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Published ahead of print on January 19, 2007, doi:10.1165/rcmb.2006-0281OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 36, pp. 633-641, 2007
© 2007 American Thoracic Society
DOI: 10.1165/rcmb.2006-0281OC

Epoxyeicosatrienoic Acid Relaxing Effects Involve Ca2+-Activated K+ Channel Activation and CPI-17 Dephosphorylation in Human Bronchi

Caroline Morin, Marco Sirois, Vincent Echave, Marcio M. Gomes and Eric Rousseau

Le Bilarium, Department of Physiology and Biophysics; Service of Thoracic Surgery; Department of Pathology, Faculty of Medicine and Health Sciences, University of Sherbrooke, Sherbrooke, Quebec, Canada

Correspondence and requests for reprints should be addressed to Eric Rousseau, Le Bilarium, Department of Physiology and Biophysics, Faculty of Medicine and Health Sciences, University of Sherbrooke, 3001 12th Avenue North, Sherbrooke, PQ, J1H 5N4 Canada. E-mail: eric.rousseau{at}usherbrooke.ca


   Abstract
Top
 Abstract
CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
The aim of the present study was to provide a mechanistic insight into how 14,15-epoxyeicosatrienoic acid (EET) relaxes organ-cultured human bronchi. Tension measurements, performed on either fresh or 3-d–cultured bronchi, revealed that the contractile responses to 1 µM methacholine and 10 µM arachidonic acid were largely relaxed by the eicosanoid regioisomer in a concentration-dependent manner (0.01–10 µM). Pretreatments with 14,15-epoxyeicosa-5(Z)-enoic acid, a specific 14,15-EET antagonist, prevented the relaxing effect, whereas iberitoxin pretreatments (10 nM) partially abolished EET-induced relaxations. In contrast, pretreatments with 1 µM indomethacin amplified relaxations in explants and membrane hyperpolarizations triggered by 14,15-EET on airway smooth muscle cells. The relaxing responses induced by 14,15-EET were likely related to reduced Ca2+ sensitivity of the myofilaments, because free Ca2+ concentration–response curves performed on beta-escin–permeabilized cultured explants were shifted toward higher [Ca2+] (lower pCa2+ values). 14,15-EET also abolished the tonic responses induced by phorbol-ester-dybutyrate (PDBu) (a protein kinase C [PKC]–sensitizing agent), on both fresh (intact) and beta-escin–permeabilized explants. Western blot analyses, using two specific primary antibodies against CPI-17 and its PKC-dependent phosphorylated isoform (p-CPI-17), confirmed that the eicosanoid interferes with this intracellular process. These data indicate that 14,15-EET hyperpolarizes airway smooth muscle cells and relaxes precontracted human bronchi while reducing Ca2+ sensitivity of fresh and cultured explants. The intracellular effects are related to a PKC-dependent process involving a lower phosphorylation level of CPI-17.

Key Words: calcium sensitivity • CPI-17 • epoxyeicosatrienoic acid • membrane potential • organ culture



   CLINICAL RELEVANCE
Top
 Abstract
 CLINICAL RELEVANCE
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
The current research and the results obtained may be highly relevant for human diseases such as asthma and chronic obstructive pulmonary disease.

 
Epoxyeicosatrienoic acids (EETs) are cytochrome P-450 epoxygenase (CYP-450) metabolites of the lipid arachidonic acid (AA) (1, 2). The epoxygenase enzymes are present in the lungs of many species, including humans (2, 3), and produce four EET regioisomers: 5,6-, 8,9-, 11,12-, and 14,15-EET (2). One of the recently explored functions of these enzymes is their ability to promote growth (4, 5) of endothelial cells (6–8). Recombinant human epoxygenase 2C9 has been shown to stimulate growth and differentiation of human lung microvascular endothelial cells in culture when introduced by virus-mediated gene transfer (7). EETs are also known for their ability to modulate vascular (9) and airway smooth muscle tone (10, 11). EET regioisomers have been proposed as a putative EpDHF, which activates Ca2+-activated K+ channels (BKCa), inducing hyperpolarization and relaxation of airway smooth muscle (ASM) cells (10). In guinea pig bronchi, EET induces relaxations and decreases Ca2+ sensitivity of ASM cells (12).

14,15-EET is the most abundant regioisomer in the lung (3, 7), whereas 8,9- and 14,15-EET have been described as angiogenic compounds (13). Moreover, 8,9- and 11,12-EET enhance mitogenesis via activation of the p38 mitogen-activated protein kinase (MAPK) pathway, whereas activation of phosphotidylinositol-3 (PI-3) kinase kinase is necessary for 14,15-EET– and 5,6-EET–induced cell proliferation (14). Hence, epoxygenase is protective against reperfusion after ischemia in the brain (15) and heart (16, 17). EETs have also been reported to inhibit apoptosis in proximal, tubule-like epithelial cells derived from pig kidney (18).

Contraction of ASM occurs via two related mechanisms: (1) a rise in cytosolic calcium concentration ([Ca2+]i), which results in the formation of calcium/calmodulin complexes and activation of the myosin light-chain (MLC) kinase (MLCK). The activated MLCK, in turn, phosphorylates the 20 kD MLC (19), resulting in ASM cell contraction. (2) A second Ca2+-independent mechanism, which requires the activation of {rho}-kinase as well as protein kinase C (PKC)–dependent phosphorylation of myosin phosphatase inhibitor protein (CPI-17) to maintain tone (20). The calcium sensitization mechanism occurs when an agonist that stimulates the activation of {rho}-kinase or PKC/CPI-17 pathway results in the inhibition of MLC phosphatase (MLCP) (21). {rho}-Kinase inhibits MLCP activity by phosphorylation of the myosin-binding subunit of MLCP. Alternatively, CPI-17 phosphorylation also results in an inhibition of MLCP activity, which in turn maintains steady-state tension in ASM (20, 22). Hence, CPI-17 dephosphorylation facilitates relaxation.

The aim of the present study was to assess the physiologic effects of 14,15-EET on human ASM at the tissue, cellular, and molecular levels using an organ culture model derived from human bronchi. Complementary approaches used included: (1) tension measurements of relaxation on human bronchial rings; (2) membrane potential measurements using the classical microelectrode technique; and (3) analyses of the effects of 14,15-EET on Ca2+ sensitivity of the mechanical apparatus. We report herein the first evidence that 14,15-EET induces concentration-dependent relaxations, as well as hyperpolarizations of the resting membrane potential of ASM derived from human bronchi. We also demonstrate that 14,15-EET decreases the Ca2+ sensitivity of myofilaments through inhibition of CPI-17 phosphorylation.


   MATERIALS AND METHODS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Isolation and Organ Culture of Human Bronchi
The study was approved by our institution's ethics committee (protocol number CRC 05–088). Human lung tissues were obtained from 27 patients undergoing surgery for lung carcinoma. After lobectomy and transport in sterile physiological saline solution, lung samples, distal from the malignant lesion, were dissected by the pathologist. The absence of tumoral infiltration was retrospectively established in all bronchi by pathologic analysis. Tissue samples were immediately placed in Krebs solution (millimolar composition: NaCl, 118; KCl, 4.7; CaCl2, 2.5; KH2PO4, 1.2; MgSO4, 1.2; NaHCO3, 25; and glucose, 11.1), previously bubbled with 95% O2 and 5% CO2 at 22°C, pH 7.4, and then immediately transported to a level-2 culture room. After removal of connective tissues and adhering parenchyma, paired rings of similar weight and length (inner diameter, 0.5–0.8 mm) were microdissected from the same bronchus segment. Bronchial rings were placed in individual wells of 12-well culture plates, containing Dulbecco's modified Eagle's medium-F12 culture medium (2 ml/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 1–3 d (23).

Isometric Tension Measurements
The mechanical effects induced by specific agonists and eicosanoids were measured as previously reported (11, 23). Bronchial rings were mounted in isolated organ baths, containing 6 ml of Krebs solution at 37°C, gassed continually with the 95% O2/5% CO2 mixture, and to which an initial load of 0.8 g was applied. Tissues were allowed to equilibrate for 1 h in Krebs solution and washed out every 15 min. Passive and active tensions were assessed using transducer systems (Radnoti Glass Tech., Monrovia, CA) coupled to Polyview software (Grass-Astro-Med Inc., West Warwick, RI) for facilitating data acquisition and analysis.

Permeabilization with beta-Escin
Bronchial rings were mounted in organ baths and stretched to 0.8 g. After measuring the contraction elicited by 1 µM methacholine chloride (MCh) in normal Krebs solution, rings were incubated for 20 min in low free-Ca2+ relaxing solution containing 87 mM KCl, 5.1 mM MgCl2, 5.2 mM NaATP, 10 mM creatine phosphate, 2 mM EGTA, and 10 mM PIPES, brought to pH 7.2 with KOH, at 23°C, followed by treatment with 50 µM beta-escin in relaxing solution for 35 min at 23°C. Ca2+ stores were depleted by addition of 10 µM A23187. The bronchial rings were washed several times with fresh relaxing solution, containing 10 mM EGTA. Calmodulin (1 µM) was present in the bathing solutions throughout the experiments, to prevent alteration of Ca2+-induced contraction. Tension developed by permeabilized bronchial rings was measured in activating solutions, containing 10 mM EGTA and specified concentrations of CaCl2 to yield the desired free-Ca2+concentration (pCa = –log [Ca2+]) (12). Step increases in free Ca2+ (pCa = 9.0–5.3) were used to induce reproducible concentration-dependent tension response curves, indicating successful permeabilization of tissues under these conditions.

Microelectrode Measurements
A longitudinal section was performed to expose the luminal face of the human bronchi. The strips were affixed, with the ASM facing up, in the middle chamber (capacity, 3 ml) of a three-compartment system, in which temperature was maintained at 37°C, as previously described (23). The tissues were superfused at a constant flow rate of 2 ml/min with standard Krebs solution, and allowed to equilibrate for 20 min. 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 (Tektroniks Inc., Beaverton, OR). The membrane potential was digitized and recorded using a Mini-Digidata interface and the Axoscope 9.0 software from Axon Instruments (Union City, CA).

SDS-PAGE and Western Blot Analysis
Human bronchi were dissected, weighed, and promptly transferred in a buffer containing 0.3 M sucrose, 20 mM K-PIPES, 4 mM EGTA, and a cocktail of protease inhibitors (protease inhibitor pellets; Roche Diagnostics, Indianapolis, IN). Tissues were then homogenized on ice, frozen in liquid nitrogen, and stored at –80°C. For SDS-PAGE, protein samples (20 µg of protein/well) from homogenate fractions were dissolved in 2% SDS and separated on 15% SDS-PAGE, using a 3% stacking gel. Gels were cast into a mini-protean III dual cell (Bio-Rad, Mississauga, ON, Canada). Western blots using specific antibodies against CPI-17, its phosphorylated form (anti–p-CPI-17) and {alpha}-actin protein were performed on homogenate fractions (24). The separated proteins from SDS-PAGE were electrophoretically transferred at 70 V onto nitrocellulose membranes (Bio-Rad) for 2 h at 4°C. Transferred membranes were blocked with Tris-buffered saline (TBS) solution containing 0.1% Tween 20 (TBS-T) plus 5% nonfat diet milk overnight, and then incubated for 180 min with 1 µg/ml of the selected specific antibody in TBS-T. After three washes in TBS-T as above, membranes were incubated for 1 h at 23°C with peroxidase-conjugated donkey anti-rabbit IgG1 antiserum (Amersham, Baie d'Urfé, PQ, Canada) and revealed by enhanced chemiluminescence (Roche, Laval, PQ, Canada).

Drugs and Chemical Reagents
14,15-EET, 8,9-EET, and AA were obtained from Cayman Chemical (Ann Arbor, MI), dissolved in 100% ethanol (EtOH) and stored as 1 mM stock solutions. phorbol-ester-dybutyrate (PDBu) and iberiotoxin were purchased from Calbiochem (VWR, Montreal, PQ, Canada). The vehicle was tested separately at the maximal concentration used in the presence of active compound. MCh and indomethacin (indo) were purchased from Sigma (St. Louis, MO). Dulbecco's modified Eagle's medium-F12 and penicillin–streptomycin were purchased from Gibco Invitrogen Corp. (Burlington, ON, Canada).

Statistical Analysis
Results are expressed as means (± SEM; where noted, n indicates the number of experiments). Statistical analyses were performed using Student's t test or a one-way ANOVA. Differences were considered significant when P < 0.05. Data curve fittings were performed using SigmaPlot 9.0 (SPSS, Chicago, IL) to determine the median effective concentration (EC50 ) and the concentration producing 50% inhibition (IC50) values (25).


   RESULTS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Effect of EETs on Bronchial Smooth Muscle Tension
Tension measurements were performed on human bronchial rings to test the effect of either 8,9- or 14,15-EET regioisomer on ASM tone. The tissues were subjected to 0.8 g basal tone and then precontracted with 1 µM MCh. When the plateau phase was reached, cumulative concentrations of 14,15-EET were added, resulting in concentration-dependent relaxing effects (Figure 1A). The relaxing effects of 14,15-EET were also assessed on AA precontracted bronchi, as illustrated in Figure 1B. Figure 1C demonstrates the concentration-dependent relaxing effects induced by 8,9-, and 14,15-EET on 1 µM MCh precontracted bronchi, either in the presence or absence of 1 µM indo pretreatment. Both regioisomers displayed significant relaxing effects on MCh-precontracted bronchi (Figure 1C). 14,15-EET proved to be more effective than the 8,9-EET regioisomer, with the relaxing effects of exogenous EETs shifted toward lower concentrations on COX inhibitor–pretreated bronchi. IC50 values for 14,15-EET were 0.43 (± 0.02) and 0.17 (± 0.02) µM in the absence and presence of indo, respectively (Figure 1C).


Figure 1
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  		Figure 1. Relaxing effects of EET on human bronchi. (A) Representative trace showing the relaxing effect induced by 14,15-EET on bronchi precontracted with 1 µM MCh. (B) Typical recording trace illustrating the relaxing response generated by 14,15-EET on bronchi precontracted with 10 µM AA. (C) Quantitative analysis of the relaxing responses induced by 8,9-EET and 14,15-EET on human bronchi precontracted with 1 µM MCh in the absence or presence of 1 µM indo pretreatment. Each point represents the mean (± SEM); n = 18 for each experimental condition; *P < 0.05.

 
EET Partially Relaxes K+-Induced Tension
Experiments were designed to assess the effects of 14,15-EET on KCl-induced tension. Figure 2A illustrates a representative trace of the concentration-dependent relaxing effects induced by 14,15-EET on human bronchial ring precontracted with 25 mM KCl in an isolated organ bath solution. Quantitative data analysis shows that 3 µM 14,15-EET induced a 65% relaxation of the maximal tension evoked by KCl addition (Figure 2B). These results confirm that the addition of 14,15-EET opposes the processes triggered by KCl depolarization, which usually involves activation of voltage-operated Ca2+ channels, Ca2+ entry, and activation of various enzymatic systems (26). Thus, the effects of EET on Ca2+ sensitivity will have to be assessed (see below). Because it was previously reported that 14,15-EET can partially activate BKCa channels in guinea pig ASM (10), the relaxing effects of EET were assessed in the absence or presence of 10 nM iberitoxin (IbTx) under normal K+ concentration. Figure 2C shows two sequential recordings in which IbTx pretreatment did not modify the muscarinic response to 1 µM MCh, but did result in a significant inhibition of the amplitude and rate of relaxation induced by 14,15-EET, as compared with the control response. Quantitative analysis of the data is summarized in Figure 2D. Note that preincubation with 10 nM IbTx had a partial inhibitory effect (26%) on the relaxation induced by 1 µM 14,15-EET, suggesting that activation of K+Ca channels would only mediate part of the responses induced by this eicosanoid in human bronchi.


Figure 2
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  		Figure 2. 14,15-EET relaxes KCl induced tensions partially via an IbTx-sensitive process. (A) Representative recording displaying the response to 14,15-EET on bronchi precontracted with 25 mM KCl. (B) Quantitative analysis of the mean concentration-dependent relaxing responses to14,15-EET on KCl responses in human bronchi (n = 17). (C) Paired responses to 1 µM 14,15-EET on a bronchial explant precontracted with 1 µM MCh, before and after 10 nM IbTx treatment. W, washout. (D) Bar histogram showing the mean inhibitory effects induced by 10 nM IbTx on 14,15-EET relaxing responses from bronchial tissues (n = 15).

 
Effect of N-Methylsulfonyl-6-[2-Propargyloxyphenyl] Hexanamide on EET Relaxation
The specific CYP-450 epoxygenase inhibitor N-methylsulfonyl-6-[2-propargyloxyphenyl] hexanamide (MS-PPOH) was used to assess the role of the endogenous production of EETs. Pretreatment with 3 µM MS-PPOH had basically no effect on basal tone or on the tonic responses induced by MCh in bronchial explants (Figure 3A). Moreover, this compound did not modify the response induced by exogenous addition of 1 µM EET, as shown in Figure 3A. Thus, epoxygenase inhibition in lower bronchi did not prevent the effects of exogenous EET, thus justifying the use of a specific EET antagonist.


Figure 3
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  		Figure 3. Effects of MS-PPOH and 14,15-EEZE on human bronchi. (A) Paired recordings of responses to 1 µM 14,15-EET on bronchi precontracted with MCh before (control) and after 3 µM MS-PPOH pretreatment. MS-PPOH was used an epoxygenase inhibitor. (B) Paired recordings of responses to 1 µM 14,15-EET on bronchi precontracted with MCh before (control) and after pretreatment with 5 µM 14,15-EEZE, an EET antagonist. (C) Bar histogram quantifying the inhibitory effect of EEZE on 14,15-EET-induced relaxations.

 
Effect of 14,15-Epoxyeicosa-5(Z)-Enoic Acid on EET Responses in Human ASM
To determine the relative efficiency of an EET antagonist on human ASM, the effect of 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE) was assessed on tone and relaxation. Paired recordings demonstrate that pretreatment with 5 µM of 14,15-EEZE induced a significant inhibition in relaxing effect induced by EET, as well as a reduction in resting tone and muscarinic responses induced by MCh (Figure 3B). The concentration-dependent inhibiting effects of 14,15-EEZE on EET relaxation are summarized in Figure 3C. Thus, human bronchi and eicosanoid-triggered relaxations are sensitive to 14,15-EEZE.

Eicosanoid Effect on ASM Membrane Potential
The effects of 14,15-EET on ASM cell membrane potential were tested after microelectrode impalement on human bronchial explants. Figure 4A illustrates a representative recording of the hyperpolarizing effects induced by 14,15-EET from a resting membrane potential of –53 mV. Upon cumulative addition of 14,15-EET concentrations (0.01–10 µM) in the mixing compartment of the experimental chamber, a hyperpolarization of the ASM cell membrane potential was consistently recorded with a short delay (Figure 4A). At the end of each experiment, the microelectrode was removed from the ASM cell to validate the recordings. The mean electrophysiologic effects of EET on ASM tissues are shown in Figure 4B. Concentration–response curves were obtained, and a maximal hyperpolarizing effect of –12 (± 3.5) mV was recorded in presence of 10 µM EET. Moreover, 10 nM iberiotoxin pretreatment resulted in a significant inhibition of the hyperpolarizing effects induced by 1µM 14,15-EET on human bronchi (Figure 2B). Complementary experiments were performed to assess the effect of indo pretreatment on EET-hyperpolarizing effect. Superimposed paired recordings illustrate the differences in hyperpolarizing effects induced by 14,15-EET on bronchi before (dashed line) and after (solid line) 1 µM indo pretreatment in the same bronchi (Figure 4C). Figure 4D displays the mean membrane potential in control tissue and after addition of 1 µM EET on bronchi pretreated, or not, with 1 µM indo. Significant increases in mean 14,15-EET responses were observed on bronchi pretreated with the COX inhibitor compared with control responses.


Figure 4
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  		Figure 4. 14,15-EET hyperpolarizes human ASM cell. (A) Representative recording of ASM membrane potential in control and after addition of cumulative concentrations of 14,15-EET. The glass microelectrode (35 M{Omega}) was filled with 3 M KCl. At the end of each experiment, the microelectrode was removed from the ASM cell to validate the recording. (B) Mean resting membrane potential values determined for 14,15-EET concentrations on ASM bronchial cells. (C) Typical superimposed recordings of ASM membrane potential following addition of 1 µM 14,15-EET in the absence (control) or presence of 1 µM indo (pretreatment). (D) Bar histogram displaying the mean resting membrane potential values determined before and after addition of 1 µM 14,15-EET on bronchial explants pretreated or not with 1 µM indo; n = 8; *P < 0.05 between control and experimental conditions for each preparation.

 
Effect of 14,15-EET on Ca2+ Sensitivity
Comparative analyses were performed on beta-escin–permeabilized preparations to assess the effect of 14,15-EET on Ca2+ sensitivity of the contractile apparatus. Figure 5A displays superimposed recordings induced by cumulative free-[Ca2+] increments in control bronchi and 1 µM 14,15-EET acutely pretreated bronchi. After 15 min, EET had a robust inhibitory effect on Ca2+-dependent tension. Cumulative concentration response curve to free-Ca2+ concentrations on cultured and permeabilized bronchial rings obtained from control and eicosanoid-treated bronchi are shown in Figure 5B. Acute (15 min) pretreatments with 1 µM 14,15-EET, as well as 48 h treatments with 0.1 µM 14,15-EET, were performed on organ culture explants. Data analysis demonstrates that EET reduced the Ca2+ sensitivity of bronchial rings, with a shift in EC50 values toward higher Ca2+ concentrations (1.19 ± 0.03 µM and 1.76 ± 0.03 µM, respectively, as compared with 0.38 ± 0.02 µM in control tissues) (Figure 5B). The mean responses demonstrate that EET pretreatments reduce the Ca2+ sensitivity of permeabilized bronchial explants.


Figure 5
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  		Figure 5. Effect of 14,15-EET on Ca2+ sensitivity. (A) Representative superimposed recordings illustrating the tension induced by cumulative increases in [Ca2+] on beta-escin–permeabilized organ culture human bronchi in control conditions (dashed line) and after 1 µM 14,15-EET pretreatment (solid line). (B) Cumulative concentration response curve to free [Ca2+] obtained from beta-escin–permeabilized organ culture bronchial rings in control conditions (closed circles; n = 21), 1 µM 14,15-EET pretreated bronchi for 15 min (open circles; n = 21) and organ culture explants treated every 12 h, for 48 h, with 0.1 µM 14,15-EET (closed triangles; n = 16). Note the rightward shift in Ca2+ sensitivity upon 14,15-EET treatments.

 
Effect of 14,15-EET on PDBu-Induced Contraction and Ca2+ Sensitivity
Complementary experiments were designed to assess the putative processes that would support the negative feedback mechanism induced by EET on this Ca2+-tension relationship (12). For example, a PKC-dependent mechanism that would involve a change in phosphorylation rate of key regulatory proteins would constitute a prime candidate. PDBu, a direct activator of conventional and novel PKCs, has been used to stimulate this pathway and to produce tone increases (27). Figure 6A shows that 1 µM 14,15-EET pretreatment of intact tissue largely prevented the contraction developed upon stimulation with 1 µM PDBu compared with the control response to this activator. This inhibitory effect of 14,15-EET on PDBu-induced tension was further quantified from mean normalized responses, and is reported in Figure 6B. A similar protocol was also performed on beta-escin–permeabilized explants under Ca2+ clamp conditions (Figures 6C and 6D). Interestingly, under these experimental conditions, 1 µM 14,15-EET also had a strong inhibitory effect on PDBu-induced tension at a pCa of 6. Data analysis clearly demonstrates that 14,15-EET had a significant inhibitory effect (Figure 6D) on the active tension triggered by the PKC activator in the presence of high intracellular Ca2+ (pCa = 6). Together, these results further support the regulatory role of EETs on ASM tone


Figure 6
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  		Figure 6. Effect of 14,15-EET on PDBu-induced tonic responses in intact and beta-escin–permeabilized organ culture bronchi. (A) Sequential traces showing the contractile response induced by 1 µM PDBu and its ensuing inhibition by 1 µM 14,15-EET. (B) Bar histogram of the relative responses to 1 µM PDBu in the absence or presence of 14,15-EET. The eicosanoid induced a large (72%) inhibition of the tone in intact tissues. (C) 14,15-EET (1 µM) also displayed a marked inhibitory effect on 1 µM PDBu-induced tension at pCa = 6. (D) Quantitative analysis of the above mean responses. Together, these results suggest that 14,15-EET is able to decrease the Ca2+ sensitivity of the myofilaments in human ASM cells; * P < 0.05.

 
Phosphorylation Level of a Regulatory Protein
CPI-17 has been reported as a PKC-dependent regulatory protein. In its phosphorylated form, p-CPI-17 displays an inhibitory effect on MLCP, which, in turn, maintains ASM tone (20, 28). Western blot analysis of homogenates from human bronchial explants challenged with PDBu alone, PDBu plus 14,15-EET, or 14,15-EET alone, revealed that CPI-17 was present in all tested fractions, whereas its phosphorylated form was reduced upon 14,15-EET treatments, and the {alpha}-actin staining was constant from one preparation to the other (Figure 7A). In contrast, PDBu had a strong activating effect on PKC-dependent CPI-17 phosphorylation according to the staining level revealed by a specific antibody against its phosphopeptide (Figure 7A). Quantitative analysis of identical immunoblot membrane areas was normalized as a function of total CPI-17 staining in the corresponding fraction. As reported in Figure 7B, pretreatments of the bronchial explants with low concentrations of 14,15-EET for 12 h reduced the staining density ratio of the phosphoprotein when compared with either control or PDBu-stimulated bronchi (Figure 7B). These data further support the results obtained from functional experiments reported previously here.


Figure 7
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  		Figure 7. Western blot analyses of homogenates derived from control, eicosanoid, and PDBu-treated human bronchi. (A) Proteins from four distinct homogenates were stained using three specific antibodies against CPI-17, p-CPI-17, and anti–{alpha}-SM actin. Note the reduced staining of p-CPI-17 bands in 0.1 µM 14,15-EET–treated tissues (third and fourth rows, after 12 h treatments, respectively), whereas PDBu treatment, used as a positive control, displays increased staining. (B) Quantitative analysis of various p-CPI-17 density ratios. Staining densities in the homogenates were expressed as a function of the CPI-17 signals. Significant differences were observed between 14,15-EET–treated tissues versus control and PDBu alone. Results are representative of five similar experiments; * P < 0.05.

 

   DISCUSSION
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
In this study, we investigated the ability of 14,15-EET to modulate ASM tone in human bronchi. This is the first report directly assessing the electrophysiologic and functional intracellular effects of these eicosanoids in human bronchi. 14,15-EET was found to relax bronchial tissues and hyperpolarize bronchial ASM cells. These effects of EETs were linked to the activation of BKCa channels, as well as a decrease in Ca2+ sensitivity of myofilaments in ASM cells, the latter being associated with a decrease in CPI-17 protein phosphorylation, as revealed by Western blot analysis. Thus, we propose that eicosanoids, such as 14,15-EET, play a key regulatory role in the fine tuning of human bronchial tone.

Effects of EET Regioisomers on Human Bronchi
Compared with previous reports in rodents (3, 10, 12), 8,9-EET as well as 14,15-EET display potent relaxing effects on human bronchi, with an IC50 value (0.43 µM) in the submicromolar range for the 14,15-EET regioisomer. To our knowledge, only one report has been cited regarding the relaxing effects of 5,6-EET methyl ester and 8,9-EET on histamine precontracted rabbit bronchi (29). Despite the fact that this CYP-450 epoxygenase has been identified in various lung tissues (7, 30, 31), a key issue has been to demonstrate the endogenous involvement of EET regioisomers in human bronchi. Recent advances in the design of pharmacologic CYP450 epoxygenase inhibitors, such as MS-PPOH (32–34), enabled us to evaluate the putative role of tissular production of this eicosanoid. In our hands, low concentrations of MS-PPOH did not modify resting tone, tonic responses to MCh, or the relaxing effect induced by 14,15-EET, suggesting that low concentration of MS-PPOH (3 µM) is not efficient in inhibiting the endogenous production of EET regioisomers. Further investigations would be required to assess the role of CYP-450 epoxygenase in human lung. By contrast, the data obtained herein with the EET antagonist were quite convincing. Because 14,15-EET is thought to play an important role in regulating tone in distal bronchi, the effects of 14,15-EEZE (35) were therefore assessed on human ASM tissues. We were able to demonstrate that EEZE prevents the relaxing effect induced by exogenous EET addition in a concentration-dependent manner. This is the first report demonstrating the effect of 14,15-EEZE on ASM tissue. It was previously shown that EET regioisomers induce partial relaxation of guinea pig ASM (10). However, large relaxations were measured in an organ culture model of guinea pig bronchi (12). Herein, the relative effects of these agents—namely, 8,9-EET and 14,15-EET—were significantly larger in human bronchi (Figures 1A–1C). The pharmacologic relaxing responses induced by 14,15-EET may, therefore, be of physiologic significance in respiratory diseases. Hence, the mode of action of these compounds may be related to their molecular interactions already quantified in human coronary and renal arteries (9).

COX Inhibition and EET Effects in Human Cultured Bronchi
One approach to minimizing the contribution of the epithelial layer is to inhibit prostaglandin production. Because epithelium removal was difficult to achieve in the present preparations, a nonselective pharmacologic inhibitor was used as an alternative. The relaxations induced by EETs were amplified by indo, thus indicating that the response to EETs is possibly modulated by an intracellular COX–derived prostanoid, as previously reported in guinea pig tissue (12).

Involvement of K+ Channel Activation in EET-Induced Hyperpolarization
In the present study, IbTx unequivocally abolished 27% of the relaxing effect induced by EET under normal K+ concentration, which suggests that the eicosanoid increases the open probability of Ca2+-activated K+ channels, which normally results in membrane hyperpolarization. Moreover, it was previously shown that EET regioisomers induce concentration-dependent relaxing effects in native and hyperresponsive guinea pig bronchi, respectively (10, 12). These effects were shown to be clearly related to a hyperpolarization of ASM cells due to activation of BKCa channels (10). Microelectrode measurements recorded herein demonstrate that 14,15-EET hyperpolarizes human ASM cells under normal K+ concentration (Figure 4), and that this effect is abolished by 10 nM IbTx. Moreover, the four EET regioisomers have previously been shown to produce vasorelaxations by causing membrane potential hyperpolarization and BKCa channel activation in vascular smooth muscle cells (36, 37). In human bronchi, the hyperpolarizing effect was amplified by indo pretreatment (Figures 4C and 4D) Consequently, COX activities modulate the electrophysiologic effects of EETs, which are linked to the activation of large, conducting, potassium-selective channels and membrane hyperpolarization, and thus explain, in part, the resulting effects (30%) on human bronchi.

14,15-EET Reduces Ca2+ Sensitivity
The inherent Ca2+ sensitivity of the MLCK, resulting in MLC phosphorylation and contraction and subsequent dephosphorylation by MLCP, is an important mechanism in the regulation of smooth muscle tone (20, 28). Modulation of this mechanism by the eicosanoids would explain their overall effects on human ASM. The present data demonstrate that in human bronchi, 14,15-EET significantly reduces Ca2+ sensitivity (Figure 5). Several studies have suggested that Ca2+ sensitizing mechanisms may also be primed under pathophysiologic conditions (21, 22, 38). It was therefore of potential clinical interest to find a lipid mediator that would be able to significantly shift the Ca2+ activation curve toward higher concentrations. In a recent publication, we reported that 14,15-EET induces a reduction in Ca2+ sensitivity in both fresh or hyperreactive guinea pig bronchi, suggesting that this eicosanoid modulates enzymatic systems, such as {rho}-kinase and/or PKC/CPI-17 (12). Moreover, long-term pretreatments (48 h) with the eicosanoid performed in the present study further altered the pharmacologic responsiveness as well as the Ca2+ sensitivity of human cultured explants (Figure 5B). We were also able to show that 14,15-EET pretreatment decreases Ca2+ sensitization induced by PDBu (a PKC activator). Based on evidence provided in the literature, CPI-17 was a likely candidate for PKC phosphorylation, involved in modulating myofilament Ca2+ sensitivity (20, 27, 39). The present Western blot analysis performed on human bronchi homogenates attest that EET pretreatments decrease CPI-17 phosphorylation levels, whereas PDBu has the opposite effect (Figure 7). In contrast, 5,6-EET has been shown to increase [Ca2+]i in pulmonary artery smooth muscle cells (40). These results suggest that eicosanoids, such as EETs, may modulate ASM tone through a shift in intracellular protein regulation, although modifications in gene expression and protein profiles cannot be ruled out (2, 41)

In summary, the responses to EET regioisomer are likely the result of an enhanced coupling between receptors and effectors in human ASM cells. The changes in smooth muscle reactivity induced by the eicosanoid tested herein are likely related to membrane hyperpolarization and a decrease in Ca2+ sensitivity of the contractile machinery. These observations point toward possible new pharmacologic targets in patients with asthma and chronic obstructive pulmonary disease.


   Acknowledgments
 
The authors thank Dr. John R. Falck for the gift of MS-PPOH and 14,15-EEZE compounds, as well as Mr. Pierre Pothier for his critical review of the manuscript.


   Footnotes
 
This work was supported by a Canadian Institutes of Health Research (CIHR) grant MOP-57677. C.M. is a recipient of a Ph.D. studentship from the Quebec Respiratory Health Training Program (QRHTP) supported by the CIHR. E.R. is a member of the Respiratory Health Network of the Fonds de la recherche en santé du Québec.

Originally Published in Press as DOI: 10.1165/rcmb.2006-0281OC on January 19, 2007

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form August 2, 2006

Accepted in final form January 4, 2007


   References
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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