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(Hypertension. 2004;43:413.)
© 2004 American Heart Association, Inc.

Scientific Contribution

Apamin-Sensitive K⁺ Currents Mediate Arachidonic Acid-Induced Relaxations of Rabbit Aorta

Kathryn M. Gauthier; Nancy Spitzbarth; Erik M. Edwards; William B. Campbell

From the Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee.

Correspondence to Kathryn M. Gauthier, PhD, Department of Pharmacology and Toxicology, Medical College of Wisconsin, 8701 Watertown Plank Rd, Milwaukee, WI 53226. kgauth{at}mcw.edu

	Abstract

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Arachidonic acid induces an endothelium-dependent relaxation of the rabbit aorta that is blocked by lipoxygenase inhibitors. The cellular vasodilatory mechanisms activated by arachidonic acid metabolites remain undefined. In rabbit thoracic aortic rings pretreated with indomethacin (10 µmol/L) and contracted with phenylephrine, arachidonic acid (0.1 to 100 µmol/L) induced concentration-dependent relaxations. Maximal relaxations averaged 45±3% and were inhibited by increasing extracellular K⁺ (30 mmol/L, 15±5%; P<0.001) or incubation with apamin (100 nmol/L, 26±7%; P<0.05) but not incubation with charybdotoxin (100 nmol/L, 41±5%). In aortic strips with an intact endothelium that were treated with phenylephrine, arachidonic acid (10 µmol/L) increased the membrane potential from -28.7±1.3 to -37.8±3.0 mV (P<0.01). Preincubation with apamin did not alter basal membrane potential but inhibited arachidonic acid-induced hyperpolarization (-31.5±1.5 mV). Incubation of rabbit aortic segments with apamin or charybdotoxin did not alter [¹⁴C]arachidonic acid metabolism. Whole-cell outward K⁺ currents from isolated rabbit aortic smooth muscle cells averaged 43.0±4.8 pA/pF at 60 mV and were significantly decreased to 35.7±4.2 pA/pF by apamin (P<0.001). Subsequent addition of charybdotoxin further decreased maximal currents to 14.4±2.3 pA/pF. Addition of 11,12,15-trihydroxyeicosatrienoic acid increased the outward whole-cell K⁺ current. In inside-out patches of aortic smooth muscle, apamin inhibited the calcium activation (100 to 300 nmol/L; P<0.001) of a small-conductance K⁺ channel (24 pS). These results suggest that arachidonic acid induces endothelium-dependent hyperpolarization and relaxation of rabbit aorta through activation of smooth muscle, apamin-sensitive K⁺ currents.

Key Words: lipoxygenase • endothelium-derived factors • potassium channels • hyperpolarization • membranes

	Introduction

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Stimulation of the vascular endothelium with acetylcholine, bradykinin, or flow induces the release of soluble mediators that act on smooth muscle to induce relaxation.^1–7 The major relaxing factors include nitric oxide (NO), prostacyclin, and a group of compounds classified as endothelium-derived hyperpolarizing factors (EDHFs). The hallmark of EDHF activity is the stimulation of smooth muscle membrane ion flux of K⁺ to induce hyperpolarization.⁸ EDHF activity and the mediators of this activity vary with vascular size, vascular bed, and species. In bovine, porcine, and canine coronary circulations, the cytochrome P450 metabolites of arachidonic acid, the epoxyeicosatrienoic acids, act as EDHFs.^9–11 However, in the rat hepatic artery and in murine and human mesenteric arteries, K⁺ and H₂O₂ serve as EDHFs.^12–14 In addition, in small arteries, electronic spread from the endothelium to the smooth muscle through gap junctions might induce smooth muscle hyperpolarization and/or propagate the EDHF response.¹⁵

In the rabbit aorta, arachidonic acid induces endothelium-dependent smooth muscle hyperpolarization and vascular relaxation.¹⁶ Arachidonic acid does not increase cAMP or cGMP of the rabbit aorta.¹⁷ Alternatively, relaxations to arachidonic acid are inhibited by lipoxygenase inhibitors and enhanced by cyclooxygenase inhibitors.¹⁸ We demonstrated previously that the rabbit aortic endothelium metabolizes arachidonic acid by 15-lipoxygenase to produce 15-hydroxyeicosatetraenoic acids and hydroxyepoxyeicosatrienoic acids (HEETAs). The HEETAs are further hydrolyzed to 11,14,15- and 11,12,15-trihydroxyeicosatrienoic acids (THETAs). Purified 11,12,15-THETA induced concentration-dependent relaxation of preconstricted aortas, which was inhibited by high extracellular K⁺.¹⁶ In addition, this compound hyperpolarized rabbit aortic smooth muscle.¹⁶ These results suggest that 11,12,15-THETA functions as an EDHF in the rabbit aorta. However, the ionic mechanisms of this activity have not been characterized.

The present study examined the effect of specific K⁺ channel inhibitors on arachidonic acid-induced relaxation and hyperpolarization. In addition, we identified specific K⁺ currents in rabbit aortic smooth muscle that might mediate the hyperpolarization response. Apamin, the small-conductance, Ca²⁺-activated K⁺ channel (SK_Ca) inhibitor, significantly inhibited arachidonic acid-induced relaxations, whereas charybdotoxin (CBTX), the intermediate- (IK_Ca) and large- (BK_Ca) conductance, Ca²⁺-activated K⁺ channel inhibitor, was without effect. Furthermore, patch-clamp evaluation revealed that rabbit aortic smooth muscle cells (SMCs) possess apamin-sensitive K⁺ currents and channels and that these currents are activated by the 15-lipoxygenase metabolite of arachidonic acid, 11,12,15-THETA. This study provides further confirmation of the role of 15-lipoxygenase metabolites of arachidonic acid as EDHFs in the rabbit aorta.

	Methods

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Vascular Reactivity
Thoracic aortas were obtained from 1- to 2-month-old, male New Zealand White rabbits (New Franken Research Rabbits, New Franken, Wis) and placed in Krebs bicarbonate buffer (in mmol/L: 118 NaCl, 4.8 KCl, 3.3 CaCl₂, 24 NaHCO₃, 1.2 KH₂PO₄, 1.2 MgSO₄, and 11 glucose), as previously described.^16–18 The tissue was cleaned of adhering fat and connective tissue and cut into rings (3 mm long) without damage to the endothelium. The aortic rings were suspended in 6-mL tissue baths containing Krebs’ bicarbonate buffer maintained at 37°C and equilibrated with 95% O₂-5% CO₂. Resting tension was slowly raised to its length tension maximum of 2 g, and the arteries were equilibrated for 1 hour. The aortic rings were stimulated with repeated applications of KCl (40 mmol/L). After stable responses to KCl were observed, the tissues were rinsed, treated with indomethacin (10 µmol/L) for 10 minutes, and contracted with phenylephrine (10 to 100 nmol/L) to 50% to 75% of the maximal KCl contraction. In some studies, the Krebs buffer was modified to increase the KCl concentration to 30 mmol/L by substituting KCl for NaCl. Cumulative concentrations of arachidonic acid (0.1 to 100 µmol/L) were added and relaxation responses measured. Similar studies were performed in vessels pretreated for 10 to 15 minutes with apamin (100 nmol/L) and/or CBTX (100 nmol/L). Basal tension represents tension before the addition of phenylephrine.

Arachidonic Acid Metabolism
Aortas were cut into 3-mm-long rings and incubated for 15 minutes at 37°C in HEPES buffer (in mmol/L: 10 HEPES, 150 NaCl, 5 KCl, 1.8 CaCl_2, 1 MgCl₂, and 5.5 glucose; pH 7.4) containing indomethacin (10 µmol/L). Aortic rings were preincubated with vehicle, apamin (100 nmol/L), or CBTX (100 nmol/L) for 10 minutes, followed by sequential addition of [¹⁴C-U]arachidonic acid (0.05 µCi, 100 nmol/L) and A23187 (10 µmol/L) with additional 10-minute incubation periods. The reaction was stopped by adding ethanol to a final concentration of 15%, the HEPES buffer was removed, acidified (pH <3.5) with glacial acetic acid and extracted on ODS solid-phase extraction columns, as previously described.^18,19 The extracted metabolites were evaporated to dryness under a stream of N₂ and stored at -40°C until analysis by high-pressure liquid chromatography (HPLC). The metabolites were resolved by reverse-phase HPLC (Nucleosil-C18 column, 5 µm, 4.6x250 mm).¹⁹ Solvent A was water, and solvent B was acetonitrile containing 0.1% glacial acetic acid, with a 40-minute linear gradient from 50% solvent B in A to 100% solvent B. Flow rate was adjusted to 1 mL/min. The column eluate was collected in 0.2-mL fractions, and radioactivity was measured by liquid scintillation spectrometry.

Membrane Potential Measurements
Rabbit aortic rings were cut open laterally and pinned to a silicone elastomer layer, with the endothelial layer exposed, in a heated (37°C) perfusion chamber. The aortic segments were perfused with a physiologic salt solution of the following composition (in mmol/L): 119 NaCl, 4.7 KCl, 1.6 CaCl₂, 1.17 MgSO₄, 5.5 glucose, 24 NaHCO₃, 1.18 NaH₂PO₄, and 0.026 EDTA, equilibrated with a 21% O₂-5% CO₂-balance N₂ gas mixture to maintain a pH of 7.4 and a PO₂ of 140 mm Hg, as previously described.²⁰ Indomethacin (10 µmol/L) and phenylephrine (100 nmol/L) were present in all perfusate solutions. Aortic segments were continually superfused for 30 minutes before initiation of experimental protocols. Impalements of SMCs were performed in a section of the artery where a small patch of endothelium had been removed by gentle rubbing with a small cotton swab. Intracellular membrane potential (E_m) values were recorded by using published methods.^16,20 In brief, glass microelectrodes were filled with 3 mol/L KCl and had estimated tip sizes of 0.1 to 0.2 µm, tip resistances of 30 to 80 M, and tip potentials of 3 mV. Electrodes were attached to a high-impedence biologic amplifier (Dagan Cell Explorer, Dagan Instruments) and digitized for analysis with a MacLab system (AD Instruments). Electrode polarization was eliminated by an Ag/AgCl half-cell. Criteria for successful cell impalement included an abrupt drop in E_m to a new steady-state value, which had been maintained for a minimum of 5 seconds, an E_m value >-20 mV, and an abrupt return to the original baseline when the electrode was retracted from the tissue. Arachidonic acid (10 µmol/L) was added to the aortic segments, and after 10 minutes E_m was measured. The arteries were rinsed and incubated with apamin (100 nmol/L) for 15 minutes, and the impalements were repeated.

Patch-Clamp Recording
Aortic SMCs were enzymatically dispersed by using published methods.²¹ Whole-cell recordings of K⁺ currents were obtained in freshly isolated, aortic SMCs with an amplifier (Axopatch 200B, Axon Instruments), appropriate software (pClamp 8, Axon Instruments), and standard methods, as previously described.²² In brief, macroscopic K⁺ currents were generated by progressive, stepwise, 10-mV depolarizing steps (500-ms duration, 5-second intervals) from a constant holding potential of -60 mV. Currents were sampled at 3 kHz and filtered at 1 kHz. After control currents were recorded, currents were obtained after the application of apamin (100 µmol/L) and apamin plus CBTX (100 µmol/L). Trials were performed in triplicate and averaged to estimate K⁺ current density. The membrane capacitance of each cell was estimated by integrating the capacitative current generated by a 10-mV hyperpolarizing pulse after electronic cancellation of pipette-patch capacitance. For experiments that evaluated the effect of 11,12,15-THETA on whole-cell currents, indomethacin (10 µmol/L) was present in all perfusate solutions. 11,12,15-THETA was synthesized and isolated as previously described.¹⁶ Whole-cell currents were measured in control cells or apamin-treated cells (100 nmol/L) after perfusion with vehicle, followed by 11,12,15-THETA.

Inside-out, single-channel K⁺ currents were recorded and analyzed by using previously described procedures.²³ Perfusate and pipette solutions contained (in mmol/L) KCl 145, MgCl₂ 1.0, EGTA 1.0, and HEPES 10 and 100 nmol/L Ca²⁺ (pH 7.4). In addition, pipette solutions contained 100 nmol/L iberiotoxin to block the BK_Ca channels. For current-voltage relations, channel activity was measured at E_ms of -80, -60, +60, and +80 mV. Mean open-state probabilities (NP_o) were determined at -60 mV in inside-out patches perfused with increasing free Ca²⁺ from 100 to 300 nmol/L. Channel recordings (2 to 4 minutes) were measured in patches without apamin or in cells incubated with 100 nmol/L apamin and pipette solutions also containing 100 nmol/L apamin.

Materials
Phenylephrine, arachidonic acid (sodium salt), iberiotoxin, apamin, CBTX, and indomethacin were obtained from Sigma Chemical Co. Indomethacin was dissolved in 95% ethanol, and all other drugs were dissolved in distilled water. All solvents were HPLC grade and were purchased from Burdick and Jackson. [¹⁴C-U]arachidonic acid (920 mCi/mmol) was obtained from New England Nuclear.

Statistical Analysis
The vascular reactivity, patch-clamp, and E_m data are expressed as mean±SEM. Statistical evaluation of the data was performed by a 1-way ANOVA followed by the Student-Newman-Keuls multiple-comparison test when significant differences were present. Alternatively, the data were analyzed by Student’s t test for paired observations. P<0.05 was considered statistically significant.

	Results

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Arachidonic acid induced a concentration-dependent relaxation of endothelium-intact rabbit aortic rings, contracted with phenylephrine, and treated with indomethacin (Figure 1A). These relaxations were inhibited by increased KCl (30 mmol/L). Preincubation with the SK_Ca channel inhibitor apamin also inhibited the arachidonic acid-induced relaxations (Figure 1B). The IK_Ca channel inhibitor CBTX did not have any effect (Figure 1B), and the combination of CBTX plus apamin did not further reduce the apamin-insensitive relaxations (data not shown). Removal of the endothelium eliminated the arachidonic acid-induced relaxations.^16,18

View larger version (13K): [in this window] [in a new window]   Figure 1. Arachidonic acid-induced relaxations of rabbit aorta: effect of 30 mmol/L KCl (A) and of apamin (100 nmol/L) and CBTX (100 nmol/L) (B). Aortic rings were pretreated with indomethacin (10 µmol/L) and contracted with phenylephrine. Values are mean±SEM, n=5 to 39. *P0.05 compared with control relaxations.

We next examined the effect of apamin on arachidonic acid-induced hyperpolarizations. E_ms of rabbit aortic smooth muscle were measured in indomethacin- and phenylephrine-treated aortic segments with an intact endothelium (Figure 2). Original recordings of E_m of an aortic strip are shown in Figure 2A, and averaged data are graphed in Figure 2B. E_m of the phenylephrine-treated aortic strips averaged -28.7±1.3 mV. Application of arachidonic acid stimulated a 10.2-mV hyperpolarization of the smooth muscle that averaged -38.9±3.0 mV (P<0.05). Incubation with apamin did not alter the basal E_m but did inhibit the arachidonic acid-induced hyperpolarization. These results suggest that endothelium-dependent, arachidonic acid-induced relaxations and hyperpolarizations of the rabbit aorta are mediated by apamin-sensitive K⁺ currents.

View larger version (13K): [in this window] [in a new window]   Figure 2. Arachidonic acid-induced hyperpolarization of rabbit aortic smooth muscle and the effect of apamin (100 nmol/L). Aortic strips were pretreated with indomethacin (10 µmol/L) and phenylephrine (100 nmol/L). A, Original recordings of impalements under various conditions. B, Averaged data from 5 experiments. Values are mean±SEM, n=45 to 55 impalements. PE indicates phenylephrine. *P0.05 compared with control.

To determine whether apamin inhibition of the arachidonic acid responses was due to altered arachidonic acid metabolism and the production and/or release of vasoactive metabolites, we evaluated the effect of apamin on arachidonic acid metabolism of rabbit aorta. Rabbit aortic rings were incubated with indomethacin and [¹⁴C]arachidonic acid. The metabolites were extracted and analyzed by reverse-phase HPLC. The radioactive metabolites comigrated with 6-keto-prostaglandin F₁, THETAs, HEETAs, and hydroxyeicosatetraenoic acids (HETES) (Figure 3). Pretreatment with apamin or CBTX did not alter the synthesis or profile of metabolism. These results indicate that apamin and CBTX do not alter the ability of the rabbit aorta to metabolize arachidonic acid.

View larger version (11K): [in this window] [in a new window]   Figure 3. Effect of apamin (100 nmol/L) and CBTX (100 nmol/L) on metabolism of [14C]arachidonic acid (AA) by rabbit aorta. Aortic rings were incubated with indomethacin (10 µmol/L). Metabolites were extracted and resolved by reverse-phase HPLC. Migration times of known standards are illustrated above each chromatogram. HETEs indicates hydroxyeicosatetraenoic acids; PG, prostaglandin. Other abbreviations are as defined in text.

Our next goal was to determine whether rabbit aortic SMCs exhibited apamin-sensitive K⁺ currents. Whole-cell K⁺ currents of isolated SMCs were elicited by 10-mV depolarizing steps from -60 to +60 mV. Figure 4A shows tracings of outward K⁺ currents from 1 cell at various voltages. The addition of apamin decreased the outward current of this cell by nearly 40%, and subsequent addition of CBTX eliminated nearly all current. Outward K⁺ currents were normalized to cell capacitance, and the averaged data are graphed in Figure 4B. Cell capacitance averaged 8.1±0.4 pF. Apamin decreased the maximum K⁺ current density at +60 mV by 17%, from 43.0±4.8 to 35.7±4.2 pA/pF. Subsequent addition of CBTX further decreased maximal currents an additional 50%, to 14.4±2.3 pA/pF. Thus, these cells show both apamin- and CBTX-sensitive outward currents, suggesting the presence of SK_Ca, IK_Ca, and BK_Ca channels.

View larger version (13K): [in this window] [in a new window]   Figure 4. Effect of apamin (100 nmol/L) and CBTX (100 nmol/L) on whole-cell outward K+ currents of rabbit aortic isolated SMCs. Currents were measured during progressive 10-mV depolarizing steps (500-ms duration, 5-second intervals) from a holding potential of -60 mV. A, Tracings of outward whole-cell K+ currents from a single SMC in the absence or presence of apamin or apamin plus CBTX. Cell capacitance was 6.4 pF. B, Averaged whole-cell current densities. Cell capacitance averaged 8.1±0.4 pF. Apamin significantly decreased current density, and subsequent addition of CBTX further reduced the outward current. Values are mean±SEM, n=10. *P0.05 compared with control.

SK_Ca channels were observed in inside-out patches of isolated rabbit aortic smooth muscle. The current-voltage relations of these small currents in symmetrical KCl (140 mmol/L) are shown in Figure 5A. Average conductance of these channels at -60 mV averaged 24±1 pS. Figure 5B shows recordings of inside-out patches with or without apamin (100 nmol/L) in the pipette solution at the various Ca²⁺ concentrations. Basal activity of the control patch at -60 mV showed rare openings that increased with increasing free Ca²⁺ concentrations (100 to 300 nmol/L) in the bath. In the presence of apamin, K⁺ channel activity was not observed until the bath Ca²⁺ concentration was increased to 300 nmol/L. Mean NP_o values of K⁺ channel activity with and without apamin are graphed in Figure 5C. In control patches, increasing the free Ca²⁺ concentrations from 100 to 300 nmol/L induced a 7-fold increase in K⁺ channel activity. Alternatively, in studies with apamin in the patch pipette, K⁺ channel activity was significantly inhibited. These results demonstrate that rabbit aortic smooth muscle possesses apamin-sensitive SK_Ca channels that might provide an effector target of arachidonic acid metabolites to induce hyperpolarization and vascular relaxation.

View larger version (10K): [in this window] [in a new window]   Figure 5. Activity of SKCa channels in inside-out patches from isolated rabbit aortic SMCs in symmetrical K+ (140 nmol/L). Pipette solutions contained iberiotoxin (100 nmol/L). A, Current-voltage relation, n=5 to 10. B, Single-channel recordings of channel activity in the presence or absence of apamin (100 nmol/L) in increasing bath Ca2+ concentrations (100 to 300 nmol/L). C, Averaged data showing effect of apamin on Ca2+ activation of the mean NPo of the SKCa channel, n=6 to 8. Values are mean±SEM. *P0.05 compared with control.

11,12,15-THETA was isolated as previously described from rabbit aorta¹⁶ and added to the bath perfusate of isolated, aortic SMCs undergoing whole-cell, patch-clamp analysis. Figure 6 shows the outward currents of a control cell (capacitance 11.0 pF) or a cell pretreated with apamin (100 nmol/L; capacitance 8.8 pF). The cells were perfused with vehicle, followed by 11,12,15-THETA. Maximal outward current at +60 mV of the control cell increased by >90%, from 250 to 490 pA, after perfusion with 11,12,15-THETA. Alternatively, the outward K⁺ current of the apamin-treated cell decreased from 380 to 350 pA after perfusion with 11,12,15-THETA. Thus, 11,12,15-THETA increases an outward, apamin-sensitive K⁺ current. This supports our previous finding that 11,12,15-THETA induces hyperpolarization of smooth muscle and therefore, functions as an EDHF.

View larger version (35K): [in this window] [in a new window]   Figure 6. Effect of 11,12,15-THETA on whole-cell outward K+ current of isolated aortic SMCs. Whole-cell currents were measured in a control cell and a cell preincubated with apamin (100 nmol/L). Cells were perfused with vehicle and 11,12,15-THETA at progressive 10-mV depolarizing steps (see Figure 4.).

	Discussion

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The original observations by Furchgott and Zawadski² have implied a role for lipoxygenase metabolites of arachidonic acid as relaxing factors in the rabbit aorta. Previously, we isolated the 15-lipoxygenase metabolite of arachidonic acid, 11,12,15-THETA, from rabbit aorta and characterized this compound as an EDHF.¹⁶ It induced hyperpolarization and vascular relaxation of the rabbit aorta. The work described in this study is the initial characterization of smooth muscle K⁺ currents activated by arachidonic acid metabolites to induce hyperpolarization and vascular relaxation of the rabbit aorta. Both relaxation and hyperpolarization responses to arachidonic acid in endothelium-intact aortas were inhibited by the SK_Ca inhibitor apamin. This clearly suggests a role for SK_Ca currents in mediating these effects. However, it was not evident whether these effects were due to inhibition of smooth muscle K⁺ currents or were secondary to the inhibition of endothelial cell K⁺ currents, resulting in the alteration of arachidonic acid metabolism and/or release of metabolites. The [¹⁴C]arachidonic acid metabolism of the rabbit aorta was not altered by apamin. Therefore, the inhibitory effects of apamin appear to occur at the vascular smooth muscle. However, some interaction of apamin with the rabbit aortic endothelium cannot be completely ruled out.

The relaxations to arachidonic acid were inhibited to a greater extent by 30 mmol/L KCl than by pretreatment with apamin. The reasons for this discrepancy are not clear but might be due to activation of apamin-insensitive K⁺ channels or incomplete inhibition of the SK_Ca current by apamin at the higher concentration of arachidonic acid.

Relaxations and hyperpolarizations induced by arachidonic acid metabolites in this study are mediated by lipoxygenase metabolites.¹⁶ Experimental protocols were performed with indomethacin to block cyclooxygenase, and under normal conditions, the rabbit aorta does not produce the cytochrome P450 dilator metabolites, the epoxyeicosatrienoic acids.²⁵ Synthesis of arachidonic acid in the rabbit aortic endothelium by 15-lipoxygenase involves synthesis of the intermediate metabolite 15-hydroperoxyeicosatetraenoic acid, which is converted to HEETA by a heme-containing hydroperoxide isomerase, which is further hydrolyzed to THETA by epoxide hydrolase.^16,19 In this and a previous study, we have shown that 11,12,15-THETA induces relaxation and hyperpolarization and increases outward K⁺ currents in the rabbit aorta.¹⁶ Other THETA isomers did not induce relaxation. It is not clear at this time whether HEETAs also contribute to vascular relaxations and hyperpolarizations induced by arachidonic acid lipoxygenase metabolites.

In numerous studies, apamin is used in conjunction with CBTX or iberiotoxin to inhibit vascular relaxations attributed to EDHF. However, in this study, apamin alone inhibited the relaxations and hyperpolarizations mediated by arachidonic acid metabolites. Apamin inhibition of EDHF was initially characterized by Murphy and Brayden²⁶ in rabbit mesenteric arteries. In that study, the non-NO, noncyclooxygenase-dependent hyperpolarizations to acetylcholine were blocked by apamin but not by tetraethylammonium (TEA), iberiotoxin, glibenclamide, or 4-aminopyridine. Apamin alone also reduced the non-NO, noncyclooxygenase hyperpolarizations to acetylcholine in the guinea pig carotid artery.²⁷ Also, in the rabbit femoral and renal artery, the non-NO, noncyclooxygenase-dependent relaxations to acetylcholine were inhibited by apamin.^28,29 Finally, in the rat mesenteric artery, non-NO-dependent relaxations to acetylcholine and histamine were also inhibited by this K⁺ channel blocker.³⁰ Therefore, apamin inhibition of relaxations and hyperpolarizations are not unique to the rabbit aorta.

In contrast to these findings, Cowan et al³¹ reported that the non-NO, noncyclooxygenase relaxations to acetylcholine in the rabbit aorta were inhibited by TEA, CBTX, or glibenclamide. Similarly, in a study by Fujimoto et al³² in rabbit mesenteric arteries, the non-NO, noncyclooxygenase relaxations to acetylcholine were inhibited by TEA, CBTX, 4-aminopyridine, or CBTX plus apamin. The reasons for these differences are not clear.

To our knowledge, this is the first study to demonstrate a single-channel current of apamin-sensitive, SK_Ca channels in vascular smooth muscle. Previously, an SK_Ca channel (37 pS) was identified in primary cultured rat aortic SMCs; however, those channels were not sensitive to apamin but were blocked by CBTX.³³ Alternatively, a Ca²⁺-activated, apamin-sensitive K⁺ channel, with a unitary conductance of 10 pS, has been identified in freshly isolated porcine endothelial cells.³⁴ SMCs of the mouse ileum possess apamin-sensitive, Ca²⁺-activated channels of 39 and 10 pS.³⁵ The 39-pS channel was also inhibited by TEA. In fresh rat hippocampal pyramidal neurons, a 21-pS, Ca²⁺-activated K⁺ channel was identified; however, the apamin sensitivity of that channel was not examined.³⁶ In comparison, the characteristics of the 24-pS, apamin-sensitive, SK_Ca channel identified in rabbit aortic smooth muscle of this study are in line with the properties of previously characterized mammalian SK_Ca channels. Because the rabbit aorta smooth muscle apamin-sensitive K⁺ channels were of small unitary conductance, their activity might be easily masked by the activity of IK_Ca and BK_Ca channels. Consequently, it is not surprising that apamin inhibited only 17% of the whole-cell, maximal outward K⁺ current, whereas the CBTX-sensitive current represented 50%.

Besides the apamin-sensitive K⁺ channel, the rabbit aortic smooth muscle membrane possesses other K⁺ currents, including BK_Ca and IK_Ca. The results from this study suggest that arachidonic acid metabolites specifically target the SK_Ca channel. At this time, it is not known whether these metabolites directly interact with the channel or whether cellular signaling cascades are required. Alternatively, global calcium increases by the metabolites can be ruled out, because this would also activate the IK_Ca and BK_Ca channels.

In summary, results from this study and previous work from our laboratory show that arachidonic acid 15-lipoxygenase metabolites of the rabbit aorta, specifically 11,12,15-THETA, activate smooth muscle, apamin-sensitive K⁺ channels to induce K⁺ efflux, membrane hyperpolarization, and vascular relaxation. Therefore, this metabolite represents an EDHF.

Perspectives
The complexity of the EDHF phenomenon increases as more compounds that mediate this vascular event are identified. The role of lipoxygenase metabolites should be recognized as a possible contributor to the EDHF response, especially when relaxations and hyperpolarizations cannot be eliminated with pharmacologic inhibitors of other traditional EDHF mediators. Also, lipoxygenase expression can be modulated by vascular pathologies, such as inflammation and atherosclerosis, and subsequently, their contribution to the regulation of vascular tone and blood flow might be altered. Further studies evaluating the role of lipoxygenase metabolites in both normal and pathologic conditions will further our understanding of EDHF and the comprehensive regulation of vascular tone.

	Acknowledgments

 
These studies were supported by the National Heart, Lung, and Blood Institute (grant HL-37981) and the Northland Affiliate of the American Heart Association. The authors thank Gretchen Barg for secretarial assistance.

Received September 29, 2003; first decision October 29, 2003; accepted November 19, 2003.

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