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Am J Physiol Lung Cell Mol Physiol 296: L130-L139, 2009. First published October 31, 2008; doi:10.1152/ajplung.90436.2008
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Relaxing effects of 17(18)-EpETE on arterial and airway smooth muscles in human lung

Caroline Morin,1 Marco Sirois,2 Vincent Echave,2 Edmond Rizcallah,3 and Eric Rousseau1

1Le Bilarium, Department of Physiology and Biophysics, 2Service of Thoracic Surgery, and 3Department of Pathology, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, Québec, Canada

Submitted 8 August 2008 ; accepted in final form 28 October 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Human cytochrome P-450 epoxygenase enzymes metabolize eicosapentaenoic acid (EPA), an {omega}-3-polyunsaturated fatty acid (PUFA), and leads to the production of 17(18)-epoxyeicosatetraenoic acid, or 17(18)-EpETE. The aim of the present study was to delineate the mode of action of 17(18)-EpETE on human pulmonary artery (HPA) and distal bronchi. Isometric tension measurements demonstrated that 17(18)-EpETE induced concentration-dependent relaxing effects in pulmonary artery and airway smooth muscles. Iberiotoxin (IbTx) and glyburide (Glyb), known BKCa and KATP channel inhibitors, respectively, reversed the relaxation induced by 17(18)-EpETE on both tissues types. Microelectrode measurements showed that exogenous addition of 17(18)-EpETE hyperpolarized the membrane potential of HPA and bronchial smooth muscle cells. These induced electrophysiological effects were reversed by the addition of 10 nM IbTx and 10 µM Glyb. Complementary experiments performed on human bronchi, using the planar lipid bilayer reconstitution technique, demonstrated that 17(18)-EpETE activated reconstituted BKCa channels at low free Ca2+ concentration. Moreover, in bronchi, the relaxing responses induced by 17(18)-EpETE were also related to reduced Ca2+ sensitivity of the myofilaments, since free Ca2+ concentration-response curves, performed on β-escin-permeabilized cultured explants, were shifted toward higher Ca2+. Together, these results provide new insight into the mode of action of 17(18)-EpETE in lung tissues and highlight this eicosanoid as a potent modulator of tone on both HPA and distal bronchi in vitro, which may be of clinical relevance in the pathophysiology of pulmonary hypertension and airway diseases.

17(18)-eicosatetraenoic acid; membrane potential; potassium channels; isometric tension; relaxation

ARACHIDONIC ACID (AA), eicosapentaenoic acid (EPA), and other {omega}-3-polyunsaturated fatty acid (PUFA) can be metabolized by cytochrome P-450 (CYP450) enzymes into several classes of oxygenated metabolites (9, 13, 22). Several of these CYP450-derived eicosanoids have potent biological activities and are recognized for playing important roles in the regulation of vascular, pulmonary, cardiac, and renal functions (25, 31, 34). It has been well-demonstrated that long-chain PUFA, such as EPA, have beneficial effects on cardiovascular function (6, 28). The underlying mechanisms behind their action, however, are incompletely understood (24). EPA may serve as an alternative substrate in CYP450-dependent epoxygenation and hydroxylation reactions as shown in rat hepatic and renal microsomes (33).

The CYP450-dependent AA metabolites, resulting from epoxygenation, include the epoxyeicosatrienoic acids (EET) 5,6-, 8,9-, 11,12-, and 14,15-EET. EETs are produced by the CYP450 subfamilies 2C and 2J (5, 37). EETs activate large-conductance calcium-activated potassium channels in vascular smooth muscle (VSM) cells and are considered as leading candidates for endothelium-derived hyperpolarizing factor (EDHF) (8, 23). In bronchial smooth muscle, EETs are hyperpolarizing factors that activate BKCa channels (3, 20). Recently, our group (20) reported that 14,15-EET decreases the Ca2+ sensitivity of human airway smooth muscle (ASM) cells by reducing the level of phosphorylated CPI-17 protein. The CYP450-dependent EPA metabolites include the epoxyeicosatetraenoic acid regioisomers 5(6)-, 8(9)-, 11(12)-, 14(15)-, and 17(18)-EpETE (Ref. 16). Specific CYP450 epoxygenase isoforms involved in EPA metabolism and which produce 17(18)-EpETE include CYP1A (27), CYP4A1, CYP4A3 (16, 22), and CYP4A12A. An additional potential source for 17(18)-EpETE are endothelial CYP450 isoforms of the 2C and 2J subfamilies that otherwise produce EETs from AA.

Recent studies have demonstrated that EPA epoxides share and even exceed the ability of AA epoxides to stimulate calcium-activated potassium (BKCa) channels (16) and mediate vasodilatation (38). CYP450 epoxygenase metabolites of docosahexaenoic acid (DHA) were shown to be potent dilators of coronary arteries and, importantly, more potent than EETs in activating BKCa channels (36). Using patch-clamp measurements, it has been demonstrated that 17(18)-EpETE stimulates K+ outward currents, displaying typical characteristics for BKCa channel activation in systemic VSM cells. Moreover, this effect is abolished by TEA (16). Recently, the BK{alpha} subunit, the pore-forming subunit of octameric BKCa channels, was shown to represent the molecular target for the principal action of 17(18)-EpETE in systemic VSM cells isolated from cerebral and mesenteric rat arteries (11).

In the present study, we assessed whether 17(18)-EpETE was able to modulate human pulmonary arteries (HPA) and bronchial smooth muscle tone. Complementary approaches were used to perform: 1) tension measurements on human arterial and bronchial rings; 2) membrane potential measurements using the classic microelectrode technique; 3) analyses of the effects of 17(18)-EpETE on unitary BKCa channels reconstituted into planar lipid bilayer (PLB); and 4) assessment of putative changes in Ca2+ sensitivity. We report herein the first evidence that 17(18)-EpETE induces concentration-dependent relaxations, as well as hyperpolarizations of resting membrane potential in vascular and ASM derived from HPA and bronchi.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Isolation and organ culture of human tissues (bronchi and pulmonary arteries). The study was approved by our local institutional ethics committee (protocol no. CRC 05-088-S1). Human lung tissues were obtained from 15 patients undergoing surgery for lung carcinoma. Following lobectomy and rapid transport in sterile physiological saline solution, lung samples, distant from the malignant lesion, were quickly dissected by the pathologist. The absence of tumoral infiltration was retrospectively established in all collected tissues by pathological analysis. Fresh tissue samples were immediately placed in Krebs solution (composition in mM: 118 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 KH2PO4, 1.2 MgSO4, 25 NaHCO3, and 11.1 glucose), previously bubbled with 95% O2-5% CO2, pH 7.4, at 22°C, and then transported to a level 2 culture room. After removal of connective tissues and adhering parenchyma, paired rings of similar weight and length (inner diameter of 0.8–2 mm) were microdissected under binocular control from the same bronchial or arterial segments. Bronchial and arterial rings were placed into individual wells of 24-well culture plates, containing DMEM/F-12 culture medium (2 ml per well) supplemented with 0.3% penicillin (100 IU/ml) and streptomycin (0.1 mg/ml). Culture plates were placed in a humidified incubator at 37°C under 5% CO2. Explants were maintained in culture for 2 days in absence of FBS (20). Explants were untreated (control) or treated (every 12 h for 48 h) with either 10 ng/ml TNF{alpha}, 10 ng/ml TNF{alpha} combined with 100 nM 17(18)-EpETE, or 100 nM 17(18)-EpETE alone before pharmacological challenge. In some experiments, the bronchial rings were treated with either 10 ng/ml TNF{alpha} in the presence of EPA or 10 ng/ml TNF{alpha} combined with EPA and the CYP450 epoxygenase inhibitor, N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide (MS-PPOH) (34).

Isometric tension measurements. The mechanical effects induced by specific agonists and eicosanoids were measured as previously reported (19, 20). Intact rings were mounted between two stirrups in isolated organ baths (Radnoti Glass Technology, Monrovia, CA) containing 6 ml of Krebs solution at 37°C, continually gassed with the 95% O2-5% CO2 mixture, and to which an initial load of 0.6 g for distal HPA and 0.8 g for human bronchi was applied as previously reported (21). Passive and active tensions were assessed using Grass transducer systems coupled to Polyview software (Grass-Astro-Med, West Warwick, RI) for facilitating data acquisition and analysis.

Microelectrode measurements. Longitudinal tissue sections were performed to expose the luminal face of the artery or bronchi. The strips were affixed with the endothelium or epithelium facing up, in the middle chamber (capacity 3 ml) of a tricompartment system, in which temperature was maintained at 37°C, as previously described (20). Membrane potential was measured using conventional intracellular borosilicate microelectrodes filled with 3 M KCl with a resistance ranging from 30 to 50 M{Omega}. Measurements were performed with a KS-G-700 amplifier from World Precision Instruments (Sarasota, FL). Electrical signals were continuously monitored on a TDS 310 oscilloscope (Tektronix, Beaverton, OR). The membrane potential was digitized and recorded using a Digidata 1200B interface and Axoscope 9.0 software from Axon Instruments (Union City, CA).

Preparation of bronchial microsomal fractions and channel reconstitution. Preparation of human crude ASM microsomal fractions and PLB were carried out as described previously (3, 21). Two chambers, denoted cis and trans, were separated by a septum with a 250 µm-diameter aperture. The aperture was pretreated with a mixture of phospholipids, namely phosphatidylethanolamine, phosphatidylserine, and phosphatidylcholine, at a ratio of 3:2:1 (in 25 mg/ml chloroform). This same mixture of phospholipids dissolved in decane was used to form the PLB. The membrane vesicles (10–60 µg of proteins) were added in the cis chamber, which was connected to the head stage of a voltage-clamp amplifier (model 8900; Dagan, Minneapolis, MN). To facilitate the fusion, the experimental chambers contained the following solutions: 250 mM KCl cis and 50 mM KCl trans plus 20 mM K-HEPES and 10 µM free Ca2+ (109 µM CaCl2 + 100 µM K-EGTA), pH 7.4. BKCa channel activities were analyzed in terms of current amplitudes and channel open probability (Po) values with Clampfit 9 software (Axon Instruments).

Permeabilization with β-escin. The measurement of resulting induced myofilament Ca2+ sensitivity was performed as recently reported (20). Bronchial rings were mounted in organ baths and incubated in low free Ca2+ relaxing solution containing (in mM): 87 KCl, 5.1 MgCl2, 5.2 sodium ATP, 10 creatine phosphate, 2 EGTA, and 10 PIPES, brought to pH 7.2 with KOH, at 22°C, followed by treatment with 50 µM β-escin in the relaxing solution for 35 min at 22°C. Ca2+ stores were depleted by addition of 10 µM A-23187. Tension developed by permeabilized bronchial rings was measured in activating solutions containing 5 mM EGTA and precalibrated aliquots of CaCl2 stock solution to yield the desired free Ca2+ concentration, pCa = –log Ca2+.

Drugs and chemical reagents. 17(18)-EpETE and AA were obtained from Cayman Chemical (Ann Arbor, MI) and dissolved in 100% ethanol (EtOH) and stored as 1 mM stock solutions. Iberiotoxin (IbTx) and glybenclamide were purchased from Calbiochem (San Diego, CA). Methacholine chloride (MCh), serotonin (5-HT), phenylephrine (PE), and histamine were purchased from Sigma (St. Louis, MO). DMEM/F-12 and penicillin-streptomycin were purchased from Gibco Invitrogen (Burlington, Ontario, Canada).

Data analysis and statistics. Results are expressed as means ± SE with n indicating the number of experiments. Statistical analyses were performed using the Student's t-test or by a one-way ANOVA. Differences were considered significant when P < 0.05. Data curve fittings were performed using SigmaPlot 9.0 (SPSS Science, Chicago, IL) to determine IC50 values.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
17(18)-EpETE relaxing effect on basal tone is sensitive to IbTx and glybenclamide. Tension measurements were performed on HPA rings to assess the effect of 17(18)-EpETE on resting tone, adjusted to 0.6 g. Cumulative concentrations of 17(18)-EpETE (0.01–10 µM) resulted in concentration-dependent relaxing effects with an IC50 value of 0.85 µM (Fig. 1, A and B). The vehicle, ethanol, had no significant effect on resting tone (Fig. 1B). A concentration of 1 µM 17(18)-EpETE yielded a mean relaxation of 1.74 ± 0.23 mN on these HPA (Fig. 1B). Since 17(18)-EpETE was recently reported to activate BKCa channels and induce vasodilatation in rat coronary microvessels (11), the relaxing effects of 17(18)-EpETE were therefore assessed in the presence of IbTx, a BKCa channel inhibitor, and glyburide (Glyb), an inhibitor of KATP channels (39). Figure 1C displays a typical recording showing that the addition of 10 µM Glyb and 10 nM IbTx both inhibited the relaxing effect induced by 1 µM 17(18)-EpETE on arterial rings. Figure 1D displays bar histograms of the mean inhibitory effects induced by either IbTx, Glyb, or IbTx plus Glyb treatments on 17(18)-EpETE-induced relaxation. Note that in some experiments, rings of pulmonary arteries of larger diameter (up to 5 mm) were dissected and mounted in the isolated organ bath system. These tissues were also relaxed by micromolar concentrations of 17(18)-EpETE. Together, these results suggest that 17(18)-EpETE induces relaxations of HPA, which involve the activation of K+ channels such as BKCa and KATP channels.


Figure 1
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  		Fig. 1. The relaxing effect of 17(18)-epoxyeicosatetraenoic acid [17(18)-EpETE] on basal tone is sensitive to iberiotoxin (IbTx) and glyburide (Glyb). A: representative trace displaying the relaxing effects induced by cumulative additions of 17(18)-EpETE on the resting tension from human pulmonary artery (HPA). B: quantitative analysis of the relaxing responses induced by 17(18)-EpETE on HPA basal tone. Each point represents the mean ± SE with n = 15 as well as the absence of effect of the vehicle [0.6% ethanol (EtOH)]. C: typical recording of the relaxing response induced by 1 µM 17(18)-EpETE and following the sequential addition of 10 µM Glyb and 10 nM IbTx on arterial rings. D: bar histogram quantifying the effect of IbTx, Glyb, and combined pretreatment on 17(18)-EpETE-induced relaxation. *P < 0.05, considered as significant by ANOVA.

 
Effect of 17(18)-EpETE on membrane potential. The results reported above suggest that 17(18)-EpETE may, in fact, modulate the membrane potential of pulmonary artery smooth muscle (PASM) cells. Thus the effect of 17(18)-EpETE on membrane potential was assessed following microelectrode impalement of PASM cells. Figure 2A illustrates a representative recording of the hyperpolarizing effects induced by three cumulative concentrations of 17(18)-EpETE from a resting membrane potential of –51 mV. At the end of each recording, the microelectrode was removed from the PASM cell to allow a return to the reference potential (0 mV), hence validating the measurements. Since this hyperpolarizing effect may involve various K+ conductances, the inhibitory effects of IbTx and Glyb on the hyperpolarization induced by 1 µM 17(18)-EpETE were therefore tested. As illustrated in Fig. 2B, IbTx (10 nM) and Glyb (10 µM) abolished the hyperpolarization induced by the eicosanoid. The mean electrophysiological effect of 17(18)-EpETE on HPA tissues is reported in Fig. 2C. The mean membrane potential was –59.4 ± 1.5 mV, yielding an average calculated hyperpolarization of –8.8 ± 1.1 mV on addition of 1 µM 17(18)-EpETE, which was significantly different from the control value. Following the combined addition of IbTx and Glyb, the mean membrane voltage (–51 ± 1.2 mV) was not significantly different from the control value (–51 mV). These results suggest that activation of BKCa and KATP channels mediates the hyperpolarizing responses induced by this eicosanoid in human PASM cells.


Figure 2
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  		Fig. 2. 17(18)-EpETE-induced hyperpolarization of pulmonary artery smooth muscle (PASM) cells. A: recording of the membrane potential from PASM cell in control and following addition of cumulative concentrations of 17(18)-EpETE. At the end of each recording, the microelectrode was removed from the PASM cell to validate the electrophysiological measurements. B: representative superimposed recordings illustrating the PASM membrane potential following addition of 1 µM 17(18)-EpETE (gray line) and the inhibitory effects of 10 nM IbTx and 10 µM Glyb on the hyperpolarizing response induced by 1 µM 17(18)-EpETE (black line). C: mean resting membrane potential values determined for 1 µM 17(18)-EpETE and following addition of either 10 nM IbTx or 10 nM IbTx plus 10 µM Glyb (n = 7 for each condition).

 
Effect of 17(18)-EpETE on human bronchi. Since the respiratory tract also contains other smooth muscle types, tension measurements were performed on human bronchial rings subjected to 0.8-g basal tone to test the putative effect of 17(18)-EpETE on ASM tone. Addition of cumulative concentrations of 17(18)-EpETE resulted in concentration-dependent relaxing effects on resting tone of human bronchi (Fig. 3A). The effects of 17(18)-EpETE were also evaluated on 1 µM MCh precontracted bronchi. On reaching the plateau phase, cumulative concentrations of 17(18)-EpETE were added and resulted in a concentration-dependent relaxing effect (Fig. 3B), yielding an IC50 value of 0.20 ± 0.03 µM for the eicosanoid in human MCh precontracted bronchi (Fig. 3C). To ascertain the involvement of K+ conductances in the relaxing effects induced by 17(18)-EpETE, experiments were performed in the absence or presence of IbTx plus Glyb on paired recordings. As summarized in Fig. 3D, quantitative analysis of the data revealed that preincubation with either 10 nM IbTx or 10 µM Glyb had partial inhibitory effects on the relaxing responses, whereas the combined addition of both K+ channel blockers induced additive inhibitory effects (up to 56.25%) on relaxation induced by 1 µM 17(18)-EpETE. This suggests that activation of both BKCa and KATP channels is likely involved in the control of the relaxing responses induced by the eicosanoid in human bronchi.


Figure 3
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  		Fig. 3. Relaxing responses to 17(18)-EpETE in human bronchi are sensitive to IbTx and Glyb. A: representative trace displaying the relaxing effects induced by cumulative additions of 17(18)-EpETE on the resting tension from a human distal bronchi. B: typical recording showing the relaxing effect induced by 17(18)-EpETE on human bronchi precontracted with 1 µM methacholine (MCh). C: quantitative analysis of the relaxing responses induced by 17(18)-EpETE on human bronchi precontracted with 1 µM MCh. Each point represents the mean ± SE with n = 19. D: bar histogram displaying the mean inhibitory effect induced by 10 nM IbTx and 10 µM Glyb on 17(18)-EpETE responses from bronchial tissues. Note that the combined addition of IbTx and Glyb abolished 56.25% of the relaxing responses (n = 21). *P < 0.05.

 
17(18)-EpETE hyperpolarizes ASM membrane potential. The effects of 17(18)-EpETE on ASM cell membrane potential were tested following microelectrode impalement on human bronchial tissues. Figure 4A illustrates a representative recording of the hyperpolarizing effects induced by 17(18)-EpETE from a resting membrane potential of –51 mV. On cumulative addition of 17(18)-EpETE concentrations (0.3, 1, and 3 µM) in the mixing compartment of the experimental chamber, an hyperpolarization of the ASM cell membrane potential was consistently recorded (Fig. 4A). At the end of each experiment, the microelectrode was removed from the ASM cell to validate the recording. The resulting concentration-response curve shown in Fig. 4B depicts a maximal hyperpolarization of –13.5 ± 1.1 mV in the presence of 3 µM 17(18)-EpETE. Moreover, 10 nM IbTx pretreatment resulted in a significant inhibition of the hyperpolarizing effects induced by 1 µM 17(18)-EpETE (Fig. 4B). Complementary experiments performed in the presence of IbTx and Glyb pretreatments demonstrated that the hyperpolarizing effect induced by 1 µM 17(18)-EpETE was completely abolished.


Figure 4
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  		Fig. 4. 17(18)-EpETE-induced hyperpolarization of human airway smooth muscle (ASM) cells. A: representative recording of ASM membrane potential in control and following addition of cumulative concentrations of 17(18)-EpETE. Addition of 10 nM IbTx reduced the hyperpolarizing response induced by 17(18)-EpETE by 56%. At the end of each experiment, the microelectrode was removed from the ASM cell to validate the recording. B: mean resting membrane potential values as a function of 17(18)-EpETE concentrations on ASM bronchial cells (n = 10). Note that in the presence of 10 nM IbTx, 10 µM Glyb alone, or as a combined pretreatment, the hyperpolarizing response to 1 µM 17(18)-EpETE was significantly reduced (n = 8).

 
Activation of reconstituted BKCa channels in PLB. The PLB technique allows us to test the direct effect of a specific compound on the activity of a single channel in the absence of cellular regulations (3). To assess the effects of 17(18)-EpETE on channel activity, BKCa channels were reconstituted from ASM vesicles in an asymmetrical KCl buffer system (50 mM trans/250 mM cis), initially containing 10 µM free Ca2+ concentration. The Ca2+ in the trans chamber (cytoplasmic side of the channel) was reduced on addition of a precalibrated amount of EGTA after which Po was monitored (21). Po values decreased as a function of Ca2+, attesting to the Ca2+ sensitivity of the reconstituted channels. Figure 5A illustrates a representative single BKCa channel recording at –20 mV in low free Ca2+ (control condition) and following addition of 1 µM 17(18)-EpETE in the cis compartment. The recording shows that 1 µM 17(18)-EpETE enhanced BKCa channel activity and that this effect was completely blocked by addition of 10 nM IbTx on the extracellular side of the channel (Fig. 5A, bottom trace), hence demonstrating the ability of the eicosanoid to activate BKCa channels. Furthermore, 17(18)-EpETE at 0.3 and 1 µM had significant effects on Po (Fig. 5B) whereby 1 µM 17(18)-EpETE induced a fourfold increase in Po from 0.15 ± 0.03 to 0.66 ± 0.06 in low 0.6 µM Ca2+.


Figure 5
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  		Fig. 5. Effect of 17(18)-EpETE on a reconstituted single human calcium-activated potassium (BKCa) channel recording. A: representative traces of a unitary current obtained at –20 mV, in asymmetrical 50:250 mM KCl buffer at low 0.6 µM free Ca2+ (top trace; control) followed by the addition of 1 µM 17(18)-EpETE in the cis compartment (middle trace) and following the addition of 10 nM IbTx (bottom trace). B: quantitative analysis of the effects of high (10 µM) and low free Ca2+ (0.6 µM) as well as 2 17(18)-EpETE concentrations (0.3 and 1 µM) on channel open probability (Po). Significant Po increases were obtained on addition of 17(18)-EpETE. *P < 0.05.

 
17(18)-EpETE reduces Ca2+ sensitivity in ASM. Comparative analyses were performed on β-escin-permeabilized preparations to assess the effect of 17(18)-EpETE on Ca2+ sensitivity of the human bronchi contractile apparatus. Figure 6A displays superimposed recordings induced by cumulative free Ca2+ (pCa 9 to 4) in control and after 15 min in 1 µM 17(18)-EpETE acutely pretreated bronchi. 17(18)-EpETE had a robust inhibitory effect on Ca2+-dependent developed tension. Cumulative concentration-response curves (CCRC) to free Ca2+ concentrations on permeabilized rings obtained from control and eicosanoid-treated bronchi demonstrate that 17(18)-EpETE reduced the Ca2+ sensitivity of contractile apparatus with a shift in EC50 values toward higher Ca2+ concentrations: 2.31 ± 0.04 µM compared with 0.38 ± 0.03 µM in controls (Fig. 6B). Together, these results attest that eicosanoid pretreatment modifies the Ca2+ sensitivity of the myofilaments and suggest a putative mode of action via the phosphorylation of regulatory proteins (20).


Figure 6
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  		Fig. 6. 17(18)-EpETE decreases Ca2+ sensitivity in ASM. A: representative superimposed recordings illustrating the tension induced by cumulative increases in Ca2+ on β-escin-permeabilized human bronchi in control conditions (black line) and following 1 µM 17(18)-EpETE pretreatment (gray line). B: cumulative concentration-response curves (CCRC) to free Ca2+ obtained from β-escin-permeabilized bronchial rings in control conditions (bullet; n = 20) and after 1 µM 17(18)-EpETE-pretreated bronchi for 15 min ({circ}; n = 21). Note the rightward shift in Ca2+ sensitivity on 17(18)-EpETE treatments. *P < 0.05.

 
Effects of 17(18)-EpETE on TNF{alpha}-pretreated bronchi. In a previous report, we (19) had shown that 10 ng/ml TNF{alpha} pretreatment induced hyperresponsiveness to contractile agonist and Ca2+ hypersensitivity of myofilaments in short-term cultured human bronchi. Herein, the pharmacomechanical properties of these preparations were investigated under various experimental conditions. Human distal bronchi were cultured for 2 days in the absence or presence of 100 nM 17(18)-EpETE and thereafter challenged with contractile agonists, methacholine and histamine. Figure 7 illustrates cumulative CCRC to methacholine from control (untreated) as well as from two series of TNF{alpha}-pretreated bronchi. Figure 7, A and B, shows that the eicosanoid treatment largely reduced the pharmacological reactivity of TNF{alpha}-pretreated tissues to methacholine and histamine. Together, these results suggest that in the presence of TNF{alpha}, a low concentration of 17(18)-EpETE significantly reduces the reactivity and sensitivity of human bronchial smooth muscles thus normalizing their responses to contractile agonists. Comparative analyses were then performed on β-escin-permeabilized preparations to measure the Ca2+ sensitivity of TNF{alpha}-pretreated bronchi (48 h) in either the absence or presence of 100 nM 17(18)-EpETE. CCRC to free Ca2+ concentrations on permeabilized bronchial rings obtained from control and treated bronchi are shown in Fig. 7C. Data demonstrates that treatment with 100 nM 17(18)-EpETE induced a shift in EC50 values (0.69 ± 0.03 µM) toward higher Ca2+ concentrations and thus reduced the Ca2+ sensitivity developed in TNF{alpha}-pretreated bronchi (0.06 ± 0.02 µM) compared with control (0.38 ± 0.03 µM) (Fig. 7C). Thus 17(18)-EpETE treatments reduced the Ca2+ sensitivity of TNF{alpha}-pretreated bronchi. This observation also suggests a functional modulation of contractile proteins.


Figure 7
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  		Fig. 7. 17(18)-EpETE reduced the effects of TNF{alpha} pretreatments in human cultured bronchi. A: CCRC to MCh on untreated (control; bullet) and TNF{alpha}-pretreated bronchi in the absence ({circ}) or presence ({blacktriangledown}) of 100 nM 17(18)-EpETE. B: CCRC to histamine generated from control and TNF{alpha}-pretreated bronchi in the absence or presence of 100 nM 17(18)-EpETE. Each point represents the mean ± SE with n = 18 for each experimental condition. C: CCRC to free Ca2+ obtained from β-escin-permeabilized bronchial rings in control conditions (bullet; n = 18), TNF{alpha}-pretreated bronchi for 48 h ({circ}; n = 20), and TNF{alpha}-treated bronchi in the presence of 100 nM 17(18)-EpETE added every 12 h for 48 h ({triangleup}; n = 18). *P < 0.05.

 
Effect of EPA in the presence of CYP450 epoxygenase inhibitor. Comparative analyses were performed to assess the effect of EPA, the 17(18)-EpETE precursor, in the absence or presence of MS-PPOH, a CYP450 epoxygenase inhibitor, on the hyperreactivity developed by human bronchi on TNF{alpha} treatments. Figure 8A displays superimposed recordings of the contractile effect induced by 1 µM MCh on untreated and TNF{alpha}-treated bronchi either in the presence of 1 µM EPA or 1 µM EPA plus 300 nM MS-PPOH or following 100 nM 17(18)-EpETE pretreatment. 17(18)-EpETE and EPA display potent inhibitory effects on the muscarinic responses, whereas the presence of MS-PPOH significantly reduced the effect of EPA. Figure 8B quantifies the mean responses to methacholine under the same experimental conditions. 17(18)-EpETE as well as EPA treatments largely decreased the pharmacological responsiveness of the sensitized tissues (TNF{alpha}-pretreated), whereas the addition of MS-PPOH largely reduced the effect of EPA (Fig. 8B), which would be consistent with a CYP450 epoxygenase inhibition and a lower production of 17(18)-EpETE from exogenous EPA addition. Together, these data suggest that an EPA metabolite, such as 17(18)-EpETE, endogenously produced by the activity of CYP450 epoxygenase could mediate the reduction of hyperresponsiveness observed on TNF{alpha}-treated human bronchi.


Figure 8
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  		Fig. 8. Effect of eicosapentaenoic acid (EPA), the 17(18)-EpETE precursor, in the absence and presence of methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide (MS-PPOH), a CYP450 epoxygenase inhibitor, on human overreactive bronchi. A: superimposed recordings illustrating the tension induced by 1 µM MCh on TNF{alpha}-pretreated bronchi either in control (top trace) or in the presence of 1 µM EPA (bottom trace) or 1 µM 17(18)-EpETE (gray line) or following 1 µM EPA plus 0.3 µM MS-PPOH pretreatment (dark gray line). B: bar histogram displaying the mean contractile responses induced by 1 µM MCh on control tissues, TNF{alpha}-pretreated bronchi, in the presence of 1 µM 17(18)-EpETE, 1 µM EPA, or 1 µM EPA plus 0.3 µM MS-PPOH. Each bar represents the mean ± SE with n = 14 for each experimental condition. *P < 0.05.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the present report, we investigated the ability of 17(18)-EpETE to modulate arterial and ASM tone in human airways. This is the first report directly assessing the functional and electrophysiological effects of this eicosanoid in human lung. It was found that 17(18)-EpETE relaxes and hyperpolarizes both pulmonary artery and bronchial smooth muscle cells. Moreover, these effects were related to the activation of BKCa and KATP channels in both tissues as well as a decrease in Ca2+ sensitivity of myofilaments from ASM cells. Thus we propose that 17(18)-EpETE, which is derived from EPA metabolism, might play a key role in regulating smooth muscle reactivity in human lung. Furthermore, a relaxing agent able to fine tune arterial as well as bronchial tone would be of significant pharmacological interest under pathophysiological conditions such as pulmonary hypertension, asthma, or acute respiratory distress syndrome (ARDS).

17(18)-EpETE relaxes and hyperpolarizes HPA. It is widely accepted that n-3 or {omega}-3-PUFA rich in fish oils protect against several types of cardiovascular diseases such as myocardial infarction, arrhythmia, atherosclerosis, or hypertension and inflammatory conditions (1, 14). EPA, DHA, or their derivatives might represent active biological components mediating these effects. Although the precise cellular and molecular mechanisms underlying these beneficial effects are still uncertain, the protective effects of PUFA are likely related to their direct effects on VSM cells (12, 18). It has been shown that these PUFA activate KATP channels and inhibit specific types of Ca2+ channels (36). These reports suggest that modulation of VSM cell functions contributes to the beneficial effects of PUFA in the systemic cardiovascular system. However, several questions remain to be addressed about the mode of action of EPA metabolite, such as 17(18)-EpETE, in human PASM. This eicosanoid was found to relax smooth muscle from distal HPA, with IC50 values in the submicromolar range, irrespective of the vasoactive agonist used. This eicosanoid displayed potent relaxing effects on both HPA resting and active tone. To our knowledge, there are only few studies concerning the relaxing effects of 17(18)-EpETE in rodents and in VSM cell culture (11, 16). In the present study, IbTx and Glyb consistently abolished the robust relaxing effect induced by 17(18)-EpETE in HPA under normal external K+ concentration, which suggests that the eicosanoid activates BKCa and KATP channels (39), hence resulting in membrane hyperpolarization. Indeed, the intracellular microelectrode measurements revealed that 17(18)-EpETE induced significant hyperpolarizations of human PASM cells. Because IbTx and Glyb prevented these hyperpolarizing effects, thereby reducing the relaxation induced by 17(18)-EpETE, BKCa and KATP channel activation thus appears to be a key determinant in the control of HPA membrane potential and tone. It has been demonstrated that EPA epoxides share and even exceed the ability of AA epoxides to stimulate BKCa channels (16) and to mediate vasodilatation in canine and porcine coronary microvessels (38). Moreover, it was recently shown that the BK{alpha} subunit, a pore-forming subunit of the BKCa channel, represents a molecular target for the principal action of 17(18)-EpETE in VSM cells isolated from cerebral and mesenteric rat arteries (11). Other eicosanoids derived from AA and produced by CYP450 epoxygenase, such as EET regioisomers, induce similar concentration-dependent relaxing effects on VSM (15, 25) and ASM (3). These effects were shown to be related to hyperpolarization of smooth muscle cells in response to activation of K+ conductances (20).

17(18)-EpETE modulates the physiological properties of ASM cell. EPA, an {omega}-3-PUFA, was shown to exert biochemical and physiological effects, which might counteract inflammatory conditions (2, 17, 29). EPA decreases TNF{alpha} production, which, in turn, would decrease airway responsiveness (7). However, the cellular and molecular mechanisms of CYP450 epoxygenase metabolite of EPA, such as 17(18)-EpETE, have never been investigated in human airway. The data herein are the first to demonstrate a concentration-dependent relaxation to 10–8 to 10–5 17(18)-EpETE on human bronchi. The relaxing effects of 17(18)-EpETE were also observed on resting tone. We (19, 20) previously reported that EET regioisomers, produced by CYP450 epoxygenase from AA, induce large relaxations on both human and guinea pig ASM. The pharmacological relaxing responses, induced by the eicosanoids, could therefore be of physiological significance in respiratory diseases. Moreover, the bronchodilatory effects of 17(18)-EpETE were partially inhibited by IbTx, strongly suggesting that the eicosanoid activates BKCa channels, which typically results in membrane hyperpolarization. In contrast, Glyb only abolished 7% of the relaxing responses to 17(18)-EpETE, indicating that KATP channels have a minor role in the control of relaxations induced by this eicosanoid in human bronchi compared with the more prominent contribution of this channel in pulmonary arteries. There is increasing evidence that TNF{alpha}, one of the proinflammatory cytokines produced by a variety of cells in the lung, is responsible for airway inflammation and hyperresponsiveness (4). A previous report demonstrated that TNF{alpha} (10 ng/ml) pretreatment induced a hyperreactivity to several pharmacological agonists in short-term cultured human bronchi (19). However, in the current study, we demonstrate that 17(18)-EpETE reduces the reactivity and sensitivity of bronchial smooth muscle subjected to TNF{alpha} pretreatment. Furthermore, in airway tissues, our data revealed that CYP450 epoxygenase-dependent EPA metabolite, 17(18)-EpETE, is likely responsible for the reduction of hyperreactivity developed by TNF{alpha}-treated bronchi compared with the mechanical responses observed in the presence of EPA and EPA plus MS-PPOH (as shown in Fig. 8). Consequently, the acute pharmacological relaxing responses triggered in human bronchi, as well as the ability of this metabolite to reduce the hyperresponsiveness, might therefore be of physiological significance in respiratory diseases.

The intracellular microelectrode technique revealed that 17(18)-EpETE induced significant hyperpolarization of human ASM cells under normal K+ concentrations and that this effect was abolished by the combined addition of IbTx and Glyb. Using the PLB reconstitution technique, we evaluated the modulation of large-conducting Ca2+-activated K+ channels by 17(18)-EpETE. This allowed us to investigate the direct action of exogenously added 17(18)-EpETE to the cis compartment (extracellular side), thus mimicking physiological release of this eicosanoid by lung cells. The results obtained confirm that 17(18)-EpETE has marked effects on gating behavior of BKCa channels by increasing their Po. These results are consistent with a direct activation of BKCa channels since they were obtained in the absence of GTP, ATP, cAMP, or other metabolites in the trans chamber (3, 21). Moreover, it has been reported that other AA metabolites, such as EET regioisomers and 20-HETE, directly activate BKCa channels derived from human distal bronchi (21). Further investigations will be required, however, to determine whether specific binding sites are indeed present on the {alpha}- or β-subunit-forming octameric BKCa channel complexes (10, 35). This aside, the electrophysiological effects of 17(18)-EpETE in human bronchi primarily involve the activation of BKCa channels and results in membrane hyperpolarizations, which, in turn, control part of the relaxing responses.

The inherent Ca2+ sensitivity of the myosin light-chain kinase (MLCK), resulting in MLC phosphorylation and contraction and subsequent dephosphorylation by MLC phosphatase, is an important determinant in the regulation of smooth muscle tone (30). Modulation of this mechanism by eicosanoids would explain their overall effect on human ASM. In a previous article, we (19) reported that a 48-h pretreatment with the eicosanoid is able to alter Ca2+ hypersensitivity developed by TNF{alpha}-stimulated bronchi by decreasing CPI-17 phosphorylation levels. The present data demonstrate that in human bronchi treated or not with TNF{alpha}, 17(18)-EpETE significantly reduces Ca2+ sensitivity. These results suggest this eicosanoid may modulate intracellular enzymatic systems, such as Rho kinase and/or PKC/CPI-17 (30). Several studies have suggested that Ca2+-sensitizing mechanisms are also primed under pathophysiological conditions (26, 32). It has been demonstrated that an increase in the expression and activation of contractile proteins, such as RhoA and CPI-17, in hyperresponsive bronchial smooth muscle from rodents, which, in turn, may be responsible for the enhanced agonist-induced Ca2+ sensitization of bronchial contraction associated with airway hyperresponsiveness (26). It was therefore of potential clinical interest to find a lipid mediator able to significantly shift the Ca2+ activation curve toward higher concentrations.

In summary, the present study provides evidence that 17(18)-EpETE is able to modulate the electrophysiological and mechanical properties of two smooth muscle tissues in human lung. These results are correlated with the activation of K+ channels and to a decrease in Ca2+ sensitivity of the contractile apparatus in preparations derived from human bronchi treated (or not) with TNF{alpha}. Collectively, the present data provide new insight regarding the mode of action of 17(18)-EpETE in lung tissues and highlight the fact that this epoxy-eicosanoid produced from EPA by CYP450 epoxygenases displays relaxing effects on both HPA and distal bronchi, which may be of key physiological and clinical relevance in pulmonary disorders.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This work is supported by Canadian Institutes of Health Research Grant MOP-57677. C. Morin is a recipient of a PhD studentship from the Natural Sciences and Engineering Research Council of Canada. E. Rousseau is a member of the Respiratory Health Network of the Fonds de la Recherche en Santé du Québec (http://rsr.chus.qc.ca).


   ACKNOWLEDGMENTS
 
We thank Pierre Pothier for critical review of the manuscript as well as Dr. Marcio M. Gomes and the members of the pathology laboratory for their technical support.


   FOOTNOTES
 

Address for reprint requests and other correspondence: E. Rousseau, Le Bilarium, Dept. of Physiology and Biophysics, Faculty of Medicine and Health Sciences, Université de Sherbrooke, 3001, 12th Ave. North, Sherbrooke, QC, Canada J1H 5N4 (e-mail: Eric.Rousseau{at}USherbrooke.ca)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 

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