Direct modulation of tracheal Clminus -channel activity by 5,6- and 11,12-EET

Dany Salvail, Marc Dumoulin, and Eric Rousseau

Le Bilarium, Department of Physiology and Biophysics, Faculty of Medicine, University of Sherbrooke, Sherbrooke, Quebec, Canada J1H 5N4

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Using microelectrode potential measurements, we tested the involvement of Cl- conductances in the hyperpolarization induced by 5,6- and 11,12-epoxyeicosatrienoic acid (EET) in airway smooth muscle (ASM) cells. 5,6-EET and 11,12-EET (0.75 µM) caused -5.4 ± 1.1- and -3.34 ± 0.95-mV hyperpolarizations, respectively, of rabbit tracheal cells (from a resting membrane potential of -53.25 ± 0.44 mV), with significant residual repolarizations remaining after the Ca2+-activated K+ channels had been blocked by 10 nM iberiotoxin. In bilayer reconstitution experiments, we demonstrated that the EETs directly inhibit a Ca2+-insensitive Cl- channel from bovine ASM; 1 µM 5,6-EET and 1.5 µM 11,12-EET lowered the unitary current amplitude by 40 (n = 6 experiments) and 44.7% (n = 4 experiments), respectively. Concentration-dependent decreases in channel open probability were observed, with estimated IC50 values of 0.26 µM for 5,6- and 1.15 µM for 11,12-EET. Furthermore, pharmacomechanical tension measurements showed that both regioisomers induced significant bronchorelaxations in epithelium-denuded ASM strips. These results suggest that 5,6- and 11,12-EET can act in ASM as epithelium-derived hyperpolarizing factors.

membrane potential; airway smooth muscle; epithelium-derived hyperpolarizing factor; epoxyeicosatrienoic acid; chloride channel; eicosanoids; tension measurements; bronchorelaxation

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

OVER THE PAST FEW YEARS, evidence has accumulated suggesting that the epithelium plays an active role in the control of membrane potential (Vm) and tone of airway smooth muscle (ASM) through the release of nitric oxide and a variety of prostanoids (2, 18, 28). Of interest here are the eicosanoids, products of the metabolism of arachidonic acid (AA) by cytochrome P-450 epoxygenase, the nature and mechanisms of action of which in ASM have yet to be identified. Research in vascular physiology has highlighted the important role played by endothelial cells in the modulation of vascular smooth muscle (VSM) tone; nitric oxide and prostacyclin (PGI2), the two main endothelium-derived relaxing factors, as well as the epoxyeicosatrienoic acid (EET) isomers, a family of molecules believed to play a role as endothelium-derived hyperpolarizing factors (EDHFs), have been found to exert their effects on VSM in a variety of organs (3, 4, 8, 21, 23, 24).

The EETs result from the olefinic epoxydation of AA by enzymes such as CYP2B4, found to produce EETs in lung homogenate (31). Pharmacological studies on ASM have revealed the role played by the epithelium in modulating cell Vm and tone. Mechanical removal of the epithelium depolarized the resting Vm (Vrest) of canine ASM by ~10 mV, a phenomenon that could be reversed by exposing the smooth muscle (SM) tissue to dispersed epithelial cells (30), suggesting that these cells release endothelium-derived relaxing factor- and/or EDHF-like substances tentatively termed epithelium-derived relaxing factors and epithelium-derived hyperpolarizing factors (EpDHFs). Several studies on rabbit (28) and guinea pig (15) airways have established the presence of isoforms of the enzymes responsible for the production of EETs in VSM. Indeed, the ability of airway epithelium to relax the underlying SM leaflet via the transformation of AA into bioactive metabolites (29) was confirmed by the observation that the relaxing effect of AA on epithelium-intact ASM reversed to a contractile effect on exposure of epithelium-denuded tracheal SM to exogenous AA (22).

A significant modulation of the contractile state of ASM is achieved through changes in Vm; in this manner, EpDHF agents induce a hyperpolarization-dependent relaxation of the ASM tissue, as do the EDHFs in the vascular system (3). Li and Campbell (16) reported the selective activation of Ca2+-activated K+ (KCa) channels by 11,12-EET in small bovine coronary arteries; their findings suggest that this effect is mediated by a stimulatory GTP-binding protein (Gs). However, this specific eicosanoid could also act on other surface membrane-bound structures. Results from our laboratory (7) have recently demonstrated that 11,12-EET can directly activate the KCa channels of bovine tracheal smooth muscle when reconstituted into planar lipid bilayers (PLBs). In addition to the hyperpolarizing K+ conductances (voltage gated, ATP sensitive, and Ca2+ dependent), other ionic fluxes such as spontaneous (12) and/or Ca2+-activated (14) Cl- conductances were shown to be involved in a depolarization of the cell membrane. Moreover, after PLB reconstitution of bovine tracheal microsomes, Salvail et al. (26) previously characterized, at the unitary level, a Ca2+-insensitive Cl- channel that displayed an almost tonic and voltage-independent activation. With a theoretical equilibrium potential (-29 mV) significantly less negative than the Vrest (-55 mV) of ASM cells (1), opening of any of these anion-selective channel populations would therefore tend to depolarize the sarcolemmal membrane.

In this study, we verified whether a simultaneous direct inhibition of the Cl- current, concomitant with the reported increase in K+ permeability, might contribute to the hyperpolarization of the cells. To test this hypothesis, vesicles derived from bovine tracheal smooth muscle were fused into PLBs, and exogenous 5,6- or 11,12-EET was applied to either side of the channel protein. Herein, we report the first evidence that both 5,6- and 11,12-EET directly inhibit Ca2+-insensitive Cl- channels from bovine ASM. This effect, in synergy with an activation of KCa channels, could contribute to the hyperpolarization and, indirectly, to the relaxation of ASM cells by these two EET regioisomers.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Microelectrode measurements. White rabbits (New Zealand, 1.5-2 kg) were injected with ketamine hydrochloride (35 mg/kg body weight) before being stunned, and their spinal cord was sectioned. The trachea was promptly removed and transferred into oxygenated (95% O2-5% CO2) Krebs solution containing (in mM) 118.0 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgSO4, 1.2 NaH2PO4, and 25.0 NaHCO3, pH 7.40 ± 0.02. Longitudinal sections were made to expose the luminal face of the epithelium. On some strips, the epithelium was mechanically removed by light brushing with a cotton applicator, and the exposed SM was cut into strips ~5 mm wide and 10-15 mm long. The strips were fixed in the middle chamber of a 5-ml bath consisting of a temperature- and solution level-controlled tricompartment system with a bubble trap in the first section and a waste outlet in the third section (superfusion rate 2 ml/min, 37°C). A constant tension was applied to the tissue, keeping it isometrically "clamped" while microelectrode measurements were performed. Conventional borosilicate microelectrodes (30-50 MOmega ) filled with either 3 M KCl or 1.5 M potassium citrate were used to measure the Vm of the cells, which were recorded on digital audiotapes (DAT, Sony). Thirty-five to forty cells per strip were penetrated. A similar protocol was performed with canine ASM; mongrel dogs were anesthetized with pentobarbital sodium, and the trachea was quickly removed and dissected as described above. The technique used to measure canine cellular Vm was identical to that employed with the rabbit tissue. We initially attempted this protocol on bovine tracheae, with significantly lower success rates; hence our use of White rabbits and dogs for this type of measurements.

Preparation of the microsomal fraction. The preparation of plasma membrane microvesicles derived from bovine tracheae was performed as reported by Savaria et al. (27). Briefly, fresh bovine tracheae obtained from a local slaughterhouse were dissected to free the layer of smooth muscle, which was weighed and promptly transferred to a buffer containing 0.3 M sucrose, 20 mM K-PIPES, 4 mM EGTA, and various protease inhibitors: 50 µM 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride (Pefabloc), 1 µM pepstatin, 1 µM leupeptin, 0.24 trypsin inhibitor unit aprotinin/100 ml, and 2 mM dithiothreitol. The tissue was then homogenized 3 × 30 s on ice, and the mixture was centrifuged for 20 min at 6,500 g at 4°C. The supernatant was recentrifuged for 60 min at 86,000 g at 4°C. The pellets containing the crude microsomal fraction were then resuspended in 0.3 M sucrose-5 mM K-PIPES, pH 7.4, frozen in liquid nitrogen, and stored at -84°C. The use of bovine organs in this type of preparation was dictated by the large amount of tissue necessary to obtain sufficient microsomal fraction.

PLB formation and vesicle fusion. PLBs were formed from a phospholipid mixture containing phosphatidylethanolamine, phosphatidylserine, and phosphatidylcholine in a ratio of 3:2:1, with a final concentration of 25 mg lipids/ml decane. A 200- to 300-µm-diameter hole drilled in Delrin cups was pretreated with the same lipid mixture dissolved in chloroform. Only PLBs with capacitance values ranging from 250 to 400 pF were retained. Aliquots of crude vesicle fractions (30-60 µg of protein) were added to the cis compartment in proximity of the PLB. Initially, both 3-ml chambers contained 50 mM CsCl, 10 µM free Ca2+ (as 109 µM CaCl2 and 100 µM EGTA), and 20 mM Tris-HEPES, pH 7.4. Before the vesicle suspension was injected, a gradient of 50 mM trans to 250 mM cis CsCl was created by adding an aliquot of 2 M CsCl buffer into the cis chamber. All solutions were made of analytic grade reagents, and all experiments were performed at room temperature (22 ± 2°C).

Recording instrumentation and signal analysis. Channel currents were recorded with a Dagan 8900 low-noise amplifier, filtered, and stored on a videotape recorder through a modified pulse code modulator (DAS/VCR 900, Unitrade). The currents were simultaneously displayed on an oscilloscope (Kikusui 5040) and a chart recorder (DASH MT, Astro Med). Current recordings were played back, filtered at 500 Hz with a four-pole Bessel filter, and sampled at 2 kHz for storage on disks. Further analysis was performed with specialized software to determine open channel probability (Po) values and obtain time histograms.

Drugs and chemical reagents. Sucrose, K-PIPES, EGTA, protease inhibitors, CaCl2, Tris-HEPES, AA, 11,12-EET, DIDS, and iberiotoxin (IbTX) were all purchased from Sigma (St. Louis, MO). 5,6-EET and a second lot of 11,12-EET were purchased from Cayman Chemical (Ann Arbor, MI). Phospholipids were obtained from Avanti Polar Lipids (Alabaster, AL). Pefabloc was ordered from Boehringer Mannheim (Laval, QC). CsCl and MgSO4 were obtained from Fischer (Nepean, ON). Because 5,6- and 11,12-EET are highly sensitive to sunlight and O2, special precautions were taken to keep the reconstitution apparatus in the dark and the EETs at -20°C until used. Injection was performed with a Hamilton syringe in which the dead volume had been saturated with ethanol (EtOH) before liquid withdrawal to avoid exposure of the stock solution of the epoxide to ambient air, followed by vigorous agitation to facilitate the dispersion of the hydrophobic substance.

Statistical analysis. Data are given as means ± SE. Statistical significance of the results was verified by paired or unpaired t-tests, with a threshold value for significance set at P < 0.05. Channel activity was monitored in terms of Po of a single reconstituted channel (Po = time spent by the channel in the open state/total time of recording) or of a group of channels [mean Po (NPo) = (a1 + 2a2 + 3a3 +...nan)/(a0 + a1 + a2 + a3 + ...an), where N is the total number of channels, a is the area under the peaks corresponding to closed and open channel states on amplitude histograms, and n is the specific channel number].

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Vm measurements. The effects of 5,6- and 11,12-EET on the Vm of rabbit and canine tracheal cells was investigated with two protocols. In a first series of experiments, we attempted to measure Vm continuously from one impaled ASM cell as the muscle strip was superfused with various drugs sequentially. Figure 1A illustrates one such continuous recording; in control conditions, the penetrated cell exhibited neither spontaneous action potentials nor oscillations in Vm. After a stable potential reading was obtained, carbamylcholine (CCh; 1 µM) was added to the chamber, and a 10.8-mV depolarization was observed. Then, exogenous 11,12-EET was added to the chamber and caused a slight repolarization of the cell to a final mean value of -44.0 mV. After the EET-induced repolarization had stabilized, the responsiveness of the cell was verified by changing the superfusate for a high-K+ (20 mM potassium gluconate) solution. As expected from a living cell, there resulted a depolarization to a Vm value more negative than -37.5 mV.


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Fig. 1.   Effect of 11,12-epoxyeicosatrienoic acid (EET) on resting membrane potential (Vm) of rabbit tracheal smooth muscle cells. A: rabbit airway smooth muscle (ASM) cell Vm changes were reconstructed from point measurements (1 point/15 s) made on a continuous recording. Carbamylcholine (CCh; 0.1 µM) depolarized the cell from a resting value of -53 to -41 mV. Addition of 0.87 µM 11,12-EET resulted in a long-lasting -3.0-mV repolarization. A further depolarization was still measurable on perfusion of a high-K+ solution. B: mean resting Vm values for rabbit tracheal cells exposed to various experimental conditions. Mechanical removal of epithelium (Epith) depolarized underlying ASM cells by +8.94 mV. 11,12-EET (0.750 µM) induced a significant -3.3-mV repolarization of the cells. Iberiotoxin (IbTX; 0.01 µM) alone depolarized cells by +3.2 mV, whereas addition of 0.750 µM 11,12-EET to IbTX-pretreated tissues caused a significant -1.5-mV repolarization (P < 0.05 by unpaired t-test; n = 30 experiments). DIDS (150 µM) induced a -2.2-mV hyperpolarization from control (CTRL) level. Application of 0.75 µM 5,6-EET, another EET regioisomer, caused a -5.4-mV polarization of the membrane, whereas acetonitrile (ACN), solvent for 5,6-EET, failed to alter Vm in a significant manner. * Significant difference from mean control Vm value, P < 0.05 by unpaired t-test.

Continuous recordings such as that in Fig. 1A proved prohibitively difficult to obtain because most cells would quickly depolarize after penetration. We were thus required to design a second protocol whereby Vm was measured in one experimental condition at a time. While the strips were continuously superfused with oxygenated physiological solution, a large number of cells were impaled and Vm was recorded from each cell for as long as it remained stable, generally 5-15 s. The substance to be tested (i.e., 5,6-EET, 11,12-EET, IbTX, ACN, or DIDS) was then added to the superfusion solution, and independent, repetitive Vm measurements were made. All data gathered in a specific condition were then averaged and represent the mean Vm reported herein (Table 1). The mean Vm of epithelium-intact rabbit ASM cells was -62.2 ± 1.1 mV (n = 21 experiments), significantly more negative than that of epithelium-denuded ASM cells, which was estimated at -53.2 ± 0.4 mV (n = 85 experiments; Fig. 1B). Superfusion of 0.75 µM 11,12-EET systematically hyperpolarized the cells by -3.4 mV to -56.6 ± 0.5 mV (n = 31 experiments), suggesting that it is capable of EpDHF-like effects in rabbit ASM. Prompted by the reported enhancement of K+-channel activity (7, 16, 17), additional experiments were planned that aimed at measuring the residual hyperpolarizing effect of 11,12-EET after the KCa channels had been selectively blocked by IbTX. The tissue was thus superfused with 10 nM IbTX for 3-5 min before the addition of 11,12-EET to the superfusate. IbTX alone caused a significant +3.4-mV depolarization of ASM cells (n = 24 experiments), an effect that could be partly reversed by 0.75 µM 11,12-EET, which significantly repolarized the cells by -1.51 mV to a Vm of -51.7 ± 0.4 mV (n = 30 experiments); this significant residual effect of 11,12-EET in the presence of IbTX suggests the existence of additional EET-dependent hyperpolarizing pathways (P < 0.05 by unpaired t-test).

                              
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Table 1.   Membrane potential measurements of rabbit and dog tracheal cells in various experimental conditions

The Cl--channel inhibitor DIDS was used to partly block the efflux of Cl- through sarcolemmal anionic channels. Superfusion with 150 µM DIDS resulted in a -2.2 ± 1.0-mV repolarization (n = 15 experiments), which, in turn, suggests that transsarcolemmal Cl- movement constitutes a depolarizing force in these cells that opposes the hyperpolarizing K+ permeability. A less stable regioisomer, 5,6-EET dissolved in acetonitrile (ACN; 0.01% aqueous), was also tested in the course of these microelectrode measurements; 0.75 µM 5,6-EET significantly hyperpolarized rabbit ASM cells by -5.3 mV (n = 30 experiments) and thereby proved to be a more potent hyperpolarizing factor in these in vitro conditions. The effects of ACN on cellular Vm were tested on the ASM cells at a concentration equivalent to that added when testing 5,6-EET (5.85 µM), without any significant effect over a 10- to 15-min superfusion period. Nonetheless, tissue strips exposed to ACN superfusion were promptly discarded such that no subsequent measurements were done on possibly poisoned tissues. The epithelial cells of the rabbit trachea were easily recognized by their significantly less negative Vrest (-31.9 ± 0.8 mV; n = 25 experiments) and generally constituted the first layer of cells penetrated by the microelectrode. Overall similar observations had initially been made from experiments performed with canine tracheal tissue in which 0.75 µM 11,12-EET induced a hyperpolarization of -1.60 ± 1.19 mV (n = 26 experiments) from a Vrest value of -54.56 ±0.54 mV (Table 1).

Characterization and inhibition of reconstituted Ca2+-insensitive Cl- channels from bovine ASM. After the fusion of a membrane-derived microvesicle into the PLB, the activity of one or more Cl- channels could be observed in the presence of a 50 mM trans-to-250 mM cis CsCl gradient. These channels have previously been characterized in greater detail under these same conditions (26). Figure 2 summarizes some of the properties of this type of channel observed after symmetrical 250 mM CsCl conditions were obtained by injecting an aliquot of CsCl into the trans chamber. The channels displayed a linear current-voltage relationship, with an average unitary conductance of 124.2 ± 7.0 pS (n = 16 experiments; Fig. 2A). Channel activity was tonically high in control {high Ca2+ concentration ([Ca2+])} conditions (Po > 0.85) and was independent of variations in holding potential or cytoplasmic [Ca2+] ([Ca2+]cyt; Fig. 2B). The representative traces illustrated in Fig. 2C were recorded at a holding potential of -40 mV in control conditions (top), after the addition of EGTA to lower the free [Ca2+]cyt < 100 nM (middle), and after 30 µM DIDS typically blocked the unitary current (bottom). These reconstituted channels were found to be sensitive to DIDS, SITS, and NPPB (IC50 = 139 nM, 10 µM, and 130 µM, respectively; Fig. 2D). On the other hand, they seemed insensitive to niflumic acid, tamoxifen, and calix[4]arene (26).


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Fig. 2.   Biophysical characteristics of reconstituted Cl- channels. A: in symmetrical 250 mM CsCl conditions, channels displayed a linear current-voltage relationship (n = 16 experiments). gamma , Mean conductance. B: channel open probability (Po) was tonically high and independent of both holding potential and free cytoplasmic Ca2+ concentration ([Ca2+]). C: representative traces of unitary current recorded in symmetrical CsCl conditions + free Ca2+, pH 7.4 (top). Channel activity and current amplitude remained unaffected with addition of 60 µM EGTA, decreasing free cytoplasmic [Ca2+] to <100 nM (middle). On the other hand, DIDS rapidly blocked channel (bottom). C, closed channel level. D: channel activity was sensitive to Cl--channel blockers DIDS, SITS, and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). NPo, mean Po, where N is total no. of channels; [inhibitor], inhibitor concentration.

Direct effects of 11,12-EET on the reconstituted Cl- channels. To determine whether 11,12-EET could exert a direct effect on this type of Cl- channel, the reconstituted protein was exposed to exogenous 11,12-EET. Control experiments were first performed to assess whether EtOH (the solvent for 11,12-EET) alone produced any effect on channel behavior. Up to concentrations of 0.5%, the maximal concentration used in this study, EtOH had no effect on channel conductance and/or Po (Fig. 3B). The multiple-channel recordings shown in Fig. 3 illustrate the Cl- current obtained in symmetrical 250 mM CsCl (control; Fig. 3A), after the addition of 0.5% EtOH trans (Fig. 3B), and after the injection of a total of 0.75 and 1.5 µM 11,12-EET (Fig. 3, C and D, respectively). Unlike EtOH, 11,12-EET decreased both the conductance and Po of the Cl- channels. In a series of reconstitution experiments aimed at quantifying this effect, concentrations ranging from 0 to 1.5 µM exogenous 11,12-EET were obtained by cumulative addition of 11,12-EET to the trans compartment every 7 min. Concentrations of 0.375, 0.75, 1.125, and 1.5 µM decreased the conductance of the channels by 4.8, 7.2, 8.7, and 44.7%, respectively (Fig. 4, A and B). Paired t-test indicated that 1.5 µM induced a significant decrease in channel conductance (P < 0.05; n = 6 experiments). Channel activity was also lowered in a concentration-dependent manner; the same concentrations respectively decreased the channel normalized average NPo by 19.5, 26.3, 46.3, and 69.5% compared with control values (Fig. 4, C and D). At -50 mV, this constant effect was found to be statistically significant at a 11,12-EET concentration >=  1.125 µM (n = 6 experiments). The IC50 value was estimated at 1.14 µM. Inhibition of the current was not voltage dependent, as can be inferred from Fig. 4C, where the decrease in NPo is similar at all tested voltages.


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Fig. 3.   Dose-dependent inhibition of multiple channels by 11,12-EET. Multichannel recordings were obtained in symmetrical CsCl at a holding potential of -40 mV. A: CTRL conditions. Three open channel (O) levels appear as downward deflections. B: addition of ethanol (EtOH), solvent for 11,12-EET, failed to affect unitary current observed in these conditions. C: after addition of 11,12-EET, Po is markedly decreased, with little effect on conductance of channels. D: 11,12-EET further reduced channel Po and significantly decreased channel conductance.


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Fig. 4.   Quantitative analysis of effect of 11,12-EET on reconstituted ASM Cl- channels. A and B: effect of increasing concentrations of 11,12-EET ([11,12-EET]) on mean conductance of reconstituted channels. In symmetrical 250 mM CsCl, channels displayed conductances of 121.2, 115.6, 112.4, 110.6, and 67.0 pS in presence of 0, 0.375, 0.75, 1.125, and 1.5 µM EET, respectively (n = 6 experiments). Paired t-tests confirmed that 1.5 µM 11,12-EET caused a significant decrease in channel conductance. C: effect of increasing [11,12-EET] on channel Po. Gradual decreases from high and voltage-independent control Po values were observed after addition of 0.375, 0.75, 1.125, and 1.5 µM 11,12-EET regardless of applied voltage. Low signal-to-noise ratios precluded Po measurement around 0 mV. D: at -50 mV, normalized NPo values declined from 0.95 in CTRL condition to 0.76, 0.70, 0.51, and 0.29 after respective concentrations of 0.375, 0.75, 1.125, and 1.5 µM 11,12-EET had been added (n = 6 experiments). * Significant variations from CTRL conditions, P < 0.05 by paired t-test.

Effect of 11,12-EET on channel gating. Despite the relatively low occurrence of single-channel reconstitutions, it was possible to perform quantitative kinetic analysis on single-channel recordings to determine whether 11,12-EET affected the gating of the Cl- channel. In control experimental conditions, the channels displayed a gating profile best described by two exponentials for the open state (short open time constant = 0.85± 0.16 ms, n = 3 experiments; long open time constant = 7.30 ± 1.85 ms, n = 3 experiments) and two exponentials for the closed state (short closed time constant = 0.5 ± 0.01 ms, n = 3 experiments; long closed time constant = 3.56 ± 1.91 ms, n = 3 experiments). Seven minutes after the addition of 0.75 µM 11,12-EET, the time necessary to reach maximal inhibition of channel activity, only the first (short) open time constant (1.64 ± 0.27 ms) remained, whereas an increase in the long closed time constant (6.06 ± 1.76 ms) could be observed, which translated into relatively less time spent by the channel in the open state. The decrease in both channel Po and unitary conductance should translate into a marked decrease in macroscopic Cl- current.

Sidedness of the effect of 11,12-EET inhibition. Given the hydrophobic nature of 11,12-EET, our initial working hypothesis was that the molecule would act on the protein from within the membrane leaflets and thus would inhibit the Cl- current regardless of the side of the membrane from which it was injected. Even when considering a reduced partition coefficient in the lipid bilayer due to the epoxide functional group and a 20°C working temperature, 11,12-EET would diffuse through the membrane in a manner of milliseconds. Contrary to our initial assumption, 11,12-EET could only alter channel behavior from a specific side of the protein; if 11,12-EET did not cause a significant decrease in channel activity within 10 min, it always failed to affect the current in any way. In such cases, addition of 11,12-EET to the side opposite that of the first injection induced the reported inhibition within 6-7 min (data not shown). Hence it appears that the site of action of 11,12-EET on the Cl- channels is located within the membrane in proximity of one of its surfaces and is not readily attainable from the opposite side of the PLB. In the absence of a functional marker such as a Ca2+ sensor, we suggest that the channels that were unaffected by the first injection of EET had been inserted into the PLB with the reverse orientation. The high probability (almost 50%) of such inverted fusions was suggested from our studies on KCa channels, which exhibit an easily determined sidedness in their sensitivity to Ca2+.

Dose-dependent inhibition of reconstituted Cl- channels by 5,6-EET. With protocols similar to those yielding the 11,12-EET data, increasing concentrations of 5,6-EET dissolved in ACN were added to the symmetrical 250 mM CsCl buffer system after reconstitution of the Cl- channels of interest. There resulted simultaneous, concentration-dependent decreases in the apparent unitary channel current and Po. Figure 5A illustrates this effect; in control conditions, this single reconstituted channel exhibited a typically high Po. Subsequent additions of 0.3 and 1 µM 5,6-EET significantly reduced current amplitude as well as channel Po, whereas addition of the same volume of solvent (7.8 µM being equivalent to the concentration of ACN applied with 1.0 µM 5,6-EET) had no significant effect (data not shown). Quantitatively, both 0.3 and 1 µM 5,6-EET induced statistically significant decreases of 24 and 40%, respectively, in unitary current amplitude measured at -40 mV (P < 0.05 by paired t-test; n = 4 experiments; Fig. 5B). In contrast, an equivalent amount of ACN had no significant effect on unitary current amplitude or channel Po (Fig. 5, B and C). 5,6-EET also diminished channel activity in a concentration-dependent manner; 0.1, 0.3, and 1 µM all induced statistically significant decreases in NPo (P < 0.05 by paired t-test; n = 4 experiments), with respective declines of 29, 56, and 67% (Fig. 5C).


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Fig. 5.   Dose-dependent modulation of channel behavior induced by 5,6-EET. A: representative recording of unitary current from a single channel in CTRL conditions (top). Addition of 0.3 (middle) and 1.0 µM (bottom) 5,6-EET diminished amplitude of current and Po of channel. B: effects of 5,6-EET and ACN (solvent) on amplitude of current measured at -40 mV. [5,6-EET], 5,6-EET concentration. 5,6-EET (0.1, 0.3 and 1.0 µM) diminished current by 15, 25, and 40%, respectively. In contrast, 7.8 µM ACN, concentration introduced when applying 1.0 µM 5,6-EET, had no significant effect. C: channel Po was similarly altered by 5,6-EET. Addition of 0.1, 0.3 and 1.0 µM 5,6-EET significantly decreased mean channel Po to 0.74, 0.44, and 0.23, respectively, compared with control value (n = 4 experiments). ACN had no significant effect on channel Po (n = 4 experiments).

Degraded 11,12-EET and AA have no effect on channel behavior. In vivo, the four EET isomers may be transformed to dihydroxyeicosatrienoic acids or hydroxyeicosatetraenoic acids, some of which are metabolically active (19). More generally, epoxides react with mildly acidic aqueous environments such as the cytoplasm to form diols. To test for an effect of degradation products of 11,12-EET, the solution was left at room temperature and exposed to light and ambient air for >2 h before being added to either side of the protein. Such additions failed to induce any change in the monitored parameters, i.e., conductance, Po, and time constants (data not shown). Moreover, the EET precursor AA, known to exert a modulatory role on Cl- channels in various cells (11, 25; see Ref. 20 for a review), failed to alter either channel conductance (Fig. 6, A and B) or Po (Fig. 6, A and C) at concentrations of AA up to twice those used for 11,12-EET (n = 5 experiments).


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Fig. 6.   Control experiments showing absence of effect of arachidonic acid (AA) on reconstituted Cl- channels. A: increasing concentrations of AA failed to alter current measured at -40 mV. B: paired t-tests indicate that current-voltage relationship of channel is unaffected by 0.75 and 1.5 µM AA with respect to CTRL conditions. C: channel activity as a function of holding potential in absence and presence of increasing concentrations of AA. Effect of 1.5 µM 11,12-EET is illustrated for comparison purposes. AA failed to alter normalized average channel Po.

Relaxation of ASM strips by 5,6- and 11,12-EET. Epithelial AA metabolites have been known to exert relaxing effects on ASM (e.g., Ref. 10). With a protocol described in detail in the companion article (7), the relaxations induced by 5,6- and 11,12-EET in epithelium-denuded CCh- or histamine (Hist)-precontracted guinea pig ASM muscle strips were monitored. Figure 7A shows representative traces obtained from one type of experiment in which 0.2 µM CCh was used to precontract the tissues before cumulative concentrations of 5,6-EET relaxed the muscle strips. Pretreatment of the tracheal strip with 50 nM IbTX partially inhibited the relaxation induced by 5,6-EET after CCh challenge (Fig. 7A'). Furthermore, pretreating the tissues with 100 µM DIDS and 50 nM IbTX to block the Cl- and KCa conductances, respectively, led to a partial recovery of the relaxation induced by 5,6-EET (Fig. 7A'') compared with that obtained in the presence of IbTX alone (Fig. 7A'). Half-maximal relaxation by 5,6-EET shifted from 0.48 µM in control conditions to 2.2 µM in the presence of 50 nM IbTX. In contrast, this value was left shifted to 1.2 µM when the inhibitory actions of IbTX, DIDS, and 5,6-EET were combined. One-way ANOVA tests indicated that both treatments (i.e., 50 nM IbTX or 50 nM IbTX + 100 µM DIDS) significantly altered the concentration-relaxation curves of these tissues to 5,6-EET (Fig. 7B). The reduced bronchorelaxant effect of 5,6-EET (vs. control condition) obtained in the presence of the specific KCa blocker suggests that the activation of KCa channels contributes to the effect of the eicosanoid in ASM. Moreover, the increased potency of 5,6-EET when DIDS also blocked the Cl- conductance and the statistically significant 5.9% relaxation induced by 100 µM DIDS alone on CCh-contracted tissues (data not shown) support a role for Cl- efflux in sustaining ASM tone. Histaminic challenge of the tracheal strips produced similar results; 3 µM 5,6-EET and 11,12-EET, which exhibited significantly lower bronchorelaxant abilities on these tissues, relaxed Hist-precontracted ASM by 129 and 19%, respectively. This effect could be partly blocked by 10 nM IbTX (Fig. 7D). Overall, these results suggest that several cellular targets exist for the EET isomers in the relaxant response of ASM tissues. Moreover, it seems that the effects of 5,6- and 11,12-EET are mechanistically distinct; whereas the response of CCh-contracted tissues to 11,12-EET appears to be based largely on KCa activation, as shown in Ref. 7, the responses of the tissues precontracted with either agonist to 5,6-EET seem to be more complex, involving KCa activation, the partial inhibition of a Cl- conductance, and other cellular cascades yet to be identified.


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Fig. 7.   Relaxant response of ASM strips induced by 5,6- and 11,12-EET in presence of IbTX and DIDS. Typical tension recordings were performed on epithelium-denuded guinea pig ASM strips after stimulation with CCh. Cumulative concentrations of 5,6-EET (nos. above traces) induced increasing relaxations of tracheal tissue (A). Preincubation with IbTX for 5 min before challenge with CCh decreased this concentration-dependent relaxation (A'). Pretreatment of tissues with DIDS and IbTX before CCh stimulation caused a partial recovery of relaxation induced by 5,6-EET (A''). Arrows, addition of CCh, EET, and IbTX and washout (W) with control Krebs solution. B: concentration-relaxation curves for 5,6-EET. In control condition, 0.48 µM 5,6-EET caused half-maximal relaxation of CCh-precontracted tissues (n = 12 experiments); this value was increased to 2.2 µM after functional inhibition of Ca2+-activated K+ (KCa) channels (n = 6 experiments; open circle ), whereas combined inhibition of Cl- conductance by DIDS and 5,6-EET led to a left shift of EC50 to 1.2 µM (n = 5 experiments; black-down-triangle ). C: effect of tested EETs on histamine (Hist)-precontracted ASM strips. Pretreatment with 10 nM IbTX decreased relaxation of Hist-contracted tissues by 5,6-EET. Similarly, 11,12-EET, dissolved in ACN, induced a 19.6% relaxation of Hist-stimulated tracheal smooth muscle, a relaxation that was decreased to 10.6% by incubating strips with 10 nM IbTX before agonist stimulation (n = 3 experiments).

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

This work is the first to highlight the direct inhibition of reconstituted ASM Cl- channels by 5,6- and 11,12-EET. These observations reveal the existence of KCa-independent pathways by which the EETs could act in mammalian ASM. This hypothesis is supported by 1) microelectrode measurements showing that both 5,6- and 11,12-EET can significantly hyperpolarize ASM cells and 2) muscle tension measurements demonstrating that the EETs can relax ASM tracheal strips in both the absence and presence of IbTX.

Using the latter, we evaluated the activation of KCa channels, examined more extensively in Ref. 7, to account for ~50% of the relaxation induced by 11,12-EET in Hist-precontracted ASM strips. This involvement falls to ~15% when 5,6-EET is added exogenously. In addition, the use of the Cl--channel blocker DIDS suggests that 24% of the relaxation induced by 3.0 µM 5,6-EET can be attributed to the inhibition of a Cl- conductance. Altogether, these pharmacomechanical data suggest that the two EET isomers tested exhibit bronchorelaxant abilities in vitro, with 5,6-EET being much more potent in these conditions. At the cellular level, Vm measurements have confirmed the ability of both EETs to act as EpDHF-like molecules in mammalian ASM. Although some of their effects are evidently KCa dependent, a significant proportion of the cellular response to the EETs appear to be achieved through other pathways. Moreover, the observation that an inhibition of the Cl- current by DIDS could mimic the hyperpolarizing effect of the EET isomers supports the involvement of this anionic conductance in the control of the Vm of ASM cells, as previously suggested by Daniel et al. (5). These measurements will have to be extended to propose a more formal link between the similar effects of the EET isomers and DIDS.

Of main interest here is the fact that the direct inhibition of Ca2+-insensitive Cl- channels by 5,6- and 11,12-EET after the incorporation of bovine tracheal SM vesicles into PLBs reveals one of the possible mechanisms by which 5,6- and 11,12-EET may act as EpDHFs: via the direct inhibition of a depolarizing sarcolemmal Cl- current. The addition of increasing doses of 5,6- and 11,12-EET alters channel behavior in a concentration-dependent manner. Even though one must be cautious when extrapolating reconstitution results to a cellular system, it can be assumed that an overall decline in Cl- current due to the direct effect of either EET isomer would be synergistic with an increase in K+-channel activity, at least in the case of 11,12-EET, thus leading to a net hyperpolarization of Vm. The involvement of a macroscopic Cl- current in ASM cell depolarization has been demonstrated by Daniel et al. (5) as well as by Janssen and Sims (14). Moreover, the direct activation of Ca2+-dependent K+ channels by 11,12-EET has been confirmed in this laboratory (7). This simplified scheme of direct ionic channel modulation explains some of the effects of 5,6- and 11,12-EET as plausible EpDHFs in ASM. However, other mechanisms have been proposed; one pathway would involve a stimulatory G protein (Gs) of the sarcolemmal membrane in the activation of vascular KCa channels (16), whereas another study (19) suggests that 11,12-EET could play a role as an intracellular messenger.

The IC50 values estimated from this reconstitution study of 5,6- and 11,12-EET inhibition were 0.26 and 1.14 µM, respectively, and fall within the range of concentrations at which EET isomers exert their effects on ionic channels in other tissues (16, 17). Of note is that concentration-response relationships are difficult to establish for AA and its active metabolites; among other factors, the consequences of working at different temperatures (22 and 37°C) must be kept in mind because the partition behavior of the eicosanoids is significantly different at 22°C than at 37°C.

All four isomers exhibit vasodilator abilities in different organs (e.g., Refs. 8, 9). Our initial choice of 11,12-EET was dictated partly by evidence gathered by Dray et al. (6), who demonstrated that pulmonary Kaposi sarcoma cells secreted predominantly 11,12-EET. Additional observations made by Campbell et al. (3) that 11,12-EET and 14,15-EET exert a more potent action on VSM K+ channels than the other isomers prompted us to consider 11,12-EET as a "potential" modulator of ASM sarcolemmal channel activity. It was beyond the immediate scope of this study to test the direct effect of all AA metabolites (EETs, hydroxyeicosatetraenoic acids, and dihydroxyeicosatrienoic acids) on our tracheal SM preparations, especially in the absence of evidence that these compounds are produced in physiologically significant amounts in this tissue. Even the chemical instability of the potent 5,6-EET raises some interrogations regarding its role in vivo; it can be assumed that it must act transiently and locally, possibly within or close to the epithelial cells where it is produced. Nonetheless, the bronchorelaxant potency of the molecule in vitro could be the basis for a challenging pharmacological venture.

The absence of potency of AA and the degradation products of EETs appear to result from their lack of an epoxide functional group and suggest a requirement for this group if EpDHF-like ability is to be exhibited. Additional incentive to test AA came from the demonstration that AA inhibits a variety of Cl- channels (11, 25), including some from airway epithelia (13); conceivable similarities among these channels and ASM Cl- could have resulted in a resembling block of the ASM channel. Similarly, the possibility that the EETs may undergo oxidation or acid-catalyzed hydrolysis in the cytoplasm before acting on their targets urged us to mimic their degradation in vitro; old, degraded 11,12-EET proved to have no effect on Cl--channel behavior (data not shown). A perplexing observation was reported in 1993 that 11,12-EET increased the amplitude of transient [Ca2+]cyt oscillations in cardiac myocytes (21). Considering the existence in tracheal SM cells of a well-described Ca2+-activated Cl- current, it would be of interest to monitor [Ca2+]cyt in tracheal myocytes after exposure to 11,12-EET to provide some insight on the additional ways by which the molecule may influence cellular behavior, intracellularly or at the surface membrane.

The main purpose of this work was to highlight the direct inhibition of bovine ASM Cl- channels by 5,6- and 11,12-EET in the absence of other cellular structures. The interactions with ionic channels revealed herein may represent a novel mechanism of action by which the EETs could exert their role as putative EpDHFs. These observations join a growing body of evidence suggesting that the eicosatrienoic acid regioisomers can modulate the membrane potential and tone of ASM cells, thus exhibiting EpDHF abilities in this tissue.

    ACKNOWLEDGEMENTS

We are endebted to Sophie Gaudreault and Dr. Alain Cadieux for assistance with the pharmacomechanical measurements reported and to Dr. Paul C. Pape for reading and discussing the manuscript.

    FOOTNOTES

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

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

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

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

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.   Aickin, C. C., and A. F. Brading. Advances in the understanding of transmembrane ionic gradients and permeabilities in smooth muscle obtained by using ion-selective micro-electrodes. Experientia 41: 879-887, 1985[Medline].

2.   Alioua, A., D. Salvail, M. Dumoulin, J. Garon, A. Cadieux, and E. Rousseau. Direct activation of KCa channels in airway smooth muscle by nitric oxide: involvement of a nitrothiosylation mechanism? Am. J. Respir. Cell Mol. Biol. 18: 1-13, 1998[Abstract/Free Full Text].

3.   Campbell, W. B., D. Gebremedhin, P. F. Pratt, and D. R. Harder. Identification of epoxyeicosatrienoic acids as endothelium-derived hyperpolarizing factors. Circ. Res. 78: 415-423, 1996[Abstract/Free Full Text].

4.   Carroll, M. A., M. Schwartzman, J. Capdevila, J. R. Falck, and J. C. McGiff. Vasoactivity of arachidonic acid epoxides. Eur. J. Pharmacol. 138: 281-283, 1987[Medline].

5.   Daniel, E. E., J. Jury, J. P. Bourreau, and L. Jager. Chloride and depolarization by acetylcholine in canine airway smooth muscle. Can. J. Physiol. Pharmacol. 71: 284-292, 1993[Medline].

6.   Dray, F., B. Vulliez-Le Normand, A. Deroussent, I. Briquet, M. M. Gabellec, S. Nakamura, L. M. Wahl, A. Gouyette, and Z. S. Salahuddin. Active metabolism of arachidonic acid by Kaposi sarcoma cells cultured from lung biopsies (KS-3); identification by HPLC and MS/MS of the predominant metabolite secreted as the 11,12-epoxy-eicosatrienoic acid. Biochim. Biophys. Acta 1180: 83-90, 1992[Medline].

7.   Dumoulin, M., D. Salvail, S. Gaudreault, A. Cadieux, and E. Rousseau. Epoxyeicosatrienoic acids relax airway smooth muscles and directly activate reconstituted KCa channels. Am. J. Physiol. 275 (Lung Cell. Mol. Physiol. 19): L423-L431, 1998[Abstract/Free Full Text].

8.   Ellis, E. F., R. J. Police, L. Yancey, J. S. McKinney, and S. C. Amruthesh. Dilation of cerebral arterioles by cytochrome P-450 metabolites of arachidonic acid. Am. J. Physiol. 259 (Heart Circ. Physiol. 28): H1171-H1177, 1990[Abstract/Free Full Text].

9.   Fukao, M., Y. Hattori, M. Kanno, I. Sakuma, and A. Kitabatake. Evidence against a role of cytochrome P450-derived arachidonic acid metabolites in endothelium-dependent hyperpolarisation by acetylcholine in rat isolated mesenteric artery. Br. J. Pharmacol. 120: 439-446, 1997[Abstract].

10.   Gao, Y., and P. M. Vanhoutte. Epithelium acts as a modulator and a diffusion barrier in the responses of canine airway smooth muscle. J. Appl. Physiol. 76: 1843-1847, 1994[Abstract/Free Full Text].

11.   Gosling, M., D. R. Poyner, and J. W. Smith. Effects of arachidonic acid upon the volume-sensitive chloride current in rat osteoblast-like (ROS 17/2.8) cells. J. Physiol. (Lond.) 493: 613-623, 1996[Abstract].

12.   Henmi, S., Y. Imaizumi, K. Muraki, and M. Watanabe. Time course of Ca2+-dependent K+ and Cl- currents in single smooth muscle cells of guinea-pig trachea. Eur. J. Pharmacol. 306: 227-236, 1996[Medline].

13.   Hwang, T. C., S. E. Guggino, and W. B. Guggino. Direct modulation of secretory chloride channels by arachidonic and other cis unsaturated fatty acids. Proc. Natl. Acad. Sci. USA 87: 5706-5709, 1990[Abstract].

14.   Janssen, L. J., and S. M. Sims. Acetylcholine activates non-selective cation and chloride conductances in canine and guinea-pig tracheal myocytes. J. Physiol. (Lond.) 453: 197-218, 1992[Abstract].

15.   Knickle, L. C., and J. R. Bend. Bioactivation of arachidonic acid by the cytochrome P450 monooxygenases of guinea pig lung: the orthologue of cytochrome P450 2B4 is solely responsible for formation of epoxyeicosatrienoic acids. Mol. Pharmacol. 45: 1273-1280, 1994[Abstract].

16.   Li, P. L., and W. B. Campbell. Epoxyeicosatrienoic acids activate K+ channels in coronary smooth muscle through a guanine nucleotide binding protein. Circ. Res. 80: 877-884, 1997[Abstract/Free Full Text].

17.   Li, P. L., A. P. Zou, and W. B. Campbell. Regulation of potassium channels in coronary arterial smooth muscle by endothelium-derived vasodilators. Hypertension 29: 262-267, 1997[Abstract/Free Full Text].

18.   Matsumoto, K., H. Aizawa, R. Inoue, S. Hamano, S. Ikeda, Z. Xie, M. Hirata, N. Hara, and Y. Ito. Effects of epithelial cell supernatant on membrane potential and contraction of dog airway smooth muscles. Am. J. Respir. Cell Mol. Biol. 10: 322-330, 1994[Abstract].

19.   McGiff, J. C. Cytochrome P450 metabolism of arachidonic acid. Annu. Rev. Pharmacol. Toxicol. 31: 339-369, 1991[Medline].

20.   Meves, H. Modulation of ion channels by arachidonic acid. Prog. Neurobiol. 43: 175-186, 1994[Medline].

21.   Moffat, M. P., C. A. Ward, J. R. Bend, T. Mock, P. Farhangkhoee, and M. Karmazyn. Effects of epoxyeicosatrienoic acids on isolated hearts and ventricular myocytes. Am. J. Physiol. 264 (Heart Circ. Physiol. 33): H1154-H1160, 1993[Abstract/Free Full Text].

22.   Nijkamp, F. P., and G. Folkerts. Reversal of arachidonic acid-induced guinea-pig tracheal relaxation into contraction after epithelium removal. Eur. J. Pharmacol. 131: 315-316, 1986[Medline].

23.   Proctor, K. G., J. R. Falck, and J. Capdevila. Intestinal vasodilation by epoxyeicosatrienoic acids: arachidonic acid metabolites produced by a cytochrome P450 monooxygenase. Circ. Res. 60: 50-59, 1987[Abstract].

24.   Rosolowsky, M., and W. B. Campbell. Synthesis of hydroxyeicosatetraenoic (HETEs) and epoxyeicosatrienoic acids (EETs) by cultured bovine coronary artery endothelial cells. Biochim. Biophys. Acta 1299: 267-277, 1996[Medline].

25.   Sakai, H., B. Kakinoki, M. Diener, and N. Takeguchi. Endogenous arachidonic acid inhibits hypotonically-activated Cl- channels in isolated rat hepatocytes. Jpn. J. Physiol. 46: 311-318, 1996[Medline].

26.   Salvail, D., A. Alioua, and E. Rousseau. Functional identification of a sarcolemmal chloride channel from bovine tracheal smooth muscle. Am. J. Physiol. 271 (Cell Physiol. 40): C1716-C1724, 1996[Abstract/Free Full Text].

27.   Savaria, D., C. Lanoue, A. Cadieux, and E. Rousseau. Large conducting potassium channel reconstituted from airway smooth muscle. Am. J. Physiol. 262 (Lung Cell. Mol. Physiol. 6): L327-L336, 1992[Abstract/Free Full Text].

28.   Szarek, J. L., M. N. Gillespie, R. J. Altiere, and L. Diamond. Reflex activation of the nonadrenergic noncholinergic inhibitory nervous system in feline airways. Am. Rev. Respir. Dis. 133: 1159-1162, 1986[Medline].

29.   Wang, Y. X., B. K. Fleischmann, and M. I. Kotlikoff. Modulation of maxi-K+ channels by voltage-dependent Ca2+ channels and methacholine in single airway myocytes. Am. J. Physiol. 272 (Cell Physiol. 41): C1151-C1159, 1997[Abstract/Free Full Text].

30.   Xie, Z., H. Hakoda, and Y. Ito. Airway epithelial cells regulate membrane potential, neurotransmission and muscle tone of the dog airway smooth muscle. J. Physiol. (Lond.) 449: 619-639, 1992[Abstract].

31.   Zeldin, D. C., J. Foley, J. Ma, J. E. Boyle, J. M. Pascual, C. R. Moomaw, K. B. Tomer, C. Steenbergen, and S. Wu. CYP2J subfamily P450s in the lung: expression, localization, and potential functional significance. Mol. Pharmacol. 50: 1111-1117, 1996[Abstract].


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