similarly activate KATP channels to hyperpolarize and relax blood vessels. In the whole-cell patch-clamp studies, IBTX completely blocked the PTPA-induced K+ current. In SMCs, COX activity is only 20% of endothelial COX activity.29 Thus, decreased COX activity may explain the lack of KATP currents activated by PTPA in our isolated SMC preparation. The effects of IBTX and indomethacin/glybenclamide on relaxation to PTPA were additive, whereas the effects of indomethacin and glybenclamide were redundant. This further confirms that PTPA COX metabolites mediate relaxations through KATP channel activation. In some vascular beds, 5,6-EET is metabolized by COX to vasoconstrictor metabolites that activate thromboxane receptors.30,31 However, bovine coronary artery endothelial cells do not produce thromboxane,16,32 and constrictor activity to 5,6-EET or its analogs was not observed in bovine coronary arteries. These data suggested that coronary artery relaxations mediated by PTPA result from 2 distinct pathways: (1) COX-dependent metabolism to a metabolite that activates KATP channels, and (2) direct activation of BKCa channels.
5,6-EET may promote the release of COX metabolites.15,33 It is not known whether the COX metabolite of 5,6-EET or activation of COX by 5,6-EET is responsible for 5,6-EET vasoactivity. We developed and tested 2 5,6-EET structural analogs: P-3,9-DPA and P-6,9-DPA. These 2 PTPA analogs lack 8 or 11 double bond, hence, they are not COX substrates. PDPAs showed decreased relaxant activity compared with PTPA but similar activity as PTPA in the presence of indomethacin. PDPA-induced relaxation was not sensitive to indomethacin, glybenclamide, or endothelium removal but was abolished by IBTX. Therefore, removal of either the 8 or 11 double bond from PTPA results in the loss of COX metabolism as well as the glybenclamide-sensitive component of its vasoactivity. This indicates that metabolism of PTPA by COX is responsible for the IBTX-resistant portion of PTPA-mediated relaxation. PDPAs retained the ability to activate IBTX-sensitive relaxation. Therefore, PDPA-mediated relaxation represents the BKCa-dependent function of 5,6-EET that is shared by all 4 EET regioisomers.
For 14,15-EET, the 8 double bond is required for full agonist activity.34 The IBTX-sensitive component of PTPA-mediated relaxation was not affected by removal of 8 double bond. Similarly, 11,12-epoxyeicosa-5-Z-enoic acid, an 11,12-EET analog lacking 8 double bond, retained nearly full agonist activity.34 Therefore, the 8 double bond requirement for agonist activity for 1 EET regioisomer may not pertain to the other regioisomers.
Perspectives
In summary, the present study showed that PTPA is a hydrolysis-resistant stable agonist of 5,6-EET. Like 5,6-EET, it causes relaxation of bovine coronary arteries by the direct activation of smooth muscle BKCa channels and by COX metabolism to a metabolite(s) that activates KATP channels. In contrast to PTPA, the 5,6-EET analogs PDPAs are not COX substrates, and their vasorelaxant activity is limited to activation of BKCa channels. Therefore, PTPA and PDPAs are useful pharmacological tools for the isolation and characterization of 5,6-EET BKCa- and COX-dependent activities in biological systems.
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Acknowledgments |
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These studies were supported by grants from the National Institutes of Health (HL-51055, GM-31278, and DK-38266) and the Robert A. Welch Foundation. The authors thank Marilyn Isbell and Sarah Hittner for their technical assistance and Gretchen Barg for secretarial assistance.
Received October 8, 2004; first decision November 3, 2004; accepted December 9, 2004.
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References |
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 B. T. Larsen, H. Miura, O. A. Hatoum, W. B. Campbell, B. D. Hammock, D. C. Zeldin, J. R. Falck, and D. D. Gutterman Epoxyeicosatrienoic and dihydroxyeicosatrienoic acids dilate human coronary arterioles via BKCa channels: implications for soluble epoxide hydrolase inhibition Am J Physiol Heart Circ Physiol, February 1, 2006; 290(2): H491 - H499. [Abstract] [Full Text] [PDF]
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