5-Oxo-ETE regulates tone of guinea pig airway smooth muscle via activation of Ca2+ pools and Rho-kinase pathway
Frederic Mercier,1
Caroline Morin,1
Martin Cloutier,1
Sonia Proteau,1
Joshua Rokach,2
William S. Powell,3 and
Eric Rousseau1
1Le Bilarium, Department of Physiology and Biophysics, Faculty of Medicine, University of Sherbrooke, Sherbrooke, Quebec, J1H 5N4; 3Meakins-Christie Laboratories, McGill University, Montreal, Quebec, Canada H2X 2P2; and 2Claude Pepper Institute and Department of Chemistry, Florida Institute of Technology, Melbourne, Florida 32901-6982
Submitted 12 January 2004
; accepted in final form 14 April 2004
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ABSTRACT
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5-Oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) is a proinflammatory mediator, but its effects on airway smooth muscle (ASM) have never been assessed. Tension measurements performed on guinea pig ASM showed that 5-oxo-ETE induced sustained concentration-dependent positive inotropic responses (EC50 = 0.89 µM) of somewhat lower amplitude than those induced by carbamylcholine and the thromboxane A2 (TXA2) agonist U-46619. Transient inotropic responses to 5-oxo-ETE were recorded in Ca2+-free medium, suggesting mobilization of intracellular Ca2+. Meanwhile, the sustained contraction, which required Ca2+ entry, was partially blocked by 1 µM nifedipine (an L-type Ca2+ channel blocker) but relatively insensitive to 100 µM Gd3+. The 5-oxo-ETE responses were also inhibited by indomethacin and SC-560 [a cyclooxygenase (COX-1) inhibitor] pretreatments but not by NS-398 (a selective COX-2 inhibitor). The contractile effects of 5-oxo-ETE on ASM were inhibited by the selective TXA2 receptor (TP receptor) antagonist SQ-29548 (75%) and by 2-(p-amylcinnamoyl) amino-4-chlorobenzoic acid pretreatment, a phospholipase A2 inhibitor (66%), suggesting that the major part of its effect is mediated by the release of TXA2. ASM responses to 5-oxo-ETE were also blocked by the Rho-kinase inhibitor Y-27632, which also partially inhibited the response to the TP receptor agonist U-46619, suggesting that the contractile response is due in part to Ca2+ sensitization of ASM cell myofilaments.
5-oxo-eicosatetraenoic acid; isometric tension; membrane potential; calcium entry; transient receptor potential; Rho-kinase
INFLAMMATORY MEDIATORS, including both lipids and peptides, induce an elaborate variety of physiological and pharmacological responses that are mediated by specific receptors coupled to various effectors (11, 21, 23, 30). A number of inflammatory lipids are derived from arachidonic acid (AA), a cell membrane component that is found esterified in the sn-2 position on the glycerol backbone of phospholipids. AA is hydrolyzed principally by cytosolic phospholipase A2 (cPLA2) and then metabolized by various enzymes, including cyclooxygenases 1 and 2 (COX-1 and COX-2), which initiate the formation of prostaglandins (PGs) and thromboxanes (TXs), and 5-lipoxygenase, which initiates the formation of leukotrienes (LTs) and 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) (8). AA is also metabolized by a number of other lipoxygenases to monohydroxy products (HETEs) and lipoxins (8) as well as by cytochrome P-450, which produces epoxyeicosatrienoic acids (EETs) and 20-HETE (35). 5-Lipoxygenase initially converts AA to 5-hydroperoxyeicosatetraenoic acid (5-HPETE), which is either further converted to LTA4 by 5-lipoxygenase or reduced to 5-HETE by peroxidase. The latter compound can then be oxidized by 5-hydroxyeicosanoid dehydrogenase to 5-oxo-ETE (31). 5-Oxo-ETE is a potent eosinophil chemoattractant (33) and can also stimulate neutrophils (32) and monocytes (38). It is active in vivo, stimulating infiltration of eosinophils into rat lungs (39) and human skin (25). In addition, it has been shown to stimulate volume reduction in intestinal epithelial cells (14) as well as the proliferation of prostatic cancer cells (10). 5-Oxo-ETE acts through a highly selective G protein-coupled receptor (29, 32) that has recently been cloned (13) and is coupled to Gi/o (13, 28).
To date, the electrophysiological effects of 5-oxo-ETE on smooth muscle have not been reported. However, this molecule is formed by the same pathway as leukotrienes, and shares structural similarities with both EETs and 20-HETE all of which are known for their ability to modulate vascular (34, 40) and airway smooth muscle tone (3, 6, 36) in asthma, chronic obstructive pulmonary disease, and acute respiratory distress syndrome patients. Alteration of bronchial wall reactivity to various eicosanoids and cytokines is a characteristic feature of chronic asthma. Hence it is now widely accepted that airway smooth muscle (ASM) of the media plays a key role in asthmatic attacks. The objective of the present study was to determine whether or not 5-oxo-ETE can modulate the pharmacological and electrophysiological responsiveness of mammalian ASM and if so what would be its mode of action? To address this question, we assessed its effects on ASM tone in a guinea pig model. Our results show that 5-oxo-ETE induces concentration-dependent contraction of ASM tissues but has no significant effect on the membrane potential of ASM cells. However, its effects are blocked by inhibition of COX and blockade of the TXA2 receptor, suggesting that it acts indirectly, through release of AA.
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MATERIALS AND METHODS
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Isometric tension measurements.
The mechanical effects of 5-oxo-ETE were measured on helically cut trachea and main bronchi taken from male and female albino guinea pigs (Hartley, weighing 250300 g), as previously reported (3, 6). A Krebs solution containing (in mM) 118.1 NaCl, 4.7 KCl, 1.2 MgSO4, 7H2O, 1.2 KH2PO4, 25 NaHCO3, 2.5 CaCl2, and 11.1 glucose, pH 7.4 upon carbogen bubbling (95% O2/5% CO2) was used as physiological medium. The effects of 5-oxo-ETE on basal tone were measured using a Radnoti organ bath (Radnoti Glass Technology, Monrovia, CA) and transducer systems coupled to Polyview software (Grass-Astro Med, West Warwick, RI) to facilitate data acquisition and analysis. The inotropic effects of 5-oxo-ETE were quantified and normalized to those induced by 0.1 µM carbamylcholine (CCh). Various pharmacological agonists, antagonists, and inhibitors have also been used as described below. All procedures involving animal tissues were performed according to current Canadian Council on Animal Care guidelines.
Microelectrode measurements.
Male and female albino guinea pigs (Hartley, 350450 g) were anesthetized with pentobarbital sodium (75 mg/kg). The trachea was rapidly removed and placed in oxygenated (95% O2-5% CO2) Krebs solution at room temperature. A longitudinal section was made to expose the luminal face of the trachea. The epithelium was mechanically removed by delicate rubbing of the surface with a cotton tipped applicator whenever required. Tissue was cut into strips 1012 mm long. The strips were affixed with the ASM facing up, in the middle chamber (capacity 3 ml) of a tricompartment system, in which the temperature (37°C) and solution level were strictly controlled as previously described (3, 6). The tissues were superfused at a constant flow rate of 2 ml/min with standard Krebs solution and allowed to equilibrate for 20 min followed by another 20 min with 5 µM wortmannin to prevent spontaneous smooth muscle contractures at the time of impalements. Tissues were kept at 20°C in oxygenated Krebs solution for several hours. Membrane potential was measured via conventional intracellular borosilicate microelectrodes filled with 3 M KCl and with resistance ranging from 30 to 50 M
. The microelectrodes were connected via an Ag/AgCl2 pellet to the head stage of an amplifier mounted on a no. 13004 micromanipulator from Narishige (Tokyo, Japan). Measurements were performed with a KS-700 amplifier from World Precision Instruments (Sarasota, FL). Electrical signals were continuously monitored on a TDS 310 oscilloscope (Tektroniks, Beaverton, OR). The membrane potential was digitized and recorded with a Digidata 1200B interface and the Axoscope 8.0 software from Axon Instruments (Union City, CA). Data were stored on disk for further analysis.
Cell culture.
Albino guinea pigs from either sex (Hartley, weighing 350450 g) were anesthetized by a lethal dose of pentobarbital sodium (75 mg/kg ip). The trachea was excised aseptically and placed immediately on ice into sterile Hanks' balanced salt solution (HBSS). The trachea was freed of connective tissue and cut longitudinally on the opposite side of the smooth muscle. The epithelial cells were mechanically removed with a sterile cotton swab. The smooth muscle tissue was minced and washed in HBSS containing 200 µM free Ca2+. The tissue was then transferred to a Falcon tube containing 10 ml of the same medium plus 950 units/ml collagenase (type IV), 10 units/ml elastase (type IV) from Sigma-Aldrich (Oakville, ON, Canada). The tissue was digested in a cell incubator at 37°C for 45 min with constant agitation. The cell suspension was then filtered through 100-µm nylon cell strainer, and the filtrate was washed with DMEM/F-12. The cells were centrifuged at 500 g for 6 min, and the pellet was resuspended in 1 ml of DMEM/F-12 supplemented by 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. The cells were plated in 25-mm dishes with
104 cells for each dish, and after 30 min incubation at 37°C, the dishes were filled with 2 ml of supplemented DMEM/F-12.
Drugs and chemical reagents.
5-Oxo-ETE was synthesized chemically as previously described (17). Moreover, most experiments were performed with highly purified 5-oxo-ETE from Cayman Chemical (Ann Arbor, MI). Similarly, U-46619, NS-398, and SQ-29548, also obtained from Cayman Chemical, were dissolved in 100% ethanol (EtOH) and stored as 1 mM stock solutions. The vehicle was tested separately at the maximal concentration used in the presence of active compound. CCh, nifedipine, 2-aminoethoxydiphenyl borate (2-APB), indomethacin and SC-560 (a specific COX-1 inhibitor) were purchased from Sigma (St. Louis, MO). Gadolinium chloride (GdCl3) was purchased from ICN Biomedicals (Cleveland, OH), and 2-(p-amylcinnamoyl) amino-4-chlorobenzoic acid (ONO-RS-082) from Biomol (Plymouth Meeting, PA). FBS, penicillin-streptomycin, and all cell media were purchased from GIBCO Invitrogen (Burlington, ON, Canada). Y-27632 was obtained from Calbiochem (San Diego, CA).
Data analysis and statistics.
Results are expressed as means ± SE; N indicates the number of animals and n the number of trials. Statistical analyses were performed by either paired or unpaired Student's t-tests, as well as ANOVA. Values of P < 0.05 were considered significant. Data curve fittings were performed with Sigma Plot 8.0 (SPSS-Science, Chicago, IL). Concentration-response curves were fitted to the equation
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where C and Cmax are the amplitude of contraction, X is the concentration of agonist, EC50 is the concentration of drug which produces half maximal amplitude of contraction, and nH is the Hill coefficient.
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RESULTS
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5-Oxo-ETE stimulates ASM contraction.
In guinea pig bronchi, exogenous addition of cumulative concentrations of 5-oxo-ETE produced sustained increases in tension (Fig. 1A). The effects were fully reversible upon washing with freshly oxygenated (95% O2/5% CO2) Krebs solution. Vehicle (EtOH) had no effect on the resting tone of guinea pig ASM (Fig. 1B). Figure 1C shows the sequential responses to CCh and 5-oxo-ETE recorded on the same bronchi. Experiments were initially performed on trachea and bronchi separately. The absolute amplitudes of the increases in tension following treatment with 5-oxo-ETE were greater with bronchial ASM, presumably due to the greater proportion of smooth muscle in these preparations. However, when the tension increases were expressed as percentages of the responses to 0.1 µM CCh, the effects of 5-oxo-ETE on bronchial and tracheal ASM were similar (data not shown). The combined data, derived from both types of ASM, gave reproducible concentration-response curves for 5-oxo-ETE with an EC50 value of 0.89 µM and a nH of 1.18 (Fig. 1D). The maximal response was
50% of that attained with CCh, suggesting that 5-oxo-ETE might be viewed as a bronchomodulator.

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Fig. 1. Concentration-response curve of 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) on isometric tension. A: typical trace showing the concentration-dependent inotropic effect of 5-oxo-ETE on guinea pig airway smooth muscle (ASM). W, washout. B: control for the vehicle used. Ethanol (EtOH, <1%) had no effect on resting tone. C: representative responses to 0.1 µM carbachol (CCh) and 1 µM 5-oxo-ETE, respectively. D: concentration-response curve: the positive inotropic effects were standardized as a percentage of the tension induced by 0.1 µM CCh on the corresponding tissue. Note that 0.1 µM CCh represents the EC30 value of the maximum response induced by this muscarinic agonist. The calculated EC50 value for 5-oxo-ETE was 0.89 µM.
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5-Oxo-ETE-induced contraction of ASM is dependent on both Ca2+ mobilization and Ca2+ entry.
Figure 2A shows that 5-oxo-ETE induces sustained contractions in the presence of 2.5 mM Ca2+ (normal Krebs solution). In contrast, 5-oxo-ETE induced only transient contractions in Ca2+-free medium (Fig. 2Ab). Subsequent addition of 2.5 mM CaCl2 during 5-oxo-ETE challenges increased ASM tone to a higher level than that attained in the initial transient contraction (Fig. 2Ab). This response was reversed upon removal of extracellular Ca2+ and 5-oxo-ETE from the physiological solution (Fig. 2Ab).

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Fig. 2. Ca2+ dependence and inhibitory effects of nifedipine, gadolinium (Gd3+), and 2-aminoethoxydiphenyl borate (2-APB) on 5-oxo-ETE-sustained responses. A, a: representative trace of the sustained response induced by 1 µM 5-oxo-ETE; b: response to 5-oxo-ETE in 0 Ca2+ extracellular medium and then upon addition of 2.5 mM CaCl2. B: relaxation induced by 1 µM nifedipine (a) and 100 µM Gd3+ (b) on guinea pig ASM precontracted with 1 µM 5-oxo-ETE. C, a: quantitative analysis of the inhibition produced by 1 µM nifedipine, 100 µM Gd3+, and their combined addition. Inset b: comparative effects of nifedipine concentrations expressed as % relaxation on CCh-induced tone (N = 5). D: sequential addition of 1 µM 5-oxo-ETE in absence (a) and then following 36 µM 2-APB (b) on the same tissue.
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Ca2+ channel blockers such as nifedipine and gadolinium (Gd3+) were used to evaluate the relative contributions of L-type calcium channels (9, 22) and nonselective cation channels (5, 6, 16, 19), respectively. Nifedipine at 1 µM partially inhibited the sustained responses induced by 5-oxo-ETE, whereas 100 µM Gd3+ had relatively little effect on its own but appeared to somewhat enhance the response to nifedipine (Fig. 2B). Gd3+ has been reported to block the channels of the transient receptor potential channel (TRPC) family (TRPC 6), which are implicated in the control of the vascular and bronchial smooth muscle tone (6, 16, 19). Gd3+ induced a 16.0 ± 4.3% relaxation on guinea pig ASM precontracted with 1 µM 5-oxo-ETE, whereas 1 µM nifedipine, used to block voltage-dependent L-type Ca2+ channels, induced a 48.1 ± 4.4% relaxation of the same preparations (Fig. 2C). These inhibitors displayed additive effects, as a combination of nifedipine and Gd3+ induced 64 ± 7% relaxation (Fig. 2C). Note that the same concentration of nifedipine (1 µM) inhibited 40% of the CCh-induced responses (Fig. 2Cb). Because
36% of the tonic response induced by 1 µM 5-oxo-ETE was not blocked by the combined effects of nifedipine and Gd3+ (Fig. 2B, a and b, and 2C), complementary intracellular mechanisms, such as Ca2+ release from intracellular stores (Fig. 2Ab), and increased sensitivity of myofilaments to free intracellular Ca2+ may also contribute to the response to 5-oxo-ETE. The role of intracellular Ca2+ to trigger the inotropic response to 1 µM 5-oxo-ETE (Fig. 2D, a and b), is further supported by the inhibitory effect of 2-APB, which has been presented as an inhibitor of inositol 1,4,5-trisphosphate (InsP3)-activated Ca2+ release channels (23, 24). Despite the fact that 2-APB was reported to interfere with nonselective channels and other transmembrane processes (26), addition of 2-APB alone resulted in an initial transient increase in ASM tension, possibly due to the activation of PLC
(20).
Is the effect of 5-oxo-ETE mediated by TXA2?
To determine whether the effects of 5-oxo-ETE on ASM could be mediated by prostanoids, we examined the effects of COX inhibitors on 5-oxo-ETE-induced ASM contraction. Preincubation of ASM with the nonselective COX inhibitor indomethacin (1 µM) for 15 min completely prevented the effect of 1 µM 5-oxo-ETE on mechanical tension (Fig. 3A). A similar result was obtained with SC-560, a specific COX-1 inhibitor. As shown in Fig. 3Ab, pretreatment with 0.3 µM SC-560 inhibited 80% of the inotropic response to 5-oxo-ETE. The inhibitory effect of SC-560, which is also detected on the resting tone, was not reversed; this result would be explained by its high affinity for the COX-1 isoform. Moreover, 1 µM indomethacin relaxed guinea pig ASM precontracted with 5-oxo-ETE (Fig. 3B). This inhibitory effect was concentration dependent and was maximal at a concentration of 1 µM indomethacin (Fig. 3Bb). In contrast, 5-oxo-ETE contraction was not inhibited by preincubation with the selective COX-2 inhibitor NS-398 (3 µM) (Fig. 3C, a and b). These results suggest that the response to 5-oxo-ETE is meditated by a COX-1-derived prostanoid and could be due to a stimulatory effect on cPLA2, as has been reported to occur in neutrophils (27). As a matter of fact, pretreatment of airway smooth muscle strips with 10 µM ONO-RS-082, a PLA2 inhibitor, largely decreases the inotropic responses induced by 1 µM 5-oxo-ETE. The presence of ONO-RS-082 inhibits 65 ± 6% (N = 3) of the mean tonic response (data not illustrated), which further supports our hypothesis.

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Fig. 3. Sensitivity of 5-oxo-ETE inotropic response to cyclooxygenase (COX) inhibitors: A, a: typical recording showing that indomethacin (Indo) pretreatment blocks the response to 3 µM 5-oxo-ETE on guinea pig ASM, but not the response to 0.1 µM CCh; b: pretreatment with 0.3 µM SC-560, a specific COX-1 inhibitor, largely inhibits the responses to 1 µM 5-oxo-ETE (N = 6). B, a: 1 µM Indo fully relaxes the contractile response induced by 3 µM 5-oxo-ETE; b: concentration-dependent inhibition of Indo on the 5-oxo-ETE inotropic response. The calculated IC50 value for Indo is 0.21 µM (N = 6). C: pretreatment with 3 µM NS-398 (b) did not prevent the inotropic effect of 5-oxo-ETE (a, nor the CCh response, data not shown).
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A likely candidate for the prostanoid mediating the contractile effect of 5-oxo-ETE seemed to be TXA2, as ASM cells are known to possess TXA2 receptors (TP receptors) for this prostanoid (7), as confirmed by the strong response to the selective TP receptor agonist U-46619 (Fig. 4A). The EC50 value for U-46619 was 0.01 µM (Fig. 4B). Unlike 5-oxo-ETE, the response to U-46619 was not affected by indomethacin (Fig. 4C) but was blocked by the selective TP receptor antagonist SQ-29548 (0.3 µM, Fig. 4D). SQ-29548 also inhibited the response induced by 5-oxo-ETE by >70% (Fig. 4, E and F).

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Fig. 4. TXA2 receptor antagonist affects both of thromboxane (TX) A2 receptor agonist and 5-oxo-ETE responses. A: representative trace showing the concentration-dependent ionotropic effect of U-46619. B: concentration-response curve expressed as a percentage of the response induced by 0.1 µM CCh. The calculated EC50 value for U-46619 was 0.01 µM (N = 3 and n = 14). C: 1 µM Indo does not prevent U-46619 response. D: 0.3 µM SQ-29548 fully reverses the inotropic response to 0.03 µM U-46619. E: 0.3 µM SQ-29548 largely inhibits the response to 5-oxo-ETE. F: quantitative and comparative analyses of the SQ-29548 inhibitory effects on 5-oxo-ETE- and U-46619-induced responses (N = 3 and n = 22, N = 2 and n = 8), respectively.
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5-Oxo-ETE does not affect ASM membrane potential.
Several eicosanoids with bronchoactive properties display electrophysiological effects, as previously reported for 20-HETE (6) and EET regioisomers (3). To determine whether this is also true for 5-oxo-ETE, we added this substance to ASM following microelectrode impalement and measured cell membrane potential continuously. Tracheal strips were superfused with Krebs solution under control condition for several minutes (as described in MATERIALS AND METHODS), and then 5-oxo-ETE was added to the perfusion buffer (Fig. 5A). The mean values for ASM cell membrane potential were 57 ± 2 mV (n = 13) in control Krebs solution and 56 ± 3 mV (n = 13) upon addition of 1 µM 5-oxo-ETE (Fig. 5B). Similarly, low concentrations (30 and 300 nM) of the TXA2 U-46619, which were shown to induce detectable and near-maximal inotropic effects, respectively, had no effect on resting membrane potential as shown in Fig. 5B (middle). Despite many attempts using either continuous recording, as illustrated in Fig. 5A, or the multi-impalement technique (3, 6), no significant depolarization was detected for either 5-oxo-ETE or low concentrations of U-46619 (Fig. 5B, middle), whereas 1 µM U-46619 depolarized the membrane potential, and 20 mM KCl induced the expected response and served as a positive control (Fig. 5B, right). Such microelectrode measurements represent a classical approach to assess the electrophysiological effects of various pharmacological compounds (3, 6) before any patch-clamp recording of transmembrane ionic currents. Nevertheless, we have verified that, in contrast to EET-regioisomers (3), 5-oxo-ETE had no effect on large-conductance calcium-activated potassium and Cl channels reconstituted into planar lipid bilayers (data not shown). These results suggest that 5-oxo-ETE has no direct effect on ionic conductances controlling resting membrane potential.

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Fig. 5. 5-Oxo-ETE and low concentration of U-46619 have no electrophysiological effect on resting membrane potential. A: representative recording of ASM membrane potential in control and following 1 µM 5-oxo-ETE superfusion for several minutes. The glass microelectrode was filled with 3 M KCl, and its resistance was 32 M . At the end of each experiment, the microelectrode was removed from the ASM cell to validate our recording. When the measured tip potential at the end of the recording was greater than ±5 mV, the experiment was eliminated from further analysis. B: mean resting membrane potential values determined during various series of experiments: in control vs. 1 µM 5-oxo-ETE (left) N = 3, control vs. 0.03, 0.3, and 1 µM U-46619 (middle, N = 9, 3, 3, and 4) and control vs. 20 mM KCl challenges (right, N = 2). Only 1 µM U-46619 and 20 mM KCl had consistent depolarizing effects.
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Rho-kinase inhibition reduces the responsiveness of ASM to 5-oxo-ETE.
Because 5-oxo-ETE had no apparent effect on membrane potential but is able to mobilize intracellular Ca2+ in various cell types and to increase ASM mechanical tension in a Ca2+-dependent manner, as shown above, we hypothesized that this eicosanoid might enhance the Ca2+ sensitivity of the myofilaments via the Rho/Rho-kinase pathway (37). We tested this hypothesis by preincubating ASM with Y-27632, a specific Rho-kinase inhibitor, before 5-oxo-ETE challenge. Figure 6 demonstrates that the inotropic responses to both 5-oxo-ETE and U-46619 were significantly decreased following Y-27632 pretreatment (Fig. 6, Ab and Bb, respectively). The inhibitory effect of Y-27632 pretreatment on 5-oxo-ETE-induced ASM contractions was concentration dependent (Table 1). The effect of Y-27632 was largely reversible upon extensive washing of the ASM tissues as attested by Fig. 6, Ac and Bc, and tension recovery values (Table 1). Addition of Y-27632 to ASM preparations previously treated with 5-oxo-ETE reversed the contractile effect of this eicosanoid. This effect was concentration dependent and resulted in a return of ASM tone to baseline at a concentration of Y-27632 of 3 µM. These observations suggest that activation of the Rho/Rho-kinase pathway is required for 5-oxo-ETE-induced ASM contraction.

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Fig. 6. Inhibitory effect of Y-27632 on 5-oxo-ETE and U-46619 inotropic responses. A: sequential recordings upon 1 µM 5-oxo-ETE repetitive challenges in control (a), following specific Rho-kinase inhibitor pretreatment (3 µM Y-27632, b), and following recovery (c). B: sequential recordings upon 0.03 µM U-46619 repetitive challenges in control (a), following 3 µM Y-27632 pretreatment (b), and following recovery (c). C: response to 1 µM 5-oxo-ETE was abolished by cumulative additions of Y-27632.
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DISCUSSION
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This is the first report assessing the inotropic effects of this eicosanoid, which has been previously identified as a chemotactic agent for leukocytes (32, 33, 38). We found that 5-oxo-ETE induced concentration-dependent responses on ASM tone, with an EC50 value of 0.89 µM. Tension increases triggered by 5-oxo-ETE are related to both Ca2+ release and Ca2+ entry. Experiments were designed to elucidate the mechanism of action of 5-oxo-ETE, which might play a role in regulation of the ASM tone under pathophysiological conditions. The data suggest that the actions of 5-oxo-ETE are dependent on PLA2 activation and TXA2 release and involve at least three components: a limited Ca2+ release from intracellular stores that, on the basis of various pharmacological maneuvers, is likely due to 1) InsP3-activated Ca2+ release channels, which are insensitive to ryanodine (23, 24), 2) Ca2+ entry via either L-type Ca2+ channels (sensitive to nifedipine) or nonselective cation channels, and 3) a myofilament sensitization to intracellular free Ca2+, likely related to the activation of the Rho-kinase pathway (35).
Prostanoid dependence of the response to 5-oxo-ETE.
The contractile effects of 5-oxo-ETE were either completely blocked or reversed by indomethacin. SC-560 pretreatments also displayed inhibitory effects, but not NS-398 (a selective COX-2 inhibitor), suggesting that they are mediated by the release of a metabolite produced via the COX-1 isozyme. In agreement with this, the selective TP receptor antagonist SQ-29548 reversed 75% of the response of ASM to 5-oxo-ETE. Pretreatment with SQ-29548 had a similar inhibitory effect on the inotropic responses to both 5-oxo-ETE and U-46619 (data not shown). These observations suggest that the major part of the response to 5-oxo-ETE is mediated through the release of TXA2 and activation of the TP receptor. However, the capacity of 5-oxo-ETE to stimulate the release of TXA2 must be limited, as the amount released must have been substantially below that required to achieve the maximal response to this substance, which, in the case of U-46619, was observed at a concentration of
0.11 µM. The fact that indomethacin completely blocked the response to 5-oxo-ETE suggests that the remaining 25% of the response may have been mediated by COX products other than TXA2. For example, both PGD2 and PGF2
are known to stimulate airway smooth muscle contraction (12). 5-Oxo-ETE has previously been shown to stimulate arachidonic acid release through activation of cPLA2 (29), the major PLA2 isoform involved in this process (8), and this could be the mechanism whereby it could stimulate prostanoid release from ASM, since ONO-RS-082 displayed a strong inhibitory effect on the inotropic responses induced by 5-oxo-ETE. It is not clear whether or not these effects of 5-oxo-ETE on ASM are mediated by the recently cloned receptor for this substance, as it was not detected on human ASM cells (15). However, it is possible that an analogous receptor could be present on guinea pig ASM cells, even if it is absent on human cells, or alternatively, the response to 5-oxo-ETE could be mediated by another receptor.
Involvement of calcium.
The transient responses triggered by 5-oxo-ETE in the absence of extracellular Ca2+ suggest that the mechanical response involved Ca2+ release from intracellular stores. This hypothesis has been addressed by different pharmacological maneuvers such as 2-APB pretreatment, which abolished the 5-oxo-ETE inotropic response. Although the effects of 5-oxo-ETE on intracellular Ca2+ levels in isolated ASM cells have not yet been assessed, it is known that this substance is a potent activator of Ca2+ mobilization in neutrophils (32). In addition to mobilization of intracellular Ca2+, Ca2+ entry was necessary to trigger and maintain the sustained inotropic responses that occurred in the presence of extracellular Ca2+ (Fig. 2Ab). In bronchi, Ca2+ entry could also result from the activation of the capacitative Ca2+ entry after depletion of intracellular Ca2+ stores, as reported previously (6). Nevertheless, the tonic response induced by 5-oxo-ETE was poorly sensitive to 100 µM Gd3+, a concentration known to block both capacitative and noncapacitative Ca2+ entry (1, 4, 6), suggesting that this process contributes only marginally to the response to 5-oxo-ETE. This result contrasts with the data recently reported to explain the inotropic effects and mode of action of 20-HETE on ASM cells, which are known to express various lipid-gated nonselective cationic TRPC channel isoforms (2, 6, 31). Among those, TRPC3 and -6 are recognized as direct modulators of resting smooth muscle tone (1, 2, 6). In contrast to Gd3+, nifedipine relaxes 48.1% the tension induced by 5-oxo-ETE, suggesting that L-type Ca2+ channels (14) are directly involved in the control of ASM tone triggered by this eicosanoid. Pharmacological and electrophysiological signals recorded from animal and human ASM have already demonstrated the existence of dihydropyridine-sensitive Ca2+ currents in this tissue (20). The cumulative use of Ca2+ channel blockers such as nifedipine and Gd3+ also reveals that a large component (36%) of the tonic response was not inhibited by a combination of these blockers (Fig. 2). This suggests that part of the inotropic responses induced by 5-oxo-ETE and U-46619 was due to activation of other signaling pathways as discussed below.
Electrophysiological effects of 5-oxo-ETE.
Using the classical microelectrode technique, we have observed that exogenous addition of 5-oxo-ETE (1 µM) had no effect on resting membrane potential. The lack of depolarizing responses to 5-oxo-ETE and low concentrations of U-46619 was somewhat surprising, since other eicosanoids known to induce bronchoconstriction, such 20-HETE, display depolarizing effects (6). Higher concentrations of U-46619 were reported to induce depolarization of vascular smooth muscle (VSM) cells. This property has now been verified for 1 µM U-46619 on ASM cells (Fig. 5B). In contrast, EETs hyperpolarize and relax VSM (35) and ASM cells (3). Apart from their location in the ASM leaflet, the cells used in our experiments were truly ASM cells, since they express large negative resting membrane potentials (59 ± 3 mV on average) and high levels of smooth muscle
-actin on the basis of staining with a selective antibody and a maker of smooth muscle cell differentiation (data not shown).
Effects of inhibition of Rho-kinases on 5-oxo-ETE- and U-46619-induced tension.
The Rho-kinase inhibitor Y-27632 blocked the tonic responses induced by 5-oxo-ETE and partially inhibited the response to U-46619, suggesting that these two molecules act in part by sensitizing myofilaments to intracellular free Ca2+ as proposed by Somlyo and Somlyo (37) for other agonists. Involvement of the Rho-kinase pathway in the response to 5-oxo-ETE would be consistent with the slow activation of the inotropic responses triggered in Ca2+ free solution (Fig. 2Ab). Interestingly, Randriamboavonjy et al. (34) has recently reported that in small endothelium-denuded coronary arteries, 20-HETE-induced contractions depend on the activation of Rho-kinase, which occurs independently of elevated intracellular free Ca2+. In addition to its involvement in Ca2+ sensitization, the Rho-kinase pathway has also been shown to be involved in agonist-induced stimulation of arachidonic acid release from fibroblasts, presumably mediated by activation of cPLA2 (18). Thus part of the inhibitory effect of Y-27632 on 5-oxo-ETE-induced ASM contraction could be due to inhibition of cPLA2-dependent TXA2 release in response to 5-oxo-ETE. This could explain the greater ability of Y-27632 to inhibit responses to 5-oxo-ETE compared with U-46619.
In conclusion, 5-oxo-ETE induces concentration-dependent positive inotropic responses in guinea pig ASM through low-affinity receptors. These responses are dependent for the most part on the formation of TXA2 and possibly other COX-1-derived products. Moreover, they were reversibly inhibited by Y-27632, suggesting that Ca2+ sensitization of myofilaments through the activation of the Rho-kinase pathway may play a significant role to explain the eicosanoid mode of action. It would be of interest to determine whether the Rho-kinase pathway is also involved in mediating other effects of 5-oxo-ETE, such as neutrophil and eosinophil migration. Collectively, our results suggest that 5-oxo-ETE may modulate the media reactivity during remodeling of the airway, which could be of pathophysiological significance.
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GRANTS
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This work was supported by a grant from the Asthma Axis of the Health Respiratory Network of the FRSQ and by Canadian Institutes of Health Research (CIHR) Grant MOP-57677. W. S. Powell was supported by CIHR Grant MOP-6254, and J. Rokach was supported by National Institutes of Health Grants DK-44730 and HL-69835 and National Science Foundation Grant CHE-90-13145.
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ACKNOWLEDGMENTS
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We thank Dr. Jean-Luc Parent for specific advice and Dr. Arthur De Brum-Fernandes for the initial gift of NS-398.
Dr. Eric Rousseau is a National Fonds de la Recherche en Santé du Québec (FRSQ) Scholar and a member of the Health Respiratory Network of the FRSQ (http://www.rsr.chus.qc.ca).
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FOOTNOTES
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Address for reprint requests and other correspondence: E. Rousseau, Le Bilarium, Dept. of Physiology and Biophysics, Faculty of Medicine, Univ. of Sherbrooke, 3001 12th Ave. No., Sherbrooke, J1H 5N4, QC, Canada (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.
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