This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Campbell, W. B. Articles by Harder, D. R. Search for Related Content PubMed PubMed Citation Articles by Campbell, W. B. Articles by Harder, D. R.

(Circulation Research. 1996;78:415-423.)
© 1996 American Heart Association, Inc.

Articles

Identification of Epoxyeicosatrienoic Acids as Endothelium-Derived Hyperpolarizing Factors

William B. Campbell, Debebe Gebremedhin, Phillip F. Pratt, David R. Harder

From the Departments of Pharmacology and Toxicology and Physiology and the Cardiovascular Research Center, Medical College of Wisconsin, 8701 Watertown Plank Rd, Milwaukee, WI 53226.

Correspondence to William B. Campbell, PhD, Department of Pharmacology and Toxicology, Medical College of Wisconsin, 8701 Watertown Plank Rd, Milwaukee, WI 53226.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Abstract Endothelial cells release several compounds, including prostacyclin, NO, and endothelium-derived hyperpolarizing factor (EDHF), that mediate the vascular effects of vasoactive hormones. The identity of EDHF remains unknown. Since arachidonic acid causes endothelium-dependent relaxations of coronary arteries through its metabolism to epoxyeicosatrienoic acids (EETs) by cytochrome P₄₅₀, we wondered if the EETs represent EDHFs. Precontracted bovine coronary arteries relaxed in an endothelium-dependent manner to methacholine. The cytochrome P₄₅₀ inhibitors, SKF 525A and miconazole, significantly attenuated these relaxations. They were also inhibited by tetraethylammonium (TEA), an inhibitor of Ca²⁺-activated K⁺ channels, and by high [K⁺]_o (20 mmol/L). Methacholine also caused hyperpolarization of coronary smooth muscle (-27±3.9 versus -40±5.1 mV), which was completely blocked by SKF 525A and miconazole. In vessels prelabeled with [³H]arachidonic acid, methacholine stimulated the release of 6-ketoprostaglandin F₁, 12-HETE, and the EETs. Arachidonic acid relaxed precontracted coronary arteries, which were also blocked by TEA, charybdotoxin, another Ca²⁺-activated K⁺ channel inhibitor, and high [K⁺]_o. 14,15-EET, 11,12-EET, 8,9-EET, and 5,6-EET relaxed precontracted coronary vessels (EC₅₀, 1x10^-6 mol/L). The four regioisomers were equally active. TEA, charybdotoxin, and high [K⁺]_o attenuated the EET relaxations. 11,12-EET hyperpolarized coronary smooth muscle cells from -37±0.2 to -59±0.3 mV. In the cell-attached mode of patch clamp, both 14,15-EET and 11,12-EET increased the open-state probability of a Ca²⁺-activated K⁺ channel in coronary smooth muscle cells. This effect was blocked by TEA and charybdotoxin. These data support the hypothesis that the EETs are EDHFs.

Key Words: cytochrome P₄₅₀ • arachidonic acid • K⁺ channels • smooth muscle • membrane potential

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Endothelial cells release a variety of compounds that regulate vascular tone, including prostacyclin, EDRF, and endothelin.^{1 2 3} It is now recognized that EDRF is NO or a compound that releases NO.⁴ Feletou and Vanhoutte⁵ showed that acetylcholine causes endothelium-dependent hyperpolarization of coronary vascular smooth muscle that results in relaxation. This effect is mediated by a soluble transferrable factor.^{5 6 7 8 9 10} This substance has been called EDHF. It is distinct and may be distinguished from EDRF or NO in several ways. The release of EDHF is mediated by M₁ muscarinic receptors, whereas the release of NO is mediated by M₂ receptors.^{8 11} EDHF is inhibited by ouabain, but NO is not.⁵ Relaxation by EDHF is more prominent in arteries with smaller diameters, unlike NO, which is greater in larger vessels.^{12 13} Hemoglobin, gossypol, and arginine analogues, such as nitro-L-arginine, inhibit NO-mediated relaxations but do not alter EDHF.^{9 14 15} Acetylcholine-induced hyperpolarization appears to be mediated by the opening of K⁺ channels, since its effect is mimicked by cromakalim, a K⁺ channel opener, and inhibited by increasing [K⁺]_o.^{15 16} In coronary and mesenteric arteries and aorta, the relaxation and hyperpolarization are blocked by TEA but not by glibenclamide.^{14 17 18} These data suggest that EDHF acts on the Ca²⁺-dependent K⁺ channels inhibited by TEA and not the ATP-sensitive K⁺ channels opened by cromakalim and inhibited by glibenclamide. Although most agree that NO and EDHF are distinct entities, several articles indicate that NO also hyperpolarizes vascular smooth muscle and stimulates K⁺ channel activity.^{19 20 21 22 23} These reports differ in the type of K⁺ channel affected by NO: two reports indicate inhibition by glibenclamide,^{19 22} whereas two other reports indicate inhibition by charybdotoxin.^{20 21}

We have recently reported that arachidonic acid causes endothelium-dependent relaxation of bovine coronary arteries.²⁴ A portion of the relaxation was inhibited by the cyclooxygenase inhibitor indomethacin, and the remainder was inhibited by an inhibitor of cytochrome P₄₅₀. Coronary endothelial cells and coronary arteries metabolize arachidonic acid via the cyclooxygenase, lipoxygenase, and cytochrome P₄₅₀ pathways.^{24 25 26 27} Prostacyclin is the major cyclooxygenase metabolite, whereas 14,15-EET, 11,12-EET, 8,9-EET, and 5,6-EET are the cytochrome P₄₅₀ metabolites. Since both prostacyclin and the EETs relax coronary arteries,^{24 26 27} they were proposed to mediate the endothelium-dependent relaxations induced by arachidonic acid. Since EETs are synthesized and released by the endothelium and relax arteries, we examined the possibility that they represent EDHFs.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Vascular Studies
Bovine hearts (2 to 4 kg) were obtained from the local slaughterhouse. The left anterior descending coronary artery was dissected, cleaned of adhering fat and connective tissue, and placed in a Krebs' bicarbonate solution containing the following (mmol/L): NaCl 119, KCl 4.8, NaHCO₃ 24, KH₂PO₄ 1.2, MgSO₄ 1.2, glucose 11, EDTA 0.02, and CaCl₂ 3.2.²⁴ The vessels were cut into rings, and care was taken not to damage the endothelium. In some vessels, the endothelium was deliberately removed by gently rubbing the lumen with forceps. The rings were suspended on a pair of stainless steel hooks in a 15-mL water-jacketed organ chamber. One hook was anchored to a steel rod, and the other was attached to a force transducer (model FT-03C, Grass Instruments). Tension was recorded on a Grass model 7D polygraph. The organ chamber was filled with Krebs' bicarbonate solution, which was bubbled with 95% O₂/5% CO₂ and maintained at 37°C. Vessels were equilibrated for 1.5 hours under 2 g of resting tension. KCl (40 mmol/L) was added until reproducible contractions were obtained. The thromboxane mimetic U46619 (1x10^-8 to 2x10^-8 mol/L) was then administered to increase basal tone to 50% of maximum. Cumulative additions of methacholine (10^-9 to 10^-6 mol/L), EETs (10^-9 to 10^-5 mol/L), or arachidonic acid (10^-9 to 10^-5 mol/L) were made. To establish the mechanisms of relaxation, some vessels were treated with SKF 525A (10^-5 mol/L), miconazole (10^-5 mol/L), TEA (0.3 or 1 mmol/L), charybdotoxin (2x10^-9 or 50x10^-9 mol/L), glibenclamide (2x10^-6 mol/L), or vehicle, and the concentration-response curve to an agonist was determined. Similar experiments were performed with [K⁺] in the Krebs' solution reduced to 0 mmol/L or increased to 20 mmol/L. Equivalent changes in [Na⁺] were made to maintain isotonicity. Results were expressed as percent relaxation, with 100% relaxation representing the basal pre-U46619 tension.

NOS Activity
Bovine coronary arteries were isolated and homogenized in a HEPES buffer containing 11 mmol/L HEPES, 0.35 mol/L sucrose, 0.1 mmol/L EDTA, 1 mmol/L dithiothreitol, 10 µg/mL leupeptin, 2 µg/mL aprotinin, 10 µg/mL SBTI, 10 µg/mL PMSF, 1% NP-40, and 10% glycerol, pH 7.4.²⁸ Bovine coronary artery endothelial cells were prepared as previously described.²⁵ Confluent monolayers were washed in HEPES buffer, scraped into fresh buffer, and sonicated. The conversion of [³H]arginine to [³H]citrulline was used to assess NOS activity.²⁸ The reaction volume was 0.1 mL in a Tris incubation buffer containing 50 mmol/L Tris-HCl (pH 7.5), 0.1 mmol/L EDTA, 0.1 mmol/L EGTA, 0.1% mercaptoethanol, 100 µmol/L leupeptin, 1 mmol/L PMSF, 10 µg/mL SBTI, 2 µg/mL aprotinin, 10 nmol/L calmodulin, 1 mmol/L NADPH, 3 µmol/L tetrahydrobiopterin, 10 µmol/L L-arginine, 0.2 µCi, 66 Ci/mmol [³H]L-arginine (New England Nuclear), 2.5 mmol/L calcium chloride, and 100 µg protein. Nitro-L-arginine (3x10^-5 mol/L), SKF 525A (10^-5 mol/L), miconazole (10^-5 mol/L), or vehicle was added. All samples were incubated for 15 minutes at 37°C in a shaking water bath, and the reaction was stopped by the addition of 1 mL of ice-cold stop buffer containing 20 mmol/L HEPES (pH 5.5), 2 mmol/L EDTA, and 2 mmol/L EGTA. The [³H]citrulline was separated from the [³H]arginine by passing the sample over a Dowex AG50W-X8 (200 to 400 mesh) column (Na⁺ form) and measured by liquid scintillation spectrometry. NOS activity was expressed as femtomoles of citrulline per milligram protein. Protein was determined by the method of Lowry et al.²⁹

Perfused Coronary Artery
Coronary arteries were isolated as described above, cannulated on both ends, and placed in a tissue bath containing Krebs' buffer at 37°C. The vessel was then perfused with Krebs' buffer at 3 mL/min in a nonrecirculating system. Perfusion pressure was maintained at 60 mm Hg by an outflow restrictor and measured continuously by a pressure transducer. The vessel was treated every 5 minutes over a period of 30 minutes with KCl (20 mmol/L). After 1 hour, [³H]arachidonic acid (5 µCi) was added to the perfusate, and the perfusate was recirculated through the vessel for 45 minutes. To remove unesterified [³H]arachidonic acid, the vessel was then perfused in a nonrecirculating system with fresh Krebs' buffer containing fatty acid–free bovine serum albumin (2 mg/mL). Once the amount of radioactivity in the perfusate stabilized, the perfusate was collected for 10 minutes (control). The vessel was then perfused with buffer containing methacholine (10^-5 mol/L), and the perfusate was collected for 10 minutes.

HPLC and Mass Spectrometry
Perfusate samples were acidified to pH 3 with glacial acetic acid and treated with ethanol to a final concentration of 15%. The lipids were then extracted from the perfusates using octadecylsilyl extraction columns (Analytichem) as previously described.^{24 25 30 31} The extracts were dried under N₂, redissolved in acetonitrile, and chromatographed on a Nucleosil C-18 reverse-phase column (5 µm, 4.5x250 mm, Phenomenex).^{24 25 30 31} Solvent A was distilled water, and solvent B contained acetonitrile/glacial acetic acid (999:1). A linear gradient from 50% solvent B in solvent A to 100% solvent B was used for 40 minutes at a flow rate of 1 mL/min. The column effluent was collected in 0.2-mL fractions, and an aliquot was analyzed for radioactivity by liquid scintillation spectrometry. Known standards were analyzed in the same manner. The fractions containing radiolabeled material comigrating with authentic EETs were collected, pooled, and dried under N₂. They were then dissolved in acetonitrile containing pentafluorobenzyl bromide (Pierce Chemical) and N,N-diisopropylethanolamine (10:1:5 [vol/vol]) and incubated for 20 minutes at room temperature.³¹ The solvents were removed under N₂, and the residue was filtered through a column of silicic acid using methylene chloride. The EET-PFB esters were analyzed on a Hewlett-Packard model 5989A GC/MS in the negative-ion chemical ionization mode.³¹ The GC used a 15-m DB-5 (0.25 µm) capillary column (J & W Scientific) with a linear gradient from 190°C to 340°C at 8°C/min. Methane was used as the reagent gas.

Membrane Potential
Bovine coronary arteries were isolated, cannulated, and perfused with Krebs' solution in the direction of flow in vivo at 37°C from a pressurized reservoir.^{10 32} The pressure of the vessel was maintained at 60 mm Hg by an outflow resistor. Smooth muscle cells of the perfused vessel were impaled with microelectrodes for the measurement of intracellular membrane potential as previously described.^{10 32} These studies used a glass microelectrode filled with 3 mol/L KCl with a tip resistance of 50 to 80 M and tip potential of <3 mV. Electrode polarization was eliminated by Ag/AgCl half cells. The main criteria for a successful impalement include a sharp drop in voltage from baseline on entry of the microelectrode into the cell, a membrane potential of <-25 mV, and a sharp return to zero of the voltage upon exit from the cell. Methacholine (10^-6 mol/L) was added to the perfusate in the presence and absence of SKF 525A or miconazole (10^-5 mol/L). Similar studies were performed in which 11,12-EET (100 nmol/L) was added to the perfusate, and changes in membrane potential were measured.

Patch-Clamp Methods
Coronary arterial smooth muscle cells were prepared as previously described³³ and placed in a 1-mL perfusion chamber on an inverted microscope stage. The bathing solution consisted of (mmol/L) KCl 145, CaCl₂ 1.8, MgCl₂ 1.1, HEPES 5, and EGTA 10 at pH 7.4, and the pipette solution contained (mmol/L) KCl 145, CaCl₂ 1.8, MgCl₂ 1.1, and HEPES 5, at pH 7.4. By using a hydraulic manipulator, the tip of a heat-polished borosilicate glass pipette was positioned on the membrane of a cell. A high-resistance seal (1 to 10 G) was formed between the pipette and the cell membrane by applying a slight suction. After the cell-attached or inside-out configuration was formed, the bath containing the cells or patches was superfused with the control solution by gravity flow from a reservoir at 5 mL/min. Cells were superfused with 14,15-EET, 11,12-EET, or arachidonic acid in various concentrations. Inside-out patches were superfused with miconazole (10^-5 mol/L) or SKF 525A (10^-5 mol/L). Single-channel currents were recorded using a List EPC-7 patch-clamp amplifier.³³ Data were low-pass–filtered at 1 kHz, digitized at a sampling rate of 3 kHz, analyzed by pClamp software (version 5.5), and stored on an IBM-compatible 386 computer for off-line analysis. Ion channels were identified by determining the current-voltage relationship, voltage-dependent activation, and Ca²⁺ sensitivity of the channel in inside-out membrane patches. The current-voltage relationship was determined by varying the patch potential between -60 and +60 mV. A large-conductance (240-pS) single K⁺ channel current, which had an open-state probability that increased with depolarization and increased with the concentration of Ca²⁺, was observed. It was blocked by TEA (1 mmol/L) and charybdotoxin (2 or 50 nmol/L). This channel, therefore, possesses the characteristics of a large-conductance Ca²⁺-activated K⁺ channel.

Cyclic Nucleotide Assays
Bovine coronary artery rings were incubated in HEPES buffer (mmol/L: HEPES 10, NaCl 150, KCl 5, CaCl₂ 1.8, MgCl₂ 1, and glucose 5.5, at pH 7.4) for 5 minutes at 37°C in the presence of isobutylmethylxanthine (0.1 mmol/L) before the addition of the EETs (10^-5 mol/L), sodium nitroprusside (10^-7 mol/L), isoproterenol (10^-6 mol/L), or vehicle.^{34 35} After a 5-minute incubation, the tissue was removed and immersed in liquid N₂. The frozen tissue was placed in 7% trichloroacetic acid in water and frozen in a dry ice/methanol bath. The samples were stored at -80°C until assayed by radioimmunoassay. Before assay, the tissues were homogenized with a Tissuemizer (Janke-Knuckel, GMBH and Co) and centrifuged, and the supernatant was removed. The supernatant was extracted with water-saturated ether three times, dried under N₂, and resuspended in assay buffer. cAMP and cGMP were measured by radioimmunoassay as previously described.^{34 35}

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bovine coronary vessels that were precontracted with U46619 produced a concentration-related relaxation to methacholine (Fig 1A). No relaxations occurred to methacholine in vessels without an intact endothelium. Pretreatment with miconazole (10^-5 mol/L) or SKF 525A (10^-5 mol/L), inhibitors of cytochrome P₄₅₀, did not alter basal tension or contractions to U46619. However, they blocked the relaxations to methacholine. The relaxations were also blocked by TEA, an inhibitor of Ca²⁺-activated K⁺ channels, and by high [K⁺]_o (20 mmol/L) (Fig 1A). Membrane potential was measured in smooth muscle cells of coronary arteries with an intact endothelium under similar conditions (Fig 1B). The resting membrane potential was -56±3.4 mV and decreased to -27±3.9 mV by pretreatment with U46619. In these pretreated vessels, methacholine increased the membrane potential to -40±5.1 mV (P<.01). The methacholine-induced hyperpolarization was blocked by miconazole as well as SKF 525A. Thus, methacholine produces endothelium-dependent relaxation and hyperpolarization of the vascular muscle, which apparently are mediated by a metabolite(s) of cytochrome P₄₅₀.

View larger version (28K): [in this window] [in a new window]   Figure 1. Effect of inhibitors of cytochrome P450 on methacholine-induced relaxations (A) and membrane hyperpolarization (B) in bovine coronary arteries. Vessels were pretreated with vehicle, TEA chloride (10-3 mol/L), SKF 525A (10-5 mol/L), or miconazole (MIC, 10-5 mol/L). In another set of vessels, the concentration of K+ in the Krebs' solution was increased from 4.8 to 20 mmol/L, and in another set of vessels, the endothelium was removed. U46619 (20 nmol/L) was then added to precontract or depolarize the vessels, and methacholine (MECH) was added. Each value represents the mean±SEM.

Coronary artery homogenates and bovine coronary endothelial cell lysates had a basal NOS activity of 27±2 and 702±13 fmol citrulline per milligram protein, respectively (Table 1). Miconazole and SKF 525A were without effect on NOS activity. For comparison, nitro-L-arginine inhibited NOS activity by 40% and 90% in the artery homogenates and cell lysates, respectively. These agents were also tested on K⁺ channel activity in inside-out patches of coronary smooth muscle cells using patch clamp (Table 1). Neither miconazole nor SKF 525A changed the open-state probability of K⁺ channels. However, charybdotoxin, an inhibitor of Ca²⁺activated K⁺ channels, significantly inhibited K⁺ channel activity. Thus, the inhibitory effects of these agents on methacholine-induced relaxations and hyperpolarization are not due to inhibition of NO synthesis or a direct action on K⁺ channels.

View this table: [in this window] [in a new window]   Table 1. Effect of Cytochrome P450 Inhibitors on NOS and K+ Channel Activity

In perfused coronary arteries in which the cellular lipids were prelabeled with [³H]arachidonic acid, the basal release of [³H]arachidonic acid and its ³H metabolites was very low (Fig 2A). Treatment with methacholine (10^-5 mol/L) greatly increased the release of ³H metabolites (Fig 2B). Specifically, methacholine stimulated the release of several arachidonic acid metabolites, including prostacyclin (measured as its stable metabolite 6-ketoprostaglandin F₁), 12-HETE, and the EETs. The fractions containing the EETs were collected, derivitized to PFB esters, and analyzed by GC/MS. The derivitized sample comigrated on GC with authentic EET-PFB standards, with both eluting at 16.1 minutes. They also gave similar mass spectra. The major ions in the mass spectrum were 319 (loss of PFB) and 301 (loss of PFB and H₂O) and indicated a molecular weight of 320 (Fig 2C). These findings confirm the release of EETs from the perfused vessel with methacholine. The synthesis of the EETs was shown previously to be inhibited by SKF 525A and miconazole.^{24 36}

View larger version (18K): [in this window] [in a new window]   Figure 2. Effect of methacholine on the release of metabolites of arachidonic acid (AA) from perfused bovine coronary arteries. Coronary arteries were prelabeled with [3H]AA as described in "Materials and Methods." Perfusate was collected in the absence and presence of methacholine (10-5 mol/L). The perfusate was extracted, and the 3H metabolites were resolved by HPLC. Migration times of known standards are shown above the chromatograph. A and B, Metabolites formed under control conditions (A) and after methacholine treatment (B). The fractions comigrating with the EETs were collected, derivitized, and analyzed by GC/MS. C, Mass spectrum.

Like methacholine, arachidonic acid relaxes coronary vessels in an endothelium-dependent manner, and these relaxations are inhibited 50% by SKF 525A.²⁴ The relaxations to arachidonic acid are also inhibited by pretreatment with TEA (Fig 3B), charybdotoxin (Fig 3B), and increasing [K⁺]_o (20 mmol/L) (Fig 3A). The cytochrome P₄₅₀ metabolites, 14,15-EET, 11,12-EET, 8,9-EET, and 5,6-EET, caused a concentration-related relaxation of precontracted coronary arteries (Fig 4A). The four regioisomers were equipotent, with a threshold relaxation occurring at 10^-8 mol/L. This effect of the EETs occurred in the presence and absence of the endothelium (control: ED₅₀, 4.3x10^-5 mol/L; denuded: ED₅₀, 4.8x10^-5 mol/L; P=NS). Additionally, 11,12-EET (100 nmol/L) increased the membrane potential from -37±0.2 to -59±0.3 mV (n=8, P<.001). Thus, EETs, like methacholine, relaxed coronary arteries and hyperpolarized coronary smooth muscle.

View larger version (20K): [in this window] [in a new window]   Figure 3. Effect of arachidonic acid on vascular tone in bovine coronary arteries. Vessels were precontracted with U46619 (20 nmol/L), and arachidonic acid was added in increasing concentrations. Changes in isometric tension were measured. A, Effect of changes in [K+]o on arachidonic acid–induced relaxations. Relaxations were measured in Krebs' solution containing 0, 4.8, and 20 mmol/L KCl. B, Effect of TEA (1 mmol/L) and charybdotoxin (CHTX, 50 nmol/L) on arachidonic acid–induced relaxations. Each value represents the mean±SEM.

View larger version (17K): [in this window] [in a new window]   Figure 4. Effects of EETs on vascular tone in bovine coronary arteries. Vessels were precontracted with U46619 (20 nmol/L), and the EETs were added in various concentrations. Changes in isometric tension were measured. A, Effects of the four regioisomeric EETs on vascular tone. B, Effects of changes in [K+]o on EET-induced relaxations. 11,12-EET–induced relaxations were measured in Krebs' solution containing 0, 4.8, and 20 mmol/L KCl. C, Effects of glibenclamide (2 µmol/L) on the 5,6-EET–induced relaxations. Each value represents the mean±SEM.

The EET-induced relaxations were inhibited by pretreatment with TEA, charybdotoxin, and increasing [K⁺]_o but not by glibenclamide. 11,12-EET caused a concentration-related relaxation (Fig 4B). Eliminating [K⁺]_o shifted the concentration-response curve to 11,12-EET to the left, whereas increasing [K⁺]_o to 20 mmol/L blocked the relaxations to EET. 5,6-EET–induced relaxations are shown in Fig 4C. Glibenclamide, an inhibitor of ATP-sensitive K⁺ channels, had no significant effect on 5,6-EET–induced relaxations. 8,9-EET also relaxed coronary arteries, and pretreatment with TEA (0.3 and 1 mmol/L) shifted the concentration-response curve to the right (Fig 5). A similar result was observed with charybdotoxin (2 or 50 nmol/L). Using the patch clamp technique in the cell-attached mode, a high-conductance single-channel K⁺ current was recorded from isolated coronary smooth muscle cells. The current-voltage relationship of the K⁺ channel was linear over a voltage range from -60 to +60 mV and had a conductance of 130 pS with physiological K⁺ in the recording pipette and 240 pS with symmetrical K⁺. The activity of this K⁺ channel was inhibited by TEA (1 mmol/L) and charybdotoxin (2 and 50 nmol/L) (Fig 6). With TEA, the amplitude of the single K⁺ channel was reduced from 10±0.2 to 2.07±0.01 pA (P<.001). The open-state probability of the channel was significantly reduced by charybdotoxin (P<.001) (Fig 6 and Table 1). Fig 7 illustrates the K⁺ channel activity under control conditions and after treatment with 14,15-EET. 14,15-EET (1 nmol/L) increased K⁺ channel activity markedly. Data from multiple cells indicate that 14,15-EET increased the number of K⁺ channel openings and the probability of channel opening in a concentration-related manner, with significant increases (P<.05) occurring with 1 nmol/L 14,15-EET. Similar results were obtained with 11,12-EET (data not shown). This K⁺ channel activity was inhibited by TEA (1 mmol/L). In contrast, arachidonic acid failed to alter K⁺ channel activity in concentrations between 10 nmol/L and 10 µmol/L (data not shown). The effect of the EETs did not appear to be due to a change in cyclic nucleotide production (Table 2). 5,6-EET increased the intracellular accumulation of cAMP slightly, whereas the other regioisomers were without effect. 11,12-EET, 8,9-EET, and 5,6-EET decreased the accumulation of cGMP slightly. In contrast, isoproterenol significantly increased the accumulation of cAMP, and sodium nitroprusside increased cGMP. When coronary arteries were incubated with ¹⁴C-labeled EETs, the only metabolites observed were their respective hydrolysis products, the vic-dihydroxyeicosatrienoic acids.²⁴ This hydrolysis was only 10% of the added EET over a 30-minute incubation. Thus, the EETs must act by a direct action on the K⁺ channel and must not involve cyclic nucleotide second messengers or the production of secondary metabolites of the EETs.

View larger version (13K): [in this window] [in a new window]   Figure 5. Effect of inhibitors of K+ channels on EET-induced relaxations. Vessels were precontracted with U46619 (20 nmol/L), and 8,9-EET was added in various concentrations. Isometric tension was measured. Vessels were pretreated with TEA (1 mmol/L) or charybdotoxin (CHTX, 50 nmol/L), and the effect of 8,9-EET was determined. Each value represents the mean±SEM.

View larger version (21K): [in this window] [in a new window]   Figure 6. Effect of TEA chloride and charybdotoxin on K+ channel activity in bovine coronary arterial smooth muscle cells. Channel activity was measured by patch clamp in inside-out patches using symmetrical KCl (145 mmol/L) at a patch potential of +40 mV. An upward deflection indicates channel opening; C, closed.

View larger version (35K): [in this window] [in a new window]   Figure 7. Effect of 14,15-EET on K+ channel activity of bovine coronary arterial smooth muscle cells. Channel activity was measured by patch clamp in the cell-attached mode using symmetrical KCl (145 mmol/L). Left, Typical tracing of K+ channel activity under control conditions (top) and after 1 (middle) and 100 (bottom) nmol/L 14,15-EET. An upward deflection indicates channel opening; o, open; and c, closed. Right, Summary data from multiple cells. Both the number of events or channel openings and probability of opening (NPo) are plotted against concentrations of 14,15-EET. Similar results were obtained with 11,12-EET. Each value represents the mean±SEM.

View this table: [in this window] [in a new window]   Table 2. Effects of EETs on cAMP and cGMP Production by Bovine Coronary Arteries

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The endothelium has emerged as an important regulator of vascular tone.^{1 2 3} Several soluble mediators released by the endothelium are involved in these vascular effects. These mediators include prostacyclin, EDRF or NO, and EDHF. The activity of EDHF may be distinguished from NO in that EDHF activity is blocked by inhibitors of Ca²⁺-activated K⁺ channels, such as TEA or charybdotoxin, or by high [K⁺]_o but is not blocked by arginine analogues that inhibit NOS or glibenclamide, an inhibitor of ATP-sensitive K⁺ channels.^{9 14 17 18} Relaxations mediated by EDRF are blocked by arginine analogues. In small coronary arteries, methacholine causes endothelium-dependent relaxations and endothelium-dependent hyperpolarization of smooth muscle cells.^{5 14 15 16 17 18} These relaxations are blocked by TEA and high [K⁺]_o. Thus, it has been proposed that methacholine stimulates coronary endothelial cells to release EDHF, which acts on coronary smooth muscle cells to open K⁺ channels, hyperpolarize the cell membrane, inhibit voltage-dependent Ca²⁺ influx, and induce relaxation. We have recently reported that arachidonic acid causes endothelium-dependent relaxation of bovine coronary arteries.²⁴ A portion of the relaxation is inhibited by a cyclooxygenase inhibitor, and the remainder is inhibited by an inhibitor of cytochrome P₄₅₀. To identify the mediators of arachidonic acid–induced relaxations, we studied the metabolism of arachidonic acid by coronary endothelial cells and coronary arteries. They metabolize arachidonic acid via the cyclooxygenase, lipoxygenase, and cytochrome P₄₅₀ pathways.^{24 25} Prostacyclin is the major cyclooxygenase metabolite, whereas 14,15-EET, 11,12-EET, 8,9-EET, and 5,6-EET are the major metabolites from cytochrome P₄₅₀. Since both prostacyclin and the EETs relax coronary arteries,^{24 26 27} they appear to mediate the endothelium-dependent relaxations induced by arachidonic acid.

Since coronary endothelial cells contain cytochrome P₄₅₀ activity³⁷ and metabolize arachidonic acid to known cytochrome P₄₅₀ products (the EETs) that relax coronary arteries,^{24 25 26 27 38} we hypothesized that the EETs may represent EDHFs. In support of this hypothesis, we found that methacholine-induced relaxation and hyperpolarization are blocked by inhibitors of cytochrome P₄₅₀. The relaxations to methacholine are also blocked by TEA and high [K⁺]_o. Methacholine stimulates the release of several arachidonic acid metabolites, including prostacyclin, 12-HETE, and the EETs. The identity of the material released by methacholine as EETs is indicated by its comigration with authentic EET standards on reverse-phase HPLC, comigration of the derivitized material with EET-PFB ester standards on GC, and a characteristic EET-PFB mass spectrum. In previous studies, the synthesis of the EETs was inhibited by SKF 525A and miconazole.^{24 36} Arachidonic acid also relaxes coronary arteries in an endothelium-dependent manner, and these relaxations are blocked by inhibitors of cytochrome P₄₅₀²⁴ as well as TEA, charybdotoxin, and high [K⁺]_o. Along these lines, others have reported that quinacrine and metyrapone, inhibitors of phospholipase and cytochrome P₄₅₀, respectively, reduce acetylcholine-induced relaxation³⁹ and that indomethacin enhances the hyperpolarization produced by acetylcholine.⁵ Similarly, the relaxations to bradykinin are inhibited by quinacrine and inhibitors of cytochrome P₄₅₀ as well as K⁺ channel blockers.³⁸ These studies also link EDHF to arachidonic acid metabolism.

These studies rely in part on inhibitors of cytochrome P₄₅₀ to assess the contribution of the EETs to the relaxations and hyperpolarizations stimulated by methacholine and arachidonic acid.²⁴ We were concerned that these inhibitors might have other actions that could contribute to the observed effects. As a result, we tested their effect on the NOS activity of coronary artery homogenates and the K⁺ channel activity of coronary smooth muscle. Both miconazole and SKF 525A were without effect on NOS activity in the concentrations used in these studies. Since both drugs were equally effective in blocking methacholine-induced relaxations and hyperpolarization in concentrations that did not alter NOS activity, inhibition of NOS could not account for these observed effects. Neither drug affected the activity of the Ca²⁺-activated K⁺ channel in inside-out patches of coronary arterial smooth muscle cells. These findings eliminate the possibility that these agents are blocking relaxations by a direct effect on K⁺ channels.

We found that the four regioisomeric EETs relax coronary arteries in a concentration-related manner. Significant relaxations to the EETs occur with nanomolar concentrations. As with methacholine and arachidonic acid, the relaxations to the EETs are blocked by TEA, charybdotoxin, and high [K⁺]_o. In contrast, the relaxations to the EETs are not endothelium dependent. Similar effects to the EETs have been reported in canine and porcine coronary arteries; however, micromolar concentrations of the EETs are required for relaxation in these studies.^{26 38} This difference in EET potency may be due to the use of large epicardial vessels in the previous studies. The EETs are more potent in relaxing smaller coronary arteries than larger ones,^{24 27} as others have reported for EDHF.^{12 13} The relaxation to the EET is associated with an increase in membrane potential and the opening of Ca²⁺-activated K⁺ channels. The threshold concentration of EET for increases in K⁺ channel activity is 1 nmol/L. The effects of the EETs on K⁺ channel activity are blocked by TEA. Thus, EETs, like EDHF, seem to act on Ca²⁺-dependent K⁺ channels to relax coronary arteries. The exact mechanism by which the EETs open K⁺ channels is not known. However, unlike NO and prostacyclin, their effects are not associated with an increase in cyclic nucleotides.

Charybdotoxin, a reversible inhibitor of the Ca²⁺-activated K⁺ channel,⁴⁰ failed to completely block the relaxations to 8,9-EET at a concentration of 50 nmol/L. Instead, the toxin caused a 10-fold shift in the EET concentration-response curve to the right. This result may be because higher concentrations of the EETs overcome the charybdotoxin blockade or because this concentration of charybdotoxin does not cause complete blockade of the channel (see Fig 6). Similar results have been reported with charybdotoxin on ß-agonist–induced relaxation of the isolated trachea and inhibition of myogenic tone in rat mesenteric arteries.^{41 42} For example, charybdotoxin (60 nmol/L) caused a 4.8-fold shift in the isoproterenol concentration-response curve in isolated trachea.

In summary, these findings indicate that EETs have many of the known properties of EDHF: they are (1) produced by the endothelium, (2) released by methacholine, (3) relax arteries, (4) hyperpolarize vascular smooth muscle, and (5) open Ca²⁺-activated K⁺ channels, and (6) their effects are blocked by TEA, charybdotoxin, and high [K⁺]_o but not glibenclamide.^{9 14 17 18 24} Thus, these data suggest that EETs represent an EDHF and that these cytochrome P₄₅₀ metabolites of arachidonic acid may mediate the endothelium-dependent relaxations to methacholine in small coronary arteries.

	Selected Abbreviations and Acronyms

 

EDHF = endothelium-derived hyperpolarizing factor EDRF = endothelium-derived relaxing factor EET = epoxyeicosatrienoic acid GC = gas chromatograph (chromatography) HPLC = high-performance liquid chromatography MS = mass spectrometer (spectrometry) NOS = NO synthase PFB = pentafluorobenzyl PMSF = phenylmethylsulfonyl fluoride SBTI = soybean trypsin inhibitor TEA = tetraethylammonium

	Acknowledgments

 
This study was supported by grants from the National Heart, Lung, and Blood Institute (HL-51055 and HL-33833) and by a grant from the Veterans Administration. The authors thank Joe James for his technical assistance, Dr Kasem Nithipatikom for his help with the GC/MS analysis, and Gretchen Barg for her secretarial assistance. They also thank Drs J.R. Falck, Sandra Pfister, and William Edgemond for their helpful discussions.

Received July 13, 1995; accepted November 8, 1995.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Furchgott RF, Vanhoutte PM. Endothelium-derived relaxing and contracting factors. FASEB J. 1989;3:2007-2018. [Abstract]
2. Rubanyi GM. Endothelium-derived relaxing and contracting factors. J Cell Biol. 1991;46:27-36. [Abstract/Free Full Text]
3. Moncada S, Palmer RMJ, Higgs EA. Nitric oxide: physiology, pathophysiology and pharmacology. Pharmacol Rev. 1991;43:109-142. [Medline] [Order article via Infotrieve]
4. Palmer RMJ, Ferrige AG, Moncada S. Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature. 1987;327:524-526. [Medline] [Order article via Infotrieve]
5. Feletou M, Vanhoutte PM. Endothelium-dependent hyperpolarization of canine coronary smooth muscle. Br J Pharmacol. 1988;93:515-524.[Medline] [Order article via Infotrieve]
6. Bolton TB, Clapp LH. Endothelial-dependent relaxant actions of carbachol and substance P in arterial smooth muscle. Br J Pharmacol. 1986;87:713-723.[Medline] [Order article via Infotrieve]
7. Komori K, Suzuki H. Electrical responses of smooth muscle cells during cholinergic vasodilation in the rabbit saphenous artery. Circ Res. 1987;61:586-593. [Abstract/Free Full Text]
8. Komori K, Suzuki H. Heterogeneous distribution of muscarinic receptors in the rabbit saphenous artery. Br J Pharmacol. 1987;92:657-664.[Medline] [Order article via Infotrieve]
9. Huang AH, Busse R, Bassenge E. Endothelium-dependent hyperpolarization of smooth muscle cells in rabbit femoral arteries is not mediated by EDRF (nitric oxide). Naunyn Schmiedebergs Arch Pharmacol. 1988;338:438-442. [Medline] [Order article via Infotrieve]
10. Kauser K, Stekiel WJ, Rubanyi G, Harder DR. Mechanism of action of EDRF on pressurized arteries: effect on K⁺ conductance. Circ Res. 1989;65:199-204. [Abstract/Free Full Text]
11. Keef KD, Bowen SM. Effect of ACh on electrical and mechanical activity in guinea pig coronary arteries. Am J Physiol. 1989;257:H1096-H1103. [Abstract/Free Full Text]
12. Nagao T, Illiano S, Vanhoutte PM. Heterogeneous distribution of endothelium-dependent relaxations resistant to N-nitro-L-arginine in rats. Am J Physiol. 1992;263:H1090-H1094. [Abstract/Free Full Text]
13. Hwa JJ, Ghibaudi L, Williams P, Chatterjii M. Comparison of acetylcholine-dependent relaxation in large and small arteries of rat mesenteric vascular bed. Am J Physiol. 1994;266:H952-H958. [Abstract/Free Full Text]
14. Chen G, Yamamoto Y, Miwa K, Suzuki H. Hyperpolarization of arterial smooth muscle induced by endothelial humoral substances. Am J Physiol. 1991;260:H1888-H1892. [Abstract/Free Full Text]
15. Fujii K, Tominaga M, Ohmori S, Kobayashi K, Koga T, Takata Y, Fujishima M. Decreased endothelium-dependent hyperpolarizations to acetylcholine in smooth muscle of the mesenteric artery of spontaneously hypertensive rats. Circ Res. 1992;70:660-669. [Abstract/Free Full Text]
16. Taylor SG, Southerton JS, Weston AH, Baker JRJ. Endothelium-dependent effects of acetylcholine in rat aorta: a comparison with sodium nitroprusside and cromakalim. Br J Pharmacol. 1988;94:853-863. [Medline] [Order article via Infotrieve]
17. Weston AH, Edwards G. Recent progress in potassium channel opener pharmacology. Biochem Pharmacol. 1992;43:47-54. [Medline] [Order article via Infotrieve]
18. Van de Voorde J, Vanheel B, Leusen I. Endothelium-dependent relaxation and hyperpolarization in aorta from control and renal hypertensive rats. Circ Res. 1992;70:1-8. [Abstract/Free Full Text]
19. Garland CJ, McPherson GA. Evidence that nitric oxide does not mediate the hyperpolarization and relaxation to acetylcholine in the rat small mesenteric artery. Br J Pharmacol. 1992;105:429-435. [Medline] [Order article via Infotrieve]
20. Bolotina VM, Najibi S, Palacino JJ, Pagano PJ, Cohen RA. Nitric oxide directly activates calcium-dependent potassium channels in vascular smooth muscle. Nature. 1994;368:850-853. [Medline] [Order article via Infotrieve]
21. Archer SL, Huang JMC, Hampl V, Nelson DP, Schultz PJ, Weir EK. Nitric oxide and cGMP cause vasorelaxation by activation of a charybdotoxin-sensitive K channel by cGMP-dependent protein kinase. Proc Natl Acad Sci U S A. 1994;91:7583-7587. [Abstract/Free Full Text]
22. Miyoshi H, Nakaya Y, Moritoki H. Nonendothelial-derived nitric oxide activates the ATP-sensitive K channel of vascular smooth muscle cells. FEBS Lett. 1994;345:47-49. [Medline] [Order article via Infotrieve]
23. Tare M, Parkington HC, Coleman HA, Neild TO, Dusting GJ. Hyperpolarization and relaxation of arterial smooth muscle caused by nitric oxide derived from the endothelium. Nature. 1990;346:69-71. [Medline] [Order article via Infotrieve]
24. Rosolowsky M, Campbell WB. Role of PGI2 and EETs in the relaxation of bovine coronary arteries to arachidonic acid. Am J Physiol. 1993;264:H327-H335. [Abstract/Free Full Text]
25. Rosolowsky M, Campbell WB. Synthesis of hydroxyeicosatetraenoic acids and epoxyeicosatrienoic acids by cultured bovine coronary artery endothelial cells. Biochem Biophys Acta. In press.
26. Rosolowsky M, Falck JR, Willerson JT, Campbell WB. Synthesis of lipoxygenase and epoxygenase products of arachidonic acid by normal and stenosed canine coronary arteries. Circ Res. 1990;66:608-621. [Abstract/Free Full Text]
27. Rosolowsky M, Falck JR, Campbell WB. Synthesis and biological activity of epoxyeicosatrienoic acids (EETs) by cultured bovine coronary artery endothelial cells. Adv Prostaglandin Thromboxane Leukot Res. 1990;21:213-216.
28. Bredt DS, Snyder SH. Isolation of nitric oxide synthetase, a calmodulin-requiring enzyme. Proc Natl Acad Sci U S A. 1990;87:682-685. [Abstract/Free Full Text]
29. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem. 1951;193:265-275. [Free Full Text]
30. Revtyak GE, Hughes MJ, Johnson AR, Campbell WB. Histamine stimulation of prostaglandin and HETE synthesis in human endothelial cells. Am J Physiol. 1988;255:C214-C225. [Abstract/Free Full Text]
31. Campbell WB, Brady MT, Rosolowsky LJ, Falck JR. Metabolism of arachidonic acid by rat adrenal glomerulosa cells: synthesis of hydroxyeicosatetraenoic acids and epoxyeicosatrienoic acids. Endocrinology. 1991;128:2183-2194. [Abstract]
32. Harder DR. Pressure-dependent membrane depolarization in cat middle cerebral artery. Circ Res. 1984;55:197-202. [Abstract/Free Full Text]
33. Gebremedhin D, Ma Y-H, Falck JR, Roman RJ, VanRollins M, Harder DR. Mechanism of action of cerebral epoxyeicosatrienoic acids on cerebral arterial smooth muscle. Am J Physiol. 1992;263:H519-H525. [Abstract/Free Full Text]
34. Pfister SL, Campbell WB. Arachidonic acid- and acetylcholine-induced relaxations of rabbit aorta. Hypertension. 1992;20:682-689. [Abstract/Free Full Text]
35. Henrich WL, McAllister EA, Smith PB, Campbell WB. Guanosine 3',5'-cyclic monophosphate as a mediator of inhibition of renin release. Am J Physiol. 1988;255:F474-F478. [Abstract/Free Full Text]
36. Harder DR, Campbell WB, Roman RJ. Role of cytochrome P450 enzymes and metabolites of arachidonic acid in the control of vascular tone. J Vasc Res. 1995;32:79-92. [Medline] [Order article via Infotrieve]
37. Abraham NG, Pinto A, Mullane KM, Levere RD, Spokas E. Presence of cytochrome P-450-dependent monooxygenase in intimal cells of the hog aorta. Hypertension. 1985;7:899-904. [Abstract/Free Full Text]
38. Hecker M, Bara AT, Bauersachs J, Busse R. Characterization of endothelium-derived hyperpolarizing factor as a cytochrome P450-derived arachidonic acid metabolite in mammals. J Physiol (Lond). 1994;481:407-414. [Medline] [Order article via Infotrieve]
39. Rubanyi GM, Vanhoutte PM. Nature of endothelium-derived relaxing factor: are there two relaxing mediators? Circ Res. 1987;61(suppl II):II-61-II-67.
40. Gimenez-Gallego M, Navia MA, Reuben JP, Katz GM, Kaczorowski GJ, Garcia ML. Purification, sequence, and model structure of charybdotoxin, a potent selective inhibitor of calcium-activated potassium channels. Proc Natl Acad Sci U S A. 1988;85:3329-3333. [Abstract/Free Full Text]
41. Jones TR, Charette L, Garcia ML, Kaczorowski GJ. Selective inhibition of relaxation of guinea-pig trachea by charybdotoxin, a potent Ca⁺⁺-activated K⁺ channel inhibitor. J Pharmacol Exp Ther. 1990;255:697-706. [Abstract/Free Full Text]
42. Asano M, Masuzawa-Ito K, Matsuda T. Charybdotoxin-sensitive K⁺ channels regulate the myogenic tone in the resting state of arteries from spontaneously hypertensive rats. Br J Pharmacol. 1993;108:214-222.[Medline] [Order article via Infotrieve]

This article has been cited by other articles:

				J.-K. Chen, J. Chen, J. D. Imig, S. Wei, D. L. Hachey, J. S. Guthi, J. R. Falck, J. H. Capdevila, and R. C. Harris Identification of Novel Endogenous Cytochrome P450 Arachidonate Metabolites with High Affinity for Cannabinoid Receptors J. Biol. Chem., September 5, 2008; 283(36): 24514 - 24524. [Abstract] [Full Text] [PDF]

				A. E. Loot, R. Popp, B. Fisslthaler, J. Vriens, B. Nilius, and I. Fleming Role of cytochrome P450-dependent transient receptor potential V4 activation in flow-induced vasodilatation Cardiovasc Res, August 27, 2008; (2008) cvn207v2. [Abstract] [Full Text] [PDF]

				N. T. Aggarwal, K. M. Gauthier, and W. B. Campbell 15-Lipoxygenase metabolites contribute to age-related reduction in acetylcholine-induced hypotension in rabbits Am J Physiol Heart Circ Physiol, July 1, 2008; 295(1): H89 - H96. [Abstract] [Full Text] [PDF]

				I. C Villar, A. J Hobbs, and A. Ahluwalia Sex differences in vascular function: implication of endothelium-derived hyperpolarizing factor J. Endocrinol., June 1, 2008; 197(3): 447 - 462. [Abstract] [Full Text] [PDF]

				A. C. Webler, R. Popp, T. Korff, U. R. Michaelis, C. Urbich, R. Busse, and I. Fleming Cytochrome P450 2C9-Induced Angiogenesis Is Dependent on EphB4 Arterioscler. Thromb. Vasc. Biol., June 1, 2008; 28(6): 1123 - 1129. [Abstract] [Full Text] [PDF]

				G. Yan, S. Chen, B. You, and J. Sun Activation of sphingosine kinase-1 mediates induction of endothelial cell proliferation and angiogenesis by epoxyeicosatrienoic acids Cardiovasc Res, May 1, 2008; 78(2): 308 - 314. [Abstract] [Full Text] [PDF]

				U. R. Michaelis, N. Xia, E. Barbosa-Sicard, J. R. Falck, and I. Fleming Role of Cytochrome P450 2C Epoxygenases in Hypoxia-Induced Cell Migration and Angiogenesis in Retinal Endothelial Cells Invest. Ophthalmol. Vis. Sci., March 1, 2008; 49(3): 1242 - 1247. [Abstract] [Full Text] [PDF]

				W. Yang, V. R. Tuniki, S. Anjaiah, J. R. Falck, C. J. Hillard, and W. B. Campbell Characterization of Epoxyeicosatrienoic Acid Binding Site in U937 Membranes Using a Novel Radiolabeled Agonist, 20-125I-14,15-Epoxyeicosa-8(Z)-Enoic Acid J. Pharmacol. Exp. Ther., March 1, 2008; 324(3): 1019 - 1027. [Abstract] [Full Text] [PDF]

				Y. Chawengsub, N. T. Aggarwal, K. Nithipatikom, K. M. Gauthier, S. Anjaiah, B. D. Hammock, J. R. Falck, and W. B. Campbell Identification of 15-hydroxy-11,12-epoxyeicosatrienoic acid as a vasoactive 15-lipoxygenase metabolite in rabbit aorta Am J Physiol Heart Circ Physiol, March 1, 2008; 294(3): H1348 - H1356. [Abstract] [Full Text] [PDF]

				K. M. Gauthier, Y. Chawengsub, D. H. Goldman, R. E. Conrow, S. Anjaiah, J. R. Falck, and W. B. Campbell 11(R),12(S),15(S)-trihydroxyeicosa-5(Z),8(Z),13(E)-trienoic acid: an endothelium-derived 15-lipoxygenase metabolite that relaxes rabbit aorta Am J Physiol Heart Circ Physiol, March 1, 2008; 294(3): H1467 - H1472. [Abstract] [Full Text] [PDF]

				P. D. Smith, S. E. Brett, K. D. Luykenaar, S. L. Sandow, S. P. Marrelli, E. J. Vigmond, and D. G. Welsh KIR channels function as electrical amplifiers in rat vascular smooth muscle J. Physiol., February 15, 2008; 586(4): 1147 - 1160. [Abstract] [Full Text] [PDF]

				P. A. Dabisch, J. T. Liles, S. R. Baber, N. H. Golwala, S. N. Murthy, and P. J. Kadowitz Analysis of L-NAME-dependent and -resistant responses to acetylcholine in the rat Am J Physiol Heart Circ Physiol, February 1, 2008; 294(2): H688 - H698. [Abstract] [Full Text] [PDF]

X. Tang, N. Aggarwal, B. B. Holmes, H. Kuhn, and W. B. Campbell