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Am J Physiol Lung Cell Mol Physiol 275: L423-L431, 1998;
1040-0605/98 $5.00
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Vol. 275, Issue 3, L423-L431, September 1998

Epoxyeicosatrienoic acids relax airway smooth muscles and directly activate reconstituted KCa channels

Marc Dumoulin1, Dany Salvail1, Sophie B. Gaudreault2, Alain Cadieux2, and Eric Rousseau1

Le Bilarium, 1 Departments of Physiology and Biophysics and 2 Pharmacology, Faculty of Medicine, University of Sherbrooke, Sherbrooke, Quebec, Canada J1H 5N4

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Epoxyeicosatrienoic acids (EETs) relax various smooth muscles by increasing outward K+ movement, but the molecular mode of action of EET regioisomers remains to be clarified. The effects of EETs were investigated on bovine airway smooth muscle tone and on reconstituted Ca2+-activated K+ (KCa) channels. 5,6-EET and 11,12-EET induced dose-dependent relaxations of precontracted bronchial spirals. These effects were partly abolished by 10 nM iberiotoxin. Bilayer experiments have shown that 0.1-10 µM 11,12-EET produced up to fourfold increases in the open probability of KCa channels from the cis (extracellular) side by enhancing the mean open time constant and reducing the long closed time constant, without affecting the unitary conductance. EET-induced activations were blocked by 10 nM iberiotoxin. Addition of vehicles or other lipids as well as of GTP and guanosine 5'-O-(3-thiotriphosphate) in the absence of EET had no effect on channel activity. Thus EETs directly activate KCa channels from airway smooth muscle through an interaction with the extracellular face of the channel. We propose that EETs could represent candidate molecules as epithelium-derived hyperpolarizing factors.

calcium-activated potassium channels; endothelium-derived hyperpolarizing factor; epithelium-derived hyperpolarizing factor; relaxation; trachea; planar lipid bilayer; eicosanoid

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

THE EPOXYEICOSATRIENOIC ACID (EET) regioisomers (5,6-, 8,9-, 11,12-, and 14,15-EET) are a family of metabolites of arachidonic acid (AA) synthesized by cytochrome P-450 epoxygenase (11, 12, 16). Cytochrome P-450 in the presence of NADPH catalyzes the formation of epoxides (EETs) from AA (10, 40) as well as the formation of midchain cis-trans conjugated dienols (hydroxyeicosatetraenoic acids) and C-19 and C-20 alcohols (19-OH-AA and 20-OH-AA). Cytochrome P-450 is present in various tissues including the liver (24), kidney (25, 26), and lung (40). AA, from exogenous sources or from sarcolemmal membrane phospholipids, is also metabolized by two additional pathways, lipoxygenase and cyclooxygenase, leading to the production of leukotrienes and PGs, respectively. Much is known about the effects of leukotrienes and PGs in the regulation of smooth muscle tone (1, 33) either in vascular smooth muscles (VSMs) or airway smooth muscles (ASMs). On the other hand, much less is known about the mechanism of action of the epoxygenase metabolites (EETs). EETs were recently shown to dilate coronary arteries (5, 9, 21, 31) as well as renal, cerebral, pial, and caudal arteries from different species (13, 17, 22, 37). Moreover, these studies demonstrated that inhibition of cytochrome P-450 in vascular endothelial cells decreased K+ outflow. Addition of exogenous EETs on endothelium-denuded arteries enhanced Ca2+-activated K+ (KCa)-channel activities without affecting ATP-sensitive K+ channels and facilitated cell repolarization and/or hyperpolarization, which, in turn, resulted in muscle relaxation. Data from a recent study (31) on VSMs suggested that KCa-channel activation by 11,12-EET occurred via a stimulatory G protein-mediated process. On the basis of these results, it was proposed that EETs could represent an endothelium-derived hyperpolarizing factor of the vascular system (9, 15, 19, 23, 31, 34, 36).

In the airways, the epithelium uses different pathways to modulate smooth muscle tone (7). The presence of epoxygenase activities has been demonstrated in Clara cells and type II pneumocytes from rabbit lung (40). Several isozymes such as CYP1A1, CYP2B4, and CYP4B1 have been purified from these cells, the latter exhibiting the majority of epoxygenase activity. Furthermore, KCa channels are widely distributed in ASM cells (2) and play an important role in the regulation of smooth muscle membrane potential and tone modulation (20, 27, 29). Despite this, little is known about the effects of EETs on ASM tone and on the electrophysiological properties of the cellular surface membrane. The aim of this study was, therefore, to investigate whether EETs could relax ASMs and, if so, whether this relaxation was partly mediated by a direct activation of KCa channels. Pharmacomechanical measurements were made on bronchial muscle strips, and a planar lipid bilayer (PLB) reconstitution technique was used to characterize the effect and mechanism of action of exogenous 11,12-EET on KCa channels. Herein, we demonstrate that 5,6-EET as well as 11,12-EET induces dose-dependent and iberiotoxin (IbTX)-sensitive relaxations on epithelium-denuded, precontracted main bronchi. Moreover, the open probability (Po) of the KCa channels was increased in a concentration-dependent manner by extracellular applications of these eicosanoids, without affecting single-channel conductance.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Bronchial smooth muscle strip preparation and isotension measurements. Male guinea pigs (Hartley, 300-350 g) were killed by exsanguination after an intraperitoneal injection of pentobarbital sodium (50 mg/kg). The lungs were quickly removed and placed in cold Krebs solution. The main bronchi were dissected and cut helically as previously described (8). Epithelial cells were removed in most experiments. Each bronchial spiral was mounted in a 4-ml jacketed organ bath containing Krebs-bicarbonate solution composed of (in mM) 118.1 NaCl, 4.7 KCl, 1.2 MgSO4, 1.2 KH2PO4, 2.5 NaHCO3, 2.5 CaCl2, and 11.1 glucose, pH 7.4, gassed with 95% O2-5% CO2 at 37°C. Isometric tension changes were measured with a Grass polygraph (model 7D). Tissues were subjected to an initial loading tension of 1 g and allowed to equilibrate for 60 min (with changes of bath medium every 15 min) before the experiments were started. After equilibration, the main bronchial spirals were precontracted with 0.2 µM carbamylcholine chloride (CCh); then EETs or vehicle was added. The relaxation responses to a specific concentration of EETs are expressed as a percentage of the maximum tension induced by CCh.

Preparation of tracheal smooth muscle microsomal fractions. The crude microsomal fraction was prepared as previously described (3, 4). The reconstitution system consisted of two experimental chambers, denoted cis and trans, separated by a septum with a 250-µm-diameter hole. The cis chamber, which was defined as the compartment to which surface membrane vesicles were added, was connected to an operational amplifier (Dagan 8900, Minneapolis, MN), and the trans chamber was connected to a virtual ground by means of two low-resistance electrodes (MERE 2, World Precision Instruments, Sarasota, FL). The hole was pretreated with a mixture of phospholipids at a concentration of 25 mg/ml chloroform in a ratio of 3:2:1 phosphatidylethanolamine-phosphatidylserine-phosphatidylcholine. PLBs were formed by the application of the same phospholipid mixture dissolved in decane. The experimental chambers contained the following solutions (in mM): 250 KCl cis-50 KCl trans plus 20 K-HEPES and 0.01 free Ca2+ (109 µM CaCl2 + 100 µM K-EGTA), pH 7.4. Microsomal fractions enriched in sarcolemmal vesicles (10-60 µg of proteins) were fused into the PLB from the cis chamber. With this approach, the cis chamber corresponds to the extracellular side and the trans chamber to the cytoplasmic side of the channel. Reconstitution of native microsomal membrane protein into a PLB is particularly suitable to test the direct effects of pharmacological and biochemical agents from either side of the bilayer in the absence of intra- or extracellular components, which is not the case with the various configurations of the patch-clamp technique.

Recording of reconstituted KCa channels. Currents were filtered at 10 kHz and recorded on videotape (DAS/VCR 900 Toshiba, Unitrade). Currents were displayed on-line on a chart recorder (DASH II model MT, Astro-Med) for trace illustrations or were played back, filtered at 1 kHz, and digitalized at 5 kHz for storage in 3-min files on a hard disk. All reconstitution studies were performed at room temperature (22 ± 2°C). KCa-channel activities were analyzed in terms of current amplitudes and channel Po values. Multiple-channel activities were routinely recorded (70%). Po values were determined as described previously (3, 4) and are expressed as mean Po (NPo), where N is the total number of channels recorded experimentally. The number of channels functionally active in the bilayer was determined at the beginning of each recording in the presence of 10 µM free cytoplasmic Ca2+ (trans) and at a holding potential (+40 mV) under which the Po of the KCa channel is maximal. KCa-channel activation by 11,12-EET was determined as NPo in the presence of 11,12-EET divided by NPo in the control condition over the same period of time.

Chemical reagents. CCh, tetraethylammonium (TEA), IbTX, GTP, and guanosine 5'-O-(3-thiotriphosphate) (GTPgamma S) were obtained from Sigma (St. Louis, MO). 11,12-EET in ethanol was obtained from Sigma, whereas other lots of 5,6- and 11,12-EETs dissolved in 0.01% acetonitrile (ACN) were obtained from Cayman Chemical (Ann Arbor, MI). All phospholipids were obtained from Avanti Polar Lipids (Alabaster, AL).

Statistical analysis. Results are means ± SE; n = 3-6 experiments. Mean values were compared by paired or unpaired (when appropriate) Student's t-test with the Sigma Plot program (SPSS, Chicago, IL). Statistical significance of the results was verified, with a threshold value for significance set at P < 0.05.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Pharmacomechanical assessment of the effect of exogenous EETs on bronchial smooth muscle tension. Tension measurements were performed on either epithelium-intact or -denuded guinea pig bronchial smooth muscle spirals to test the effect of either 5,6- or 11,12-EET on ASMs. The muscles were initially precontracted with 0.2 µM CCh; after a plateau phase was reached, various concentrations of 11,12-EET in ethanol were added. They resulted in a slightly significant relaxation compared with the vehicle (ethanol). To overcome the effects induced by ethanol as a solvent, a second vehicle, ACN, was tested in parallel with the 5,6- and 11,12-EET molecules. Aqueous solutions of 0.01% ACN, used to dissolve EET regioisomers, had no effect on precontracted bronchi, as shown in Fig. 1A, except at high, toxic concentrations (in the molar range). Thus the dose-dependent relaxing effects of 11,12- and 5,6-EET dissolved in ACN were assessed on precontracted bronchial strips after muscarinic stimulations (Fig. 1, B and C). These two regioisomers also had relaxing effects on histamine-precontracted bronchi (as shown in Fig. 1, D and D', respectively) or tracheal spirals (data not shown). 5,6-EET proved much more potent than the 11,12-isomer on both muscarinic- and histaminic-induced tensions. Hence the effects of exogenous EETs were more important on epithelium-denuded strips (recording not shown).


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  		Fig. 1.   Effects of 2 regioisomers of epoxyeicosatrienoic acid (EET) on precontracted guinea pig main bronchi. Epithelium-denuded bronchial spirals were precontracted with either carbamylcholine chloride (CCh) or histamine (Hist) and then challenged with various concentrations of 5,6- or 11,12-EET (nos. above traces) dissolved in 0.01% acetonitrile. Arrows, addition of EET and vehicle or washout (W) with control Krebs solution. A: control digitized recording showing absence of effects of cumulative concentrations of acetonitrile (vehicle used in subsequent experiments). A wide range of concentrations up to 1 M, which is toxic for tissues, was tested. B: dose-dependent relaxations induced by cumulative addition of 11,12-EET (1-8 µM) dissolved in 0.01% acetonitrile. C: dose-dependent relaxations induced by 2 concentrations of 5,6-EET after addition of vehicle, which had no effect, as in A. D and D': effects of 5,6-EET and 11,12-EET, respectively, on bronchial spirals precontracted with Hist. Note that effects of EETs as well as those of muscarinic and histaminic agonists were fully reversible.

IbTX affects EET-induced relaxations. Experiments were designed to test the concentration-dependent efficacy of EETs on precontracted bronchi as well as the effect of IbTX, a specific blocker of large-conducting KCa channels. Figure 2A' shows that preincubation of bronchial strips with 10 nM IbTX affected the amplitude and time course of the relaxing responses induced by 2 µM 5,6-EET compared with the control condition (Fig. 2A). Quantitative analysis of the data is summarized in Fig. 2, B and C. The concentration-dependent effect of EETs from 0.03 to 3 µM confirmed the greater potency of 5,6-EET versus 11,12-EET (Fig. 2C) on relaxation amplitude. Moreover, preincubation with 10 nM IbTX consistently inhibited part of the relaxing responses induced by the EET molecules, suggesting that activation of the KCa channel might be involved in the responses induced by 5,6- as well as by 11,12-EET. However, IbTX was much more effective in inhibiting the relaxing responses induced by 2 µM 11,12-EET than that caused by 2 µM 5,6-EET (Fig. 2, B and C). This observation would be consistent with the idea that the effect of exogenous EETs might affect other conductances or membrane processes. Because previous studies (9, 19, 22, 31) on VSMs have also shown that 11,12-EET induced relaxations by acting on ionic conductances, subsequent experiments were performed on unitary channel proteins with the PLB reconstitution technique to ascertain the relevance of this mechanism in ASMs and to test whether EET isomers can act directly on KCa channels.


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  		Fig. 2.   Effects of iberiotoxin (IbTX) pretreatment on relaxing responses induced by 5,6- and 11,12-EET after muscarinic stimulations. A: consecutive and representative recordings showing effects of the same concentration of 5,6-EET before (A) and after (A') pretreatment of tissues with IbTX before CCh challenge. Arrows, addition of CCH, EET, and IbTX, or W. Amplitude as well as time course of relaxations induced by 5,6-EET were both altered in presence of IbTX. B: quantitative analysis of effect of 5,6-EET on amplitude of relaxation in absence (control) or presence of IbTX, a specific blocker of large-conductance Ca2+-activated K+ (KCa) channels. C: IbTX pretreatment also modifies relaxing effects of 11,12-EET. Both isomers were dissolved in acetonitrile. Data are means ± SE; n = 4 and 5 experiments for each set of experimental conditions. Note that 10 nM IbTX had a proportionally larger effect on 11,12-EET-induced relaxation. * Significant variation from respective control condition, P < 0.05.

Characterization of reconstituted KCa channels from bovine ASMs. KCa-channel activities were characterized in asymmetrical (50/250 mM) or symmetrical (250/250 mM) KCl buffer systems containing, initially, 10 µM free Ca2+. Single-channel recordings in Fig. 3A show the influence of holding potential on the behavior of a large-conducting channel and its Po. The channel Po-holding potential relationship is best described by a Boltzmann equation (Fig. 3B) in a range of -60 to +60 mV in symmetrical conditions. The unitary current is activated by steady-state membrane depolarizations; the curve reaches a plateau above +30 mV.


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  		Fig. 3.   Voltage-dependent behavior of reconstituted single K+ channels from bovine airway smooth muscle (ASM). A: single-channel current at different holding potentials. Traces were obtained in symmetrical KCl buffer plus free Ca2+, pH 7.4. O, open state; C, closed state. Channel open probability (Po) decreases as holding potential changes from positive to negative values. B: voltage dependence of channel Po in symmetrical condition. Data were fitted with a Boltzmann equation, where Vm is membrane potential, V1/2 is holding potential when Po = 0.5 and was estimated at -43 mV, and K is a constant (13.94). Po values in B were calculated by amplitude histogram analysis and are means ± SE; n = 6 experiments.

The Ca2+ sensitivity of the large-conducting K+-selective channel (280 pS) was confirmed by varying the free Ca2+ concentration ([Ca2+]) in the trans chamber ([Ca2+]trans), which generally corresponds to the cytoplasmic face of the channel, at a constant holding potential (+20 mV) in symmetrical conditions. Channel activity was recorded at various [Ca2+]trans values ranging from 0.01 to 10 µM. Po was reduced as [Ca2+]trans was lowered (Fig. 4A). The average Ca2+ sensitivity of the reconstituted channels was normalized and quantified (Fig. 4B). The NPo-free [Ca2+]trans relationship was fitted with a Hill equation where the half-maximal activation [dissociation constant (Kd)] was obtained at 0.7 µM free [Ca2+]trans with a Hill coefficient of 2.81, which suggests the existence of at least three Ca2+-activating sites. Thus the voltage and Ca2+ dependence of this large-conducting K+-selective channel attest of its identity as a KCa channel, as previously reported (4, 32). A brief pharmacological characterization of these KCa channels with TEA and IbTX, both known blockers of this type of channel (18, 20), was performed. The addition of TEA to the cis chamber of the bath induced a block of the channel that significantly decreased the unitary current amplitude (from 13 to 4 pA at 0 mV), without altering the channel Po (Fig. 5A). The results obtained after the addition of IbTX in the cis chamber are shown in Fig. 5B; representative traces were obtained in symmetrical condition at +20 mV, where 10 nM IbTX almost completely blocked the channel activity. All data in Figs. 3-5 are consistent with typical KCa-channel characteristics (20, 32).


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  		Fig. 4.   Ca2+ sensitivity of large-conducting K+ channels from bovine ASM. A: multiple-channel recording at different intracellular free Ca2+ concentration ([Ca2+]) levels. Current traces were obtained in symmetrical KCl buffer as in Fig. 3 at +20 mV. After cumulative additions of EGTA, free [Ca2+]trans was sequentially decreased from 10 to 0.6 µM; normalized mean Po (NPo, where N is total no. of channels recorded experimentally) was accordingly reduced from 1.0 to 0.05. B: dependence of channel Po on [Ca2+]. Data were fitted with a Hill equation, where Po max is maximal Po and Kd (dissociation constant) is half-maximal activation obtained at 0.7 µM [Ca2+] with Hill coefficient (n) = 2.81. Po values for B were calculated from amplitude histogram analysis and are means ± SE; n = 3-5 experiments.


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  		Fig. 5.   Effects of tetraethylammonium (TEA) and IbTX on bovine ASM KCa channels reconstituted in planar lipid bilayers. A: unitary currents were recorded in asymmetrical KCl buffer with free Ca2+ at 0 mV in control (top) and after addition of TEA to cis (extracellular) side (bottom). Analysis indicated a decrease in unitary current amplitude after application of drug. B: representative traces obtained in symmetrical KCl buffer as described above at +20 mV and in presence of free Ca2+ in control (top) and after addition of µM IbTX to cis side (bottom). Addition of IbTX at this concentration resulted in a complete block of channel activity. Nos. on right, apparent conductance (gamma ) and Po in A and B, respectively.

Activating effect of 11,12-EET on reconstituted KCa channels. KCa-channel recordings in control conditions and after the addition of increasing concentrations of 11,12-EET in the cis chamber (Fig. 6A) demonstrated that cumulative 11,12-EET concentration ([11,12-EET]) enhanced channel Po. A concentration-dependent activation was consistently observed in a narrow range of concentrations: the lowest concentration showing a significant effect was 0.9 µM (P < 0.05), whereas 3 µM produced a fourfold increase in Po (Fig. 6B). The activating effects of EET molecules on KCa channels were observed at various voltages (data not shown). Of note is that the concentrations required to increase the Po of reconstituted ASM KCa channels remained higher than those determined from similar patch-clamp studies on ASMs (9, 31). In addition, current-voltage curves in control conditions and in the presence of various [11,12-EET] levels (Fig. 6C) show that the eicosanoid had no effect on the unitary conductance because no change of slope was observed.


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  		Fig. 6.   Effect of 11,12-EET on single-channel recordings and concentration-dependent activation of reconstituted KCa channels. A: representative recordings of KCa channels obtained in symmetrical KCl buffer at +20 mV in low free [Ca2+]trans and after addition of cumulative concentrations of 11,12-EET on cis side. B: quantification of effect of 11,12-EET in same conditions as in A on addition of cumulative concentrations of 11,12-EET. Significant increases were obtained after addition of concentrations >=  0.9 µM 11,12-EET. C: current-voltage curve in control conditions (symmetrical KCl buffer plus 0.6 µM free Ca2+) and in presence of indicated concentrations of 11,12-EET. 11,12-EET had no effect on current amplitude and unitary conductance of KCa channels. All data are means ± SE; n = 5 experiments.

Specificity and mode of action of EET molecules on KCa channel. It was reported that extracellular AA as well as G proteins activated by intracellular GTP analogs might modify the gating of various K+ channels including KCa channels (29). The results obtained from control experiments performed in the presence of bioactive lipids and GTP analogs are shown in Fig. 7, A and B, respectively. The addition of AA (1-3 µM) on the cis side had no effect on channel activity, although activation was observed for higher concentrations of the EET precursor. In contrast, platelet-activating factor (PAF) had no effect in the concentration range tested (3-10 µM) from the cis side. This absence of effect of PAF and the high concentrations of AA (>5 µM) required to significantly enhance channel activity support the inherent and specific potency of 11,12-EET added to the cis side. Note that neither 0.6% ethanol in the cis chamber nor 3 µM 11,12-EET in the trans chamber had any effect on single-channel activity.


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  		Fig. 7.   Activating effects of 11,12-EET were compared with effect of 2 bioactive lipids, GTP and guanosine 5'-O-(3-thiotriphosphate) (GTPgamma S). A: quantitative analysis of effects of arachidonic acid (AA) and platelet-activating factor (PAF). Effects of these lipids were assessed on KCa-channel activity and compared with effects of various concentrations of 11,12-EET and ethanol. Neither vehicle or 11,12-EET added to trans chamber nor PAF had any effect on channel behavior. However, AA, which had basically no effect at low concentrations, activated KCa channels at higher concentrations (5-10 µM). All data are means ± SE; n = 5, 4, and 3 experiments for each set, respectively. B: results from complementary experiments show that GTP and GTPgamma S (up to 100 µM trans) had no activating (or synergistic) effect on KCa-channel behavior compared with direct activation induced by 1.5 µM 11,12-EET from cis chamber. Average (±SE) values were calculated from n = 5, 3, 3, and 4 experiments, respectively. *P < 0.05.

It has been suggested that the effects of EET molecules in VSMs could be due to an activation of G proteins (31). Because G proteins could be present in our bilayers on fusion of native membrane vesicles, the effects of GTP and its nonhydrolyzable analog GTPgamma S were tested on ASM KCa channels alone and in the presence of EET. Figure 7B shows that GTP and GTPgamma S had no effect when added alone in the trans chamber, corresponding to the intracellular face of the channels. In addition, when 1.5 µM 11,12-EET was applied from the cis chamber either before or after 100 µM GTP, the KCa channels were activated to an extent similar to that observed in the absence of GTP (Fig. 7B). These data strongly suggest that EET molecules directly activated KCa channels reconstituted into PLB and that GTP had no direct or synergistic effect before or after EET addition. Note that, in this work, cholera toxin activation was not assessed due to technical (the large volume of the experimental chamber) and cost limitations. Because quantitative analysis demonstrated that 11,12-EET had no direct effect on channel Po when added to the trans (intracellular) side of the channel protein (Fig. 7A), we therefore postulate that 11,12-EET acts from the extracelluar side and interacts with transmembrane segments or phospholipids within the external bilayer leaflet to induce channel activation.

Multiple-channel activations by EET and blockage by IbTX. In Fig. 8, the effects of EET were further assessed on multiple-channel recordings; up to three unitary current levels were observed in the presence of 1 µM free Ca2+ trans (Fig. 8, A and A'). Addition of EGTA decreased the free [Ca2+] to 0.3 µM and reduced channel activities (Fig. 8, B and B'), whereas addition of 3 µM 11,12-EET reactivated the functional channels present in the PLBs (Fig. 8, C and C'). All three channels, which were sensitive to voltage and Ca2+, were sequentially blocked by 50 nM IbTX (Fig. 8, D and D'). Altogether, these results confirmed that EET molecules are able to reactivate all reconstituted functional KCa channels, even at low [Ca2+] values and that this activation can be reversed by IbTX.


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  		Fig. 8.   IbTX blocked ASM KCa channels activated by 11,12-EET. Multiple-channel recordings were obtained in symmetrical KCl buffer at +30 mV under various experimental conditions. A: in presence of free Ca2+ trans. B: at low free Ca2+ concentration on addition of EGTA. C: after addition of 11,12-EET, channels reactivated in absence of Ca2+ and pH changes. D: addition of IbTX sequentially blocked channel activity and thus reversed EET activation. Complete inhibition by IbTX was obtained within 10 min. A', B', C', and D', corresponding amplitude histograms calculated from 2-min data files of representative steady-state recordings.

Time analysis of a single KCa channel on 11,12-EET activation. Figure 9 shows the open and closed time distributions determined from a single-channel recording before and after the addition of 11,12-EET. The distributions of the open or closed events were fitted by either a single exponential (left) or the sum of two exponentials (right), respectively. The mean open time constant was increased by 53% on addition of 1.5 µM 11,12-EET, whereas the long closed time constant was subjected to a fivefold decrease (231 ms in control condition and 46 ms in the presence of EET). These results indicate that 11,12-EET affects the gating behavior of the KCa channel by reducing the duration of long closed events and stabilizing the duration of the open events. This, in turn, resulted in an increase in the average number of transition-by-time units, a lengthening of the open events, and thus an increase in Po values.


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  		Fig. 9.   Time analysis of single KCa-channel activity before and after addition of 11,12-EET. A: open (left) and closed (right) time distributions obtained under control conditions in presence of free [Ca2+]trans. Distributions were fitted either by a single exponential or sum of 2 exponentials. tau op, Open time constant; tau cl1, short closed time constant; tau cl2, long closed time constant. B: identical analyses were performed on the same single KCa-channel recordings after addition of 11,12-EET. Value of mean open time constant was increased by exogenous EET, whereas long closed time constant was decreased fivefold.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

This work is the first to report in vitro relaxations of muscarinic- and histaminic-precontracted ASMs by EETs. This modulation appears to be partly induced by a direct activation of KCa channels by EETs, as demonstrated by their sensitivity to IbTX. The most important contribution of the present work was therefore to show that EETs directly activate reconstituted KCa channels. Although it has previously been shown that all EET regioisomers have similar effects on K+ conductance (9), the present work focused directly on the molecular mechanism of action of two metabolites of the epoxygenase pathway, 5,6- and 11,12-EET.

The results of this study are in-line with recent reports showing the modulation of smooth muscle tone by EETs on various mammalian arteries (13-15, 19, 22, 34-36) as well as on guinea pig bronchi (40). Moreover, it has been shown that EET molecules activate K+ currents in VSMs (9, 22, 31). Single-channel measurements have demonstrated an increase in the Po of KCa channels but failed to provide precise information on the mechanism of action of the EETs (9).

In this study, pharmacomechanical measurements revealed the strong relaxing effects of 5,6-EET and the concentration-dependent action of 5,6- as well as of 11,12-EET when dissolved in an ACN aqueous solution. Because our data demonstrated a significant effect of 5,6- and 11,12-EET only on epithelium-denuded bronchi (Fig. 1, A-D), we suggest that the epithelial layer could play the role of a biochemical barrier to exogenous EET molecules. However, ASM spirals were much more sensitive to 5,6-EET than to 11,12-EET when low concentrations of each regioisomer diluted in ACN were tested, as demonstrated in Figs. 1, B and C, and 2B. Similar differences in EET sensitivities were also observed on histamine-precontracted strips (Fig. 1, D and D'). These results would argue in favor of stereo- or regio-specific effects of EETs on ASM tone. This is of interest because it was shown that EET isomers were produced in various ratios by microsomal epoxygenases (10). Furthermore, tension measurements revealed that 5,6- and 11,12-EET-induced relaxations were both partially inhibited by IbTX pretreatment (Fig. 2, B and C), with a greater percentage of inhibition induced by 10 nM IbTX on 11,12-EET- than on 5,6-EET-induced relaxations. Such results support a contribution of the KCa channels to ASM relaxation (4, 28, 29). On CCh-induced tonic contractions, the KCa channels must display a low Po despite membrane depolarization and an increase in cytosolic free [Ca2+]. In fact, it has been demonstrated that the maintenance of ASM contraction involves KCa-channel inhibition (28), thus momentarily reducing the contribution of these channels in the control of membrane potential and, consequently, in the control of relaxation. Because 10 nM IbTX partly inhibits EET-induced relaxations on precontracted strips, it was postulated that 5,6- as well as 11,12-EET might activate a cationic conductance, although other membrane processes may also be involved.

Using the PLB reconstitution technique, we evaluated the modulation of KCa channels by 5,6- and 11,12-EET after performing electrophysiological and pharmacological characterizations of the channels. Single- and multiple-channel reconstitutions were performed to evaluate channel sensitivities to voltage and to the intracellular free [Ca2+] (Figs. 3 and 4) as well as to known blockers such as TEA and IbTX (Fig. 5). We then investigated the direct action of exogenously added 11,12-EET to the cis (extracellular) compartment (Fig. 7), thus mimicking the physiological release of EETs by epithelial cells and/or other lung cells. These results are consistent with a direct activation of KCa channels because they have been obtained in the absence of GTP, ATP, cAMP, or other metabolites in the trans chamber. A direct activation implies interaction of EET molecules with channel proteins or neighboring proteins or with phospholipids, without the implication of intracellular cascades such as the activation of G proteins. Li and Campbell (31) recently reported that EETs activated KCa channels, presumably via the stimulation of a Gs protein that could, in turn, modulate channel gating. They postulated that EETs could bind to a receptor, activating the Gs proteins, or act directly on the Gs proteins themselves. The activated G proteins, hence, appeared to affect KCa channels without the involvement of second messengers (cAMP and cGMP) or kinases such as protein kinases A and G, two kinases known to directly phosphorylate KCa channels (3, 27, 30). The present results do not support and basically refute the requirement for the involvement of a G protein because GTP had no stimulating action or synergistic effect on reconstituted ASM KCa channels, whereas EET molecules were still very effective. Hence we suggest that, in ASM, the EETs tested can alter K+ efflux via a direct interaction with the KCa channel.

The lowest concentration of EET inducing a significant activation was 0.9 µM (P < 0.05), whereas a fourfold activation was obtained with the addition of 3 µM 11,12-EET. These results suggest a lower sensitivity of the reconstituted ASM channels to 11,12-EET than that previously reported for arterial KCa channels where as little as 100 nM 11,12-EET induced a 10-fold NPo activation (31). The lower sensitivity of the ASM K+ channels may be an artifact of the reconstitution technique. PLBs are made of very specific phospholipid composition and completely eliminate intracellular cascades. Nevertheless, one cannot rule out the possibility of a dual activation or multiple regulation of ASM KCa channels, already foreseen as multisensor ionic channels, being sensitive to voltage, intracellular free [Ca2+], phosphorylation (3, 27), oxydo-reduction, and nitric oxide (4).

Complementary experiments have also demonstrated that PAF and low concentrations (<= 3 µM) of AA, the precursor of 11,12-EET in vivo, had no direct effect on the reconstituted KCa channel unless much larger AA concentrations were used. Hence it appears that the epoxide group of the 11,12-EET molecule is important in the direct modulation of ASM KCa channels. All experiments involving various [11,12-EET] levels demonstrated that the molecules did not affect the unitary conductance of the KCa channels (Fig. 6C), which is in agreement with the results of previous work (9, 31). Meanwhile, we report the first quantitative time analysis of KCa-channel gating on 11,12-EET activation, which demonstrates an increase in mean open time and a reduction in the duration of the long closed state (Fig. 9). Finally, control experiments showed that vehicle alone as well as trans (intracellular) application of 11,12-EET did not affect KCa-channel behavior (Fig. 7). The side-specific effect of EETs argues in favor of the presence of activation sites on the extracellular face of the KCa-channel proteins to which these highly hydrophobic molecules could bind.

Recently, it was reported that a high-affinity binding site for 14(R),15(S)-EET was present in guinea pig mononuclear cell membranes (39). Other authors (31) have postulated the existence of a binding site for 11,12-EET on bovine coronary arterial cell membranes. It is also known that EETs bind to the phospholipids of the surface membrane (6). Further investigation is necessary to determine whether specific binding sites are present on the KCa channel or on functional neighboring proteins such as their beta -subunit in ASM cell membranes or whether EET molecules only bind to particular specific phospholipid acyl chains or membrane microdomains. Interestingly, it has been reported that all four EET regioisomers activate high-conductance, Ca2+-dependent K+ channels on pig coronary arterial endothelial cells (15). This possibility remains to be proven for other cell membranes of the respiratory tract.

In conclusion, EETs induced dose-dependent relaxations of ASM in vitro, which were partially inhibited by IbTX and partly by a direct activation of the KCa channels present in the sarcolemmal membrane of ASM cells. The evidence reported herein reveals a direct activation of ASM KCa channels, unlike that reported in a study (31) performed on coronary arteries. Moreover, we have shown that 11,12-EET affects the gating but not the unitary conductance of KCa channels. It is reasonable to postulate the presence of a specific extracellular binding site on the channel proteins or within surrounding phospholipid microdomains. Note that the present study does not rule out the possibility that EET molecules affect other conductances. Because 11,12-EET induces an increase in KCa-channel activity, it should cause a repolarization or hyperpolarization of ASM cells. We therefore propose that EETs represent potential endothelium-derived hyperpolarizing factors in ASM. This hypothesis is further tested in the companion paper (38).

    ACKNOWLEDGEMENTS

We thank Drs. Paul Pape and E. Ruiz Petrich for discussing and revising the manuscript.

    FOOTNOTES

This work was supported by Grant MT 12287 from the Medical Research Council of Canada and by the Association Pulmonaire du Québec.

E. Rousseau is a Senior Fonds de la Recherche en Santé du Québec (FRSQ) Scholar. D. Salvail is a recipient of an FRSQ-Fonds pour la Formation de Chercheurs et d'Aide à la Recherche Student Fellowship. Both laboratories are members of the Health Respiratory Network of the FRSQ.

Address for reprint requests: E. Rousseau, Le Bilarium, Department of Physiology and Biophysics, Faculty of Medicine, Univ. of Sherbrooke, 3001, 12th Ave. North, Sherbrooke, Quebec, Canada J1H 5N4.

Received 29 October 1997; accepted in final form 1 May 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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