Epoxyeicosatrienoic acids relax airway smooth muscles and
directly activate reconstituted
KCa channels
Marc
Dumoulin1,
Dany
Salvail1,
Sophie B.
Gaudreault2,
Alain
Cadieux2, and
Eric
Rousseau1
Le Bilarium, 1 Departments of
Physiology and Biophysics and
2 Pharmacology, Faculty of
Medicine, University of Sherbrooke, Sherbrooke, Quebec, Canada J1H 5N4
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ABSTRACT |
Epoxyeicosatrienoic acids (EETs) relax various smooth
muscles by increasing outward K+
movement, but the molecular mode of action of EET regioisomers remains
to be clarified. The effects of EETs were investigated on bovine airway
smooth muscle tone and on reconstituted
Ca2+-activated
K+
(KCa) channels. 5,6-EET
and 11,12-EET induced dose-dependent relaxations of precontracted
bronchial spirals. These effects were partly abolished by 10 nM
iberiotoxin. Bilayer experiments have shown that 0.1-10 µM
11,12-EET produced up to fourfold increases in the open probability of
KCa channels from the
cis (extracellular) side by enhancing
the mean open time constant and reducing the long closed time constant,
without affecting the unitary conductance. EET-induced activations were
blocked by 10 nM iberiotoxin. Addition of vehicles or other lipids as
well as of GTP and guanosine
5'-O-(3-thiotriphosphate) in the
absence of EET had no effect on channel activity. Thus EETs directly
activate KCa channels from
airway smooth muscle through an interaction with the extracellular face
of the channel. We propose that EETs could represent candidate
molecules as epithelium-derived hyperpolarizing factors.
calcium-activated potassium channels; endothelium-derived
hyperpolarizing factor; epithelium-derived hyperpolarizing factor; relaxation; trachea; planar lipid bilayer; eicosanoid
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INTRODUCTION |
THE EPOXYEICOSATRIENOIC ACID (EET) regioisomers (5,6-, 8,9-, 11,12-, and 14,15-EET) are a family of metabolites of arachidonic acid (AA) synthesized by cytochrome
P-450 epoxygenase (11, 12, 16).
Cytochrome P-450 in the presence of
NADPH catalyzes the formation of epoxides (EETs) from AA (10, 40) as
well as the formation of midchain
cis-trans conjugated dienols
(hydroxyeicosatetraenoic acids) and C-19 and C-20 alcohols (19-OH-AA
and 20-OH-AA). Cytochrome P-450 is
present in various tissues including the liver (24), kidney (25, 26),
and lung (40). AA, from exogenous sources or from sarcolemmal membrane
phospholipids, is also metabolized by two additional pathways,
lipoxygenase and cyclooxygenase, leading to the production of
leukotrienes and PGs, respectively. Much is known about the effects of
leukotrienes and PGs in the regulation of smooth muscle tone (1, 33)
either in vascular smooth muscles (VSMs) or airway smooth muscles
(ASMs). On the other hand, much less is known about the mechanism of
action of the epoxygenase metabolites (EETs). EETs were recently shown
to dilate coronary arteries (5, 9, 21, 31) as well as renal, cerebral, pial, and caudal arteries from different species (13, 17, 22, 37).
Moreover, these studies demonstrated that inhibition of cytochrome
P-450 in vascular endothelial cells
decreased K+ outflow. Addition of
exogenous EETs on endothelium-denuded arteries enhanced
Ca2+-activated
K+
(KCa)-channel activities
without affecting ATP-sensitive K+
channels and facilitated cell repolarization and/or
hyperpolarization, which, in turn, resulted in muscle relaxation. Data
from a recent study (31) on VSMs suggested that
KCa-channel activation by 11,12-EET occurred via a stimulatory G protein-mediated process. On the
basis of these results, it was proposed that EETs could represent an
endothelium-derived hyperpolarizing factor of the vascular system (9,
15, 19, 23, 31, 34, 36).
In the airways, the epithelium uses different pathways to modulate
smooth muscle tone (7). The presence of epoxygenase activities has been
demonstrated in Clara cells and type II pneumocytes from rabbit lung
(40). Several isozymes such as CYP1A1, CYP2B4, and CYP4B1 have been
purified from these cells, the latter exhibiting the majority of
epoxygenase activity. Furthermore,
KCa channels are widely
distributed in ASM cells (2) and play an important role in the
regulation of smooth muscle membrane potential and tone modulation (20,
27, 29). Despite this, little is known about the effects of EETs on ASM
tone and on the electrophysiological properties of the cellular surface
membrane. The aim of this study was, therefore, to investigate whether
EETs could relax ASMs and, if so, whether this relaxation was partly
mediated by a direct activation of
KCa channels.
Pharmacomechanical measurements were made on bronchial muscle strips,
and a planar lipid bilayer (PLB) reconstitution technique was used to
characterize the effect and mechanism of action of exogenous 11,12-EET
on KCa channels. Herein, we
demonstrate that 5,6-EET as well as 11,12-EET induces dose-dependent and iberiotoxin (IbTX)-sensitive relaxations on epithelium-denuded, precontracted main bronchi. Moreover, the open probability
(Po) of the
KCa channels was increased
in a concentration-dependent manner by extracellular applications of
these eicosanoids, without affecting single-channel conductance.
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MATERIALS AND METHODS |
Bronchial smooth muscle strip preparation and
isotension measurements. Male guinea pigs (Hartley,
300-350 g) were killed by exsanguination after an intraperitoneal
injection of pentobarbital sodium (50 mg/kg). The lungs were quickly
removed and placed in cold Krebs solution. The main bronchi were
dissected and cut helically as previously described (8). Epithelial
cells were removed in most experiments. Each bronchial spiral was
mounted in a 4-ml jacketed organ bath containing Krebs-bicarbonate
solution composed of (in mM) 118.1 NaCl, 4.7 KCl, 1.2 MgSO4, 1.2 KH2PO4,
2.5 NaHCO3, 2.5 CaCl2, and 11.1 glucose, pH 7.4, gassed with 95% O2-5%
CO2 at 37°C. Isometric tension
changes were measured with a Grass polygraph (model 7D). Tissues were
subjected to an initial loading tension of 1 g and allowed to
equilibrate for 60 min (with changes of bath medium every 15 min)
before the experiments were started. After equilibration, the main
bronchial spirals were precontracted with 0.2 µM carbamylcholine
chloride (CCh); then EETs or vehicle was added. The relaxation
responses to a specific concentration of EETs are expressed as a
percentage of the maximum tension induced by CCh.
Preparation of tracheal smooth muscle microsomal
fractions. The crude microsomal fraction was prepared
as previously described (3, 4). The reconstitution system consisted of
two experimental chambers, denoted cis
and trans, separated by a septum with
a 250-µm-diameter hole. The cis
chamber, which was defined as the compartment to which surface membrane
vesicles were added, was connected to an operational amplifier (Dagan
8900, Minneapolis, MN), and the trans
chamber was connected to a virtual ground by means of two
low-resistance electrodes (MERE 2, World Precision Instruments,
Sarasota, FL). The hole was pretreated with a mixture of phospholipids
at a concentration of 25 mg/ml chloroform in a ratio of 3:2:1
phosphatidylethanolamine-phosphatidylserine-phosphatidylcholine. PLBs
were formed by the application of the same phospholipid mixture dissolved in decane. The experimental chambers contained the following solutions (in mM): 250 KCl cis-50 KCl
trans plus 20 K-HEPES and 0.01 free
Ca2+ (109 µM
CaCl2 + 100 µM K-EGTA), pH 7.4. Microsomal fractions enriched in sarcolemmal vesicles (10-60 µg
of proteins) were fused into the PLB from the
cis chamber. With this approach, the
cis chamber corresponds to the
extracellular side and the trans
chamber to the cytoplasmic side of the channel. Reconstitution of
native microsomal membrane protein into a PLB is particularly suitable to test the direct effects of pharmacological and biochemical agents
from either side of the bilayer in the absence of intra- or
extracellular components, which is not the case with the various configurations of the patch-clamp technique.
Recording of reconstituted KCa channels.
Currents were filtered at 10 kHz and recorded on videotape (DAS/VCR 900 Toshiba, Unitrade). Currents were displayed on-line on a chart recorder
(DASH II model MT, Astro-Med) for trace illustrations or were played
back, filtered at 1 kHz, and digitalized at 5 kHz for storage in 3-min
files on a hard disk. All reconstitution studies were performed at room
temperature (22 ± 2°C).
KCa-channel activities were
analyzed in terms of current amplitudes and channel Po values.
Multiple-channel activities were routinely recorded (70%).
Po values were
determined as described previously (3, 4) and are expressed as mean
Po
(NPo), where
N is the total number of channels
recorded experimentally. The number of channels functionally active in
the bilayer was determined at the beginning of each recording in the
presence of 10 µM free cytoplasmic
Ca2+
(trans) and at a holding potential
(+40 mV) under which the
Po of the
KCa channel is maximal.
KCa-channel activation by
11,12-EET was determined as
NPo in the
presence of 11,12-EET divided by NPo in the
control condition over the same period of time.
Chemical reagents. CCh,
tetraethylammonium (TEA), IbTX, GTP, and guanosine
5'-O-(3-thiotriphosphate)
(GTP
S) were obtained from Sigma (St. Louis, MO). 11,12-EET in
ethanol was obtained from Sigma, whereas other lots of 5,6- and
11,12-EETs dissolved in 0.01% acetonitrile (ACN) were obtained from
Cayman Chemical (Ann Arbor, MI). All phospholipids were obtained from
Avanti Polar Lipids (Alabaster, AL).
Statistical analysis. Results are
means ± SE; n = 3-6
experiments. Mean values were compared by paired or unpaired (when
appropriate) Student's t-test with
the Sigma Plot program (SPSS, Chicago, IL). Statistical significance of
the results was verified, with a threshold value for significance set
at P < 0.05.
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RESULTS |
Pharmacomechanical assessment of the effect of
exogenous EETs on bronchial smooth muscle tension.
Tension measurements were performed on either epithelium-intact or
-denuded guinea pig bronchial smooth muscle spirals to test the effect
of either 5,6- or 11,12-EET on ASMs. The muscles were initially
precontracted with 0.2 µM CCh; after a plateau phase was reached,
various concentrations of 11,12-EET in ethanol were added. They
resulted in a slightly significant relaxation compared with the vehicle
(ethanol). To overcome the effects induced by ethanol as a solvent, a
second vehicle, ACN, was tested in parallel with the 5,6- and 11,12-EET molecules. Aqueous solutions of 0.01% ACN, used to dissolve EET regioisomers, had no effect on precontracted bronchi, as shown in Fig.
1A,
except at high, toxic concentrations (in the molar range). Thus the
dose-dependent relaxing effects of 11,12- and 5,6-EET dissolved in ACN
were assessed on precontracted bronchial strips after muscarinic
stimulations (Fig. 1, B and
C). These two regioisomers also had
relaxing effects on histamine-precontracted bronchi (as shown in Fig.
1, D and
D', respectively) or tracheal spirals (data not shown). 5,6-EET proved much more potent than the
11,12-isomer on both muscarinic- and histaminic-induced tensions. Hence
the effects of exogenous EETs were more important on epithelium-denuded strips (recording not shown).

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Fig. 1.
Effects of 2 regioisomers of epoxyeicosatrienoic acid (EET) on
precontracted guinea pig main bronchi. Epithelium-denuded bronchial
spirals were precontracted with either carbamylcholine chloride (CCh)
or histamine (Hist) and then challenged with various concentrations of
5,6- or 11,12-EET (nos. above traces) dissolved in 0.01% acetonitrile.
Arrows, addition of EET and vehicle or washout (W) with control Krebs
solution. A: control digitized
recording showing absence of effects of cumulative concentrations of
acetonitrile (vehicle used in subsequent experiments). A wide range of
concentrations up to 1 M, which is toxic for tissues, was tested.
B: dose-dependent relaxations induced
by cumulative addition of 11,12-EET (1-8 µM) dissolved in 0.01%
acetonitrile. C: dose-dependent
relaxations induced by 2 concentrations of 5,6-EET after addition of
vehicle, which had no effect, as in A.
D and
D': effects of 5,6-EET and
11,12-EET, respectively, on bronchial spirals precontracted with Hist.
Note that effects of EETs as well as those of muscarinic and histaminic
agonists were fully reversible.
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IbTX affects EET-induced relaxations.
Experiments were designed to test the concentration-dependent efficacy
of EETs on precontracted bronchi as well as the effect of IbTX, a
specific blocker of large-conducting KCa channels. Figure
2A'
shows that preincubation of bronchial strips with 10 nM IbTX affected
the amplitude and time course of the relaxing responses induced by 2 µM 5,6-EET compared with the control condition (Fig.
2A). Quantitative analysis of the data is summarized in Fig. 2, B and
C. The concentration-dependent effect
of EETs from 0.03 to 3 µM confirmed the greater potency of 5,6-EET
versus 11,12-EET (Fig. 2C) on
relaxation amplitude. Moreover, preincubation with 10 nM IbTX
consistently inhibited part of the relaxing responses induced by the
EET molecules, suggesting that activation of the
KCa channel might be
involved in the responses induced by 5,6- as well as by 11,12-EET.
However, IbTX was much more effective in inhibiting the relaxing
responses induced by 2 µM 11,12-EET than that caused by 2 µM
5,6-EET (Fig. 2, B and C). This observation would be
consistent with the idea that the effect of exogenous EETs might affect
other conductances or membrane processes. Because previous studies (9,
19, 22, 31) on VSMs have also shown that 11,12-EET induced relaxations
by acting on ionic conductances, subsequent experiments were performed
on unitary channel proteins with the PLB reconstitution technique to
ascertain the relevance of this mechanism in ASMs and to test whether
EET isomers can act directly on
KCa channels.

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Fig. 2.
Effects of iberiotoxin (IbTX) pretreatment on relaxing responses
induced by 5,6- and 11,12-EET after muscarinic stimulations.
A: consecutive and representative
recordings showing effects of the same concentration of 5,6-EET before
(A) and after
(A') pretreatment of tissues
with IbTX before CCh challenge. Arrows, addition of CCH, EET, and IbTX,
or W. Amplitude as well as time course of relaxations induced by
5,6-EET were both altered in presence of IbTX.
B: quantitative analysis of effect of
5,6-EET on amplitude of relaxation in absence (control) or presence of
IbTX, a specific blocker of large-conductance
Ca2+-activated
K+
(KCa) channels.
C: IbTX pretreatment also modifies
relaxing effects of 11,12-EET. Both isomers were dissolved in
acetonitrile. Data are means ± SE;
n = 4 and 5 experiments for each set
of experimental conditions. Note that 10 nM IbTX had a proportionally
larger effect on 11,12-EET-induced relaxation. * Significant
variation from respective control condition,
P < 0.05.
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Characterization of reconstituted KCa
channels from bovine ASMs.
KCa-channel activities were
characterized in asymmetrical (50/250 mM) or symmetrical
(250/250 mM) KCl buffer systems containing, initially, 10 µM free Ca2+. Single-channel
recordings in Fig.
3A show
the influence of holding potential on the behavior of a
large-conducting channel and its Po. The channel
Po-holding
potential relationship is best described by a Boltzmann equation (Fig.
3B) in a range of
60 to +60
mV in symmetrical conditions. The unitary current is activated by steady-state membrane depolarizations; the curve reaches a plateau above +30 mV.

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Fig. 3.
Voltage-dependent behavior of reconstituted single
K+ channels from bovine airway
smooth muscle (ASM). A: single-channel
current at different holding potentials. Traces were obtained in
symmetrical KCl buffer plus free
Ca2+, pH 7.4. O, open state; C,
closed state. Channel open probability
(Po) decreases
as holding potential changes from positive to negative values.
B: voltage dependence of channel
Po in symmetrical
condition. Data were fitted with a Boltzmann equation, where
Vm is membrane
potential, V1/2 is
holding potential when
Po = 0.5 and was
estimated at 43 mV, and K is a
constant (13.94).
Po values in
B were calculated by amplitude
histogram analysis and are means ± SE;
n = 6 experiments.
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The Ca2+ sensitivity of the
large-conducting K+-selective
channel (280 pS) was confirmed by varying the free
Ca2+ concentration
([Ca2+]) in the
trans chamber
([Ca2+]trans),
which generally corresponds to the cytoplasmic face of the channel, at
a constant holding potential (+20 mV) in symmetrical conditions.
Channel activity was recorded at various [Ca2+]trans
values ranging from 0.01 to 10 µM.
Po was reduced as
[Ca2+]trans
was lowered (Fig.
4A). The
average Ca2+ sensitivity of the
reconstituted channels was normalized and quantified (Fig.
4B). The
NPo-free
[Ca2+]trans
relationship was fitted with a Hill equation where the half-maximal
activation [dissociation constant
(Kd)] was
obtained at 0.7 µM free
[Ca2+]trans
with a Hill coefficient of 2.81, which suggests the existence of at
least three Ca2+-activating sites.
Thus the voltage and Ca2+
dependence of this large-conducting
K+-selective channel attest of its
identity as a KCa channel,
as previously reported (4, 32). A brief pharmacological
characterization of these
KCa channels with TEA and
IbTX, both known blockers of this type of channel (18, 20), was
performed. The addition of TEA to the
cis chamber of the bath induced a
block of the channel that significantly decreased the unitary current
amplitude (from 13 to 4 pA at 0 mV), without altering the channel
Po (Fig.
5A). The
results obtained after the addition of IbTX in the
cis chamber are shown in Fig.
5B; representative traces were
obtained in symmetrical condition at +20 mV, where 10 nM IbTX almost
completely blocked the channel activity. All data in Figs. 3-5 are
consistent with typical
KCa-channel characteristics
(20, 32).

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Fig. 4.
Ca2+ sensitivity of
large-conducting K+ channels from
bovine ASM. A: multiple-channel
recording at different intracellular free
Ca2+ concentration
([Ca2+]) levels.
Current traces were obtained in symmetrical KCl buffer as in Fig. 3 at
+20 mV. After cumulative additions of EGTA, free
[Ca2+]trans
was sequentially decreased from 10 to 0.6 µM; normalized mean
Po
(NPo, where
N is total no. of channels recorded
experimentally) was accordingly reduced from 1.0 to 0.05. B: dependence of channel
Po on
[Ca2+]. Data were
fitted with a Hill equation, where
Po max is
maximal Po and
Kd (dissociation
constant) is half-maximal activation obtained at 0.7 µM
[Ca2+] with Hill
coefficient (n) = 2.81. Po values for
B were calculated from amplitude
histogram analysis and are means ± SE;
n = 3-5 experiments.
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Fig. 5.
Effects of tetraethylammonium (TEA) and IbTX on bovine ASM
KCa channels reconstituted
in planar lipid bilayers. A: unitary
currents were recorded in asymmetrical KCl buffer with free
Ca2+ at 0 mV in control
(top) and after addition of TEA to
cis (extracellular) side
(bottom). Analysis indicated a
decrease in unitary current amplitude after application of drug.
B: representative traces obtained in
symmetrical KCl buffer as described above at +20 mV and in presence of
free Ca2+ in control
(top) and after addition of µM
IbTX to cis side
(bottom). Addition of IbTX at this
concentration resulted in a complete block of channel activity. Nos. on
right, apparent conductance ( ) and
Po in
A and
B, respectively.
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Activating effect of 11,12-EET on reconstituted
KCa channels.
KCa-channel recordings in
control conditions and after the addition of increasing concentrations
of 11,12-EET in the cis chamber (Fig.
6A)
demonstrated that cumulative 11,12-EET concentration ([11,12-EET]) enhanced channel
Po. A
concentration-dependent activation was consistently observed in a
narrow range of concentrations: the lowest concentration showing a
significant effect was 0.9 µM (P < 0.05), whereas 3 µM produced a fourfold increase in
Po (Fig.
6B). The activating effects of EET
molecules on KCa channels were observed at various voltages (data not shown). Of note is that the
concentrations required to increase the
Po of
reconstituted ASM KCa
channels remained higher than those determined from similar patch-clamp
studies on ASMs (9, 31). In addition, current-voltage curves in control
conditions and in the presence of various [11,12-EET] levels (Fig. 6C) show that the
eicosanoid had no effect on the unitary conductance because no change
of slope was observed.

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Fig. 6.
Effect of 11,12-EET on single-channel recordings and
concentration-dependent activation of reconstituted
KCa channels.
A: representative recordings of
KCa channels obtained in
symmetrical KCl buffer at +20 mV in low free
[Ca2+]trans
and after addition of cumulative concentrations of 11,12-EET on
cis side.
B: quantification of effect of
11,12-EET in same conditions as in A
on addition of cumulative concentrations of 11,12-EET.
Significant increases were obtained after addition of concentrations 0.9 µM 11,12-EET. C: current-voltage
curve in control conditions (symmetrical KCl buffer plus 0.6 µM free
Ca2+) and in presence of
indicated concentrations of 11,12-EET. 11,12-EET had no effect on
current amplitude and unitary conductance of
KCa channels. All data are
means ± SE; n = 5 experiments.
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Specificity and mode of action of EET molecules on
KCa channel.
It was reported that extracellular AA as well as G proteins activated
by intracellular GTP analogs might modify the gating of various
K+ channels including
KCa channels (29). The
results obtained from control experiments performed in the presence of
bioactive lipids and GTP analogs are shown in Fig.
7, A and
B, respectively. The
addition of AA (1-3 µM) on the
cis side had no effect on channel activity, although activation was observed for higher concentrations of
the EET precursor. In contrast, platelet-activating factor (PAF) had no
effect in the concentration range tested (3-10 µM) from the
cis side. This absence of effect of
PAF and the high concentrations of AA (>5 µM) required to
significantly enhance channel activity support the inherent and
specific potency of 11,12-EET added to the
cis side. Note that neither 0.6%
ethanol in the cis chamber nor 3 µM
11,12-EET in the trans chamber had any
effect on single-channel activity.

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Fig. 7.
Activating effects of 11,12-EET were compared with effect of 2 bioactive lipids, GTP and guanosine
5'-O-(3-thiotriphosphate)
(GTP S). A: quantitative analysis of
effects of arachidonic acid (AA) and platelet-activating factor (PAF).
Effects of these lipids were assessed on
KCa-channel activity and
compared with effects of various concentrations of 11,12-EET and
ethanol. Neither vehicle or 11,12-EET added to
trans chamber nor PAF had any effect
on channel behavior. However, AA, which had basically no effect at low
concentrations, activated
KCa channels at higher
concentrations (5-10 µM). All data are means ± SE;
n = 5, 4, and 3 experiments for each
set, respectively. B: results from
complementary experiments show that GTP and GTP S (up to 100 µM
trans) had no activating (or
synergistic) effect on
KCa-channel behavior
compared with direct activation induced by 1.5 µM 11,12-EET from
cis chamber. Average (±SE) values
were calculated from n = 5, 3, 3, and
4 experiments, respectively. *P < 0.05.
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It has been suggested that the effects of EET molecules in VSMs could
be due to an activation of G proteins (31). Because G proteins could be
present in our bilayers on fusion of native membrane vesicles, the
effects of GTP and its nonhydrolyzable analog GTP
S were tested on
ASM KCa channels alone and
in the presence of EET. Figure 7B
shows that GTP and GTP
S had no effect when added alone in the
trans chamber, corresponding to the
intracellular face of the channels. In addition, when 1.5 µM
11,12-EET was applied from the cis
chamber either before or after 100 µM GTP, the
KCa channels were activated
to an extent similar to that observed in the absence of GTP (Fig.
7B). These data strongly suggest
that EET molecules directly activated
KCa channels reconstituted
into PLB and that GTP had no direct or synergistic effect before or after EET addition. Note that, in this work, cholera toxin activation was not assessed due to technical (the large volume of the experimental chamber) and cost limitations. Because quantitative analysis
demonstrated that 11,12-EET had no direct effect on channel
Po when added to the trans (intracellular) side of the
channel protein (Fig. 7A), we
therefore postulate that 11,12-EET acts from the extracelluar side and
interacts with transmembrane segments or phospholipids within the
external bilayer leaflet to induce channel activation.
Multiple-channel activations by EET and blockage by
IbTX. In Fig. 8, the
effects of EET were further assessed on multiple-channel recordings; up
to three unitary current levels were observed in the presence of 1 µM
free Ca2+
trans (Fig. 8,
A and
A'). Addition of EGTA decreased
the free [Ca2+] to 0.3 µM and reduced channel activities (Fig. 8,
B and
B'), whereas addition of 3 µM
11,12-EET reactivated the functional channels present in the PLBs (Fig.
8, C and
C'). All three channels, which
were sensitive to voltage and
Ca2+, were sequentially blocked by
50 nM IbTX (Fig. 8, D and
D'). Altogether, these results
confirmed that EET molecules are able to reactivate all reconstituted
functional KCa channels,
even at low [Ca2+]
values and that this activation can be reversed by IbTX.

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Fig. 8.
IbTX blocked ASM KCa
channels activated by 11,12-EET. Multiple-channel recordings were
obtained in symmetrical KCl buffer at +30 mV under various experimental
conditions. A: in presence of free
Ca2+
trans.
B: at low free
Ca2+ concentration on addition of
EGTA. C: after addition of 11,12-EET,
channels reactivated in absence of
Ca2+ and pH changes.
D: addition of IbTX sequentially
blocked channel activity and thus reversed EET activation. Complete
inhibition by IbTX was obtained within 10 min.
A',
B',
C', and
D', corresponding amplitude
histograms calculated from 2-min data files of representative
steady-state recordings.
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Time analysis of a single KCa channel on
11,12-EET activation.
Figure 9 shows the open and closed time
distributions determined from a single-channel recording before and
after the addition of 11,12-EET. The distributions of the open or
closed events were fitted by either a single exponential
(left) or the sum of two exponentials (right), respectively.
The mean open time constant was increased by 53% on addition of 1.5 µM 11,12-EET, whereas the long closed time constant was subjected to
a fivefold decrease (231 ms in control condition and 46 ms in the
presence of EET). These results indicate that 11,12-EET affects the
gating behavior of the KCa
channel by reducing the duration of long closed events and stabilizing
the duration of the open events. This, in turn, resulted in an increase
in the average number of transition-by-time units, a lengthening of the
open events, and thus an increase in
Po values.

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Fig. 9.
Time analysis of single
KCa-channel activity before
and after addition of 11,12-EET. A:
open (left) and closed
(right) time distributions obtained
under control conditions in presence of free
[Ca2+]trans.
Distributions were fitted either by a single exponential or sum of 2 exponentials. op, Open time
constant; cl1, short closed
time constant; cl2, long closed
time constant. B: identical analyses
were performed on the same single
KCa-channel recordings
after addition of 11,12-EET. Value of mean open time constant was
increased by exogenous EET, whereas long closed time constant was
decreased fivefold.
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DISCUSSION |
This work is the first to report in vitro relaxations of muscarinic-
and histaminic-precontracted ASMs by EETs. This modulation appears to
be partly induced by a direct activation of
KCa channels by EETs, as
demonstrated by their sensitivity to IbTX. The most important
contribution of the present work was therefore to show that EETs
directly activate reconstituted
KCa channels. Although it
has previously been shown that all EET regioisomers have similar effects on K+ conductance (9), the
present work focused directly on the molecular mechanism of action of
two metabolites of the epoxygenase pathway, 5,6- and 11,12-EET.
The results of this study are in-line with recent reports showing the
modulation of smooth muscle tone by EETs on various mammalian arteries
(13-15, 19, 22, 34-36) as well as on guinea pig bronchi (40).
Moreover, it has been shown that EET molecules activate
K+ currents in VSMs (9, 22, 31).
Single-channel measurements have demonstrated an increase in the
Po of
KCa channels but failed to
provide precise information on the mechanism of action of the EETs (9).
In this study, pharmacomechanical measurements revealed the strong
relaxing effects of 5,6-EET and the concentration-dependent action of
5,6- as well as of 11,12-EET when dissolved in an ACN aqueous solution.
Because our data demonstrated a significant effect of 5,6- and
11,12-EET only on epithelium-denuded bronchi (Fig. 1,
A-D),
we suggest that the epithelial layer could play the role of a
biochemical barrier to exogenous EET molecules. However, ASM spirals
were much more sensitive to 5,6-EET than to 11,12-EET when low
concentrations of each regioisomer diluted in ACN were tested, as
demonstrated in Figs. 1, B and
C, and
2B. Similar differences in EET
sensitivities were also observed on histamine-precontracted strips
(Fig. 1, D and
D'). These results would argue
in favor of stereo- or regio-specific effects of EETs on ASM tone. This
is of interest because it was shown that EET isomers were produced in
various ratios by microsomal epoxygenases (10). Furthermore, tension
measurements revealed that 5,6- and 11,12-EET-induced relaxations were
both partially inhibited by IbTX pretreatment (Fig. 2,
B and
C), with a greater percentage of
inhibition induced by 10 nM IbTX on 11,12-EET- than on 5,6-EET-induced relaxations. Such results support a contribution of the
KCa channels to ASM
relaxation (4, 28, 29). On CCh-induced tonic contractions, the
KCa channels must display a
low Po despite
membrane depolarization and an increase in cytosolic free
[Ca2+]. In fact, it
has been demonstrated that the maintenance of ASM contraction involves
KCa-channel inhibition
(28), thus momentarily reducing the contribution of these channels in
the control of membrane potential and, consequently, in the control of
relaxation. Because 10 nM IbTX partly inhibits EET-induced relaxations
on precontracted strips, it was postulated that 5,6- as well as
11,12-EET might activate a cationic conductance, although other
membrane processes may also be involved.
Using the PLB reconstitution technique, we evaluated the modulation of
KCa channels by 5,6- and
11,12-EET after performing electrophysiological and pharmacological
characterizations of the channels. Single- and multiple-channel
reconstitutions were performed to evaluate channel sensitivities to
voltage and to the intracellular free
[Ca2+] (Figs. 3 and 4)
as well as to known blockers such as TEA and IbTX (Fig. 5). We then
investigated the direct action of exogenously added 11,12-EET to the
cis (extracellular) compartment (Fig.
7), thus mimicking the physiological release of EETs by epithelial cells and/or other lung cells. These results are consistent
with a direct activation of
KCa channels because they
have been obtained in the absence of GTP, ATP, cAMP, or other
metabolites in the trans chamber. A
direct activation implies interaction of EET molecules with channel
proteins or neighboring proteins or with phospholipids, without the
implication of intracellular cascades such as the activation of G
proteins. Li and Campbell (31) recently reported that EETs activated
KCa channels, presumably
via the stimulation of a Gs
protein that could, in turn, modulate channel gating. They postulated
that EETs could bind to a receptor, activating the
Gs proteins, or act directly on
the Gs proteins themselves. The
activated G proteins, hence, appeared to affect
KCa channels without the
involvement of second messengers (cAMP and cGMP) or kinases such as
protein kinases A and G, two kinases known to directly phosphorylate
KCa channels (3, 27, 30).
The present results do not support and basically refute the requirement
for the involvement of a G protein because GTP had no stimulating action or synergistic effect on reconstituted ASM
KCa channels, whereas EET
molecules were still very effective. Hence we suggest that, in ASM, the
EETs tested can alter K+ efflux
via a direct interaction with the
KCa channel.
The lowest concentration of EET inducing a significant activation was
0.9 µM (P < 0.05), whereas a
fourfold activation was obtained with the addition of 3 µM 11,12-EET.
These results suggest a lower sensitivity of the reconstituted ASM
channels to 11,12-EET than that previously reported for arterial
KCa channels where as
little as 100 nM 11,12-EET induced a 10-fold
NPo activation (31). The lower sensitivity of the ASM
K+ channels may be an artifact of
the reconstitution technique. PLBs are made of very specific
phospholipid composition and completely eliminate intracellular
cascades. Nevertheless, one cannot rule out the possibility of a dual
activation or multiple regulation of ASM
KCa channels, already
foreseen as multisensor ionic channels, being sensitive to voltage,
intracellular free
[Ca2+],
phosphorylation (3, 27), oxydo-reduction, and nitric oxide (4).
Complementary experiments have also demonstrated that PAF and low
concentrations (
3 µM) of AA, the precursor of 11,12-EET in vivo,
had no direct effect on the reconstituted
KCa channel unless much
larger AA concentrations were used. Hence it appears that the epoxide
group of the 11,12-EET molecule is important in the direct modulation
of ASM KCa channels. All
experiments involving various [11,12-EET] levels
demonstrated that the molecules did not affect the unitary conductance
of the KCa channels (Fig. 6C), which is in agreement with the
results of previous work (9, 31). Meanwhile, we report the first
quantitative time analysis of
KCa-channel gating on
11,12-EET activation, which demonstrates an increase in mean open time
and a reduction in the duration of the long closed state (Fig. 9).
Finally, control experiments showed that vehicle alone as well as
trans (intracellular) application of
11,12-EET did not affect
KCa-channel behavior (Fig.
7). The side-specific effect of EETs argues in favor of the presence of activation sites on the extracellular face of the
KCa-channel proteins to
which these highly hydrophobic molecules could bind.
Recently, it was reported that a high-affinity binding site for
14(R),15(S)-EET
was present in guinea pig mononuclear cell membranes (39). Other
authors (31) have postulated the existence of a binding site for
11,12-EET on bovine coronary arterial cell membranes. It is also known
that EETs bind to the phospholipids of the surface membrane (6).
Further investigation is necessary to determine whether specific
binding sites are present on the KCa channel or on
functional neighboring proteins such as their
-subunit in ASM cell
membranes or whether EET molecules only bind to particular specific
phospholipid acyl chains or membrane microdomains. Interestingly, it
has been reported that all four EET regioisomers activate
high-conductance, Ca2+-dependent
K+ channels on pig coronary
arterial endothelial cells (15). This possibility remains to be proven
for other cell membranes of the respiratory tract.
In conclusion, EETs induced dose-dependent relaxations of ASM in vitro,
which were partially inhibited by IbTX and partly by a direct
activation of the KCa
channels present in the sarcolemmal membrane of ASM cells. The evidence
reported herein reveals a direct activation of ASM
KCa channels, unlike that
reported in a study (31) performed on coronary arteries. Moreover, we
have shown that 11,12-EET affects the gating but not the unitary
conductance of KCa
channels. It is reasonable to postulate the presence of a specific
extracellular binding site on the channel proteins or within
surrounding phospholipid microdomains. Note that the present study does
not rule out the possibility that EET molecules affect other
conductances. Because 11,12-EET induces an increase in
KCa-channel activity, it
should cause a repolarization or hyperpolarization of ASM cells. We
therefore propose that EETs represent potential endothelium-derived
hyperpolarizing factors in ASM. This hypothesis is
further tested in the companion paper (38).
 |
ACKNOWLEDGEMENTS |
We thank Drs. Paul Pape and E. Ruiz Petrich for discussing and
revising the manuscript.
 |
FOOTNOTES |
This work was supported by Grant MT 12287 from the Medical Research
Council of Canada and by the Association Pulmonaire du Québec.
E. Rousseau is a Senior Fonds de la Recherche en Santé du
Québec (FRSQ) Scholar. D. Salvail is a recipient of an FRSQ-Fonds pour la Formation de Chercheurs et d'Aide à la Recherche Student Fellowship. Both laboratories are members of the Health Respiratory Network of the FRSQ.
Address for reprint requests: E. Rousseau, Le Bilarium, Department of
Physiology and Biophysics, Faculty of Medicine, Univ. of Sherbrooke,
3001, 12th Ave. North, Sherbrooke, Quebec, Canada J1H 5N4.
Received 29 October 1997; accepted in final form 1 May 1998.
 |
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