Le Bilarium, Faculty of Medicine, Department of Physiology and Biophysics, University of Sherbrooke, Sherbrooke, Quebec, Canada J1H 5N4
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ABSTRACT |
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Epoxyeicosatrienoic
acids (EETs) are produced from arachidonic acid via the cytochrome
P-450 epoxygenase pathway. EETs are able to modulate
smooth muscle tone by increasing K+ conductance, hence
generating hyperpolarization of the tissues. However, the molecular
mechanisms by which EETs induce smooth muscle relaxation are not fully
understood. In the present study, the effects of EETs on airway smooth
muscle (ASM) were investigated using three electrophysiological
techniques. 8,9-EET and 14,15-EET induced concentration-dependent
relaxations of the ASM precontracted with a muscarinc agonist
(carbamylcholine chloride), and these relaxations were partly inhibited
by 10 nM iberiotoxin (IbTX), a specific large-conductance
Ca2+-activated K+ (BKCa) channel
blocker. Moreover, 3 µM 8,9- or 14,15-EET induced hyperpolarizations
of 12 ± 3.5 and
16 ± 3 mV, with EC50 values of 0.13 and 0.14 µM, respectively, which were either reversed or
blocked on addition of 10 nM IbTX. These results indicate that BKCa channels are involved in hyperpolarization and
participate in the relaxation of ASM. In addition, complementary
experiments demonstrated that 8,9- and 14,15-EET activate reconstituted
BKCa channels at low free Ca2+ concentrations
without affecting their unitary conductance. These increases in channel
activity were IbTX sensitive and correlated well with the
IbTX-sensitive hyperpolarization and relaxation of ASM. Together these
results support the view that, in ASM, the EETs act through an
epithelium-derived hyperpolarizing factorlike effect.
epoxyeicosatrienoic acids; bronchorelaxation; membrane potential; potassium conductance; eicosanoids; planar lipid bilayers; epithelium-derived hyperpolarizing factors; large-conductance calcium-activated potassium channel
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INTRODUCTION |
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THE
METABOLISM OF ARACHIDONIC ACID (AA) in the respiratory tract
system has been well investigated with regard to lipoxygenase and
cyclooxygenase (COX) pathways that, respectively, produce leukotrienes
(LT) and prostaglandins (PG) (19, 28, 36). Certain
products of both lipoxygenase (LTC4, LTD4,
LTE4) and COX (PGD2, PGF2,
thromboxane A2) induce potent bronchocontriction associated with pathophysiological conditions such as asthma
(37). In contrast, other COX products (PGE2,
PGI2) as well as nitric oxide (NO) synthase (NOS) products
are considered as relaxing agents in the vascular system
(32).
Despite the reported presence of cytochrome P-450 (CYP450)
monoxygenases in liver (21, 22, 39), kidney (21,
31), and lung (11, 23, 43) of many species such as
human, rabbit, mouse, hamster, and guinea pig, the role of the third AA
pathway is less understood. This pathway involves -hydroxylase and
the NADPH-dependent epoxygenase enzymes, which respectively produce hydroxyeicosatetranoic acids (HETEs) and the four epoxyeicosatrienoic acid regioisomers (5,6-, 8,9-, 11,12-, and 14,15-EET) (8, 9, 19,
28, 36). The physiological roles and modes of action of these
bioactive compounds have yet to be fully elucidated. Recent studies in
vascular smooth muscle (VSM) have shown that EETs can dilate
renal, cerebral, and coronary arteries by acting as an
endothelium-derived relaxant factor (EDRF) (7, 16, 29, 30,
40). The four EET regioisomers produce vasorelaxation by
causing membrane potential hyperpolarization in VSM cells (3, 13,
14). Hyperpolarizing effects do not involve either COX or
NOS-dependent pathways but rather CYP450 epoxygenase metabolites (15). Indeed, there is good evidence that these effects
could be mediated in the vascular bed by an EET-dependent mechanism. The activation of large-conductance Ca2+-activated
K+ (BKCa) channels by the EETs
(17) reveals their possible roles as an
endothelium-derived hyperpolarizing factor (EDHF).
The presence of CYP450 epoxygenase and EET production in epithelial and
Clara cells of the airways has been verified (11). Recently, Scarborough et al. (35) also reported the
expression of a CYP2J2 isoform in non-Clara cells from human and rat
lung epithelium. Investigation of the possible roles of EETs in airway smooth muscle (ASM) revealed that 5,6- and 8,9-EET relax precontracted rabbit bronchial tissues (43) and can directly activate
BKCa (12) and inhibit
Cl-selective channels (33). Despite this,
few studies have focused their attention on the efficiency of EETs as
relaxing factors and their exact mode of action in airways. Despite the
fact that there were only a few reports demonstrating variations in the cellular responses to the different isomers (44), the
rationale for the present work was based on the observations made by
this laboratory that the EETs might have a different order of potency in the airways than in the vascular bed (6).
This study was aimed at assessing the electrophysiological effects of EETs on ASM at the tissular, cellular, and molecular levels. Three complementary approaches were used: 1) tension measurements of relaxation on epithelium-denuded guinea pig ASM precontracted with a muscarinic agonist, 2) membrane potential measurements using the classical microelectrode technique and quantification of the pharmacological effects of the EETs, and 3) analyses of the direct activating effects of two EETs on unitary BKCa channels reconstituted into planar lipid bilayer. We demonstrate that 8,9- and 14,15-EET induced concentration-dependent and iberiotoxin (IbTX)-sensitive relaxations as well as a hyperpolarization of the resting membrane potential of ASM from tracheae of rabbits. These effects were correlated with a direct activation of BKCa channels from ASM cells as shown by single-channel analysis.
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MATERIALS AND METHODS |
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Tension measurements. Albino guinea pigs (Hartley, weighing 300-350 g) were anesthetized with a lethal dose of pentobarbital sodium (50 mg/kg). The trachea and lungs were quickly removed and placed in an oxygenated (95% O2-5% CO2) Krebs-bicarbonate solution at room temperature containing (in mM) 118.1 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 12.5 NaHCO3, and 11.1 dextrose, pH 7.4 ± 0.05 on bubbling. The trachea and external main bronchi were then isolated from the lung and cut helically as described previously (5). The epithelial cells were mechanically removed. Each tissue was placed in a 4-ml jacketed organ bath containing Krebs-bicarbonate solution at 37°C. Changes in isometric tension were measured with a Grass polygraph (model 7D). Before the experiments were started, the tissues were subjected to an initial loading tension of 1 g and then allowed to equilibrate for 60 min, with replacement of the Krebs-bicarbonate solution in the bath every 15 min. At the end of the equilibration period, the tissues were precontracted with 0.2 µM carbamylcholine chloride (CCh), followed by the addition of EETs or vehicle to the bath. The cumulative concentration-response curves of the various compounds are expressed as percentage of relaxation.
Electrophysiological recording.
Male albino rabbits (1.5-2.5 kg) were anesthetized with
pentobarbital sodium (35 mg/kg). The trachea was rapidly removed and placed in oxygenated (95% O2-5% CO2) Tyrode
solution at room temperature containing (in mM) 136 NaCl, 4 KCl, 1.8 CaCl2, 0.5 MgCl2, 0.35 Na2HPO4, 12.5 NaHCO3, and 11 dextrose, pH 7.4 ± 0.05. A longitudinal section was made to
expose the luminal face of the trachea. The epithelium was mechanically
removed by delicate rubbing of the surface with a cotton applicator,
and the tissues were cut into strips 10-15 mm long and 5 mm wide.
The strips were affixed with the ASM facing up in the middle chamber
(capacity 3 ml) of a tricompartment system in which the temperature
(37°C) and solution level were strictly controlled as previously
described (33, 36). The tissues were perfused at a
constant flow rate of 2 ml/min with standard (or modified whenever
needed) Tyrode solution and were allowed to equilibrate for 20 min
followed by another 20 min with 5 µM wortmannin to prevent smooth
muscle cell contractures at the time of impalement. Nonexperimental
tissues were conserved at room temperature in oxygenated Tyrode
solution for several hours. The transmembrane potential was measured by
ASM cell impalement from the luminal side using conventional
intracellular borosilicate microelectrodes filled with 3 M KCl. Their
electrical resistance ranged from 20 to 50 M. The microelectrodes
were connected via a Ag-AgCl2 pellet to the head stage of
an amplifier mounted on a Narishige No13004 micromanipulator.
Measurements were performed with a KS-700 amplifier from World
Precision Instruments. Electrical signals were continuously monitored
on an oscilloscope (Tektroniks, TDS 310). The membrane potentials were
recorded using a Digidata 1200B interface and Axoscope 7.0 software
(Axon Intruments). Data were stored on disk for further analysis.
Preparation of tracheal smooth muscle microsomal fractions and channel reconstitution. Preparation of bovine crude ASM microsomal fractions and planar lipid bilayers (PLB) was carried out exactly as described previously (12, 34). Two chambers, denoted cis and trans, were separated by a septum with a 250-µm-diameter hole. The hole was pretreated with a mixture of phospholipids (25 mg/ml chloroform): phosphatidylethanolamine-phosphatidylserine-phosphatidylcholine in a ratio of 3:2:1. The same mixture of phospholipids dissolved in decane was used to form the PLB. The membrane vesicles (10-60 µg of proteins) were added in the cis chamber, which was connected to the head stage of voltage-clamp amplifier (model 8900; Dagan, Minneapolis, MN), whereas the trans chamber was connected to virtual ground, both by means of low-resistance electrodes (MERE 2; World Precision Instruments, Sarasota, FL). To facilitate the fusion, the experimental chambers contained the following solution (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. However, most current traces were recorded in symmetrical condition, if not specified otherwise. The unitary currents were filtered at 1 kHz and recorded on videotape (DAS/VCR 900 Toshiba, Unitrade) and then digitized at 4 kHz for storage in 3-min files on hard disk. All reconstitution studies were performed at room temperature (22 ± 2°C). BKCa channel activities were analyzed in terms of current amplitudes and channel open probability (Po) values with pCLAMP 6 (Axon instruments).
Drugs and chemical reagents. CCh, IbTX, and wortmannin were all purchased from Sigma (St Louis, MO). 5,6-EET dissolved in 100% acetonitrile (ACN) and 11,12-, 8,9- and 14,15-EET dissolved in 100% ethanol were purchased from Cayman Chemical (Ann Arbor, MI) and stored as 312 µM stock solutions. All phospholipids were obtained from Avanti Polar Lipids (Albaster, AL). For microelectrode measurements, EETs and IbTX were dissolved in Tyrode solution at the required concentration, and wortmannin was dissolved and stored in DMSO. For each experimental procedure, the vehicles were tested separately at the maximal concentration used in the presence of active compound.
Data analysis and statistics.
The concentration-response curves were fitted to the Hill equation
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RESULTS |
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Relaxing effect of EETs.
Epithelium-denuded guinea pig bronchial smooth muscle spirals were used
to assess the relative and putative effects of the four EETs on ASM
tension. The tissues were initially precontracted with 0.2 µM CCh,
and once the plateau phase was reached, cumulative concentrations of
EETs were added. As shown in Fig. 1, all
EET regioisomers had relaxing effects, with 5,6-EET being the most potent isomer. However, since the EETs were dissolved in either ACN
(5,6-EET) or ethanol (8,9-, 11,12-, and 14,15-EET) and considering the
concentration-dependent effect of ACN (Fig. 1, inset), the specific effect of the 5,6-EET was relatively small after the solvent
effect was subtracted. Hence 8,9- and 14,15-EET exhibited greater net
relaxing effects, inducing relaxation of 42% and 82% at the maximal
10 µM concentration tested (Fig. 1, solid symbols).
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IbTX sensitivity of EET-induced relaxations.
Figure 2A shows the effects of
pretreating the bronchial strips with 10 nM IbTX, a specific blocker of
BKCa channels on ASM responsiveness to EETs. The ASM
spirals were pretreated with 10 nM IbTX before muscarinic stimulation
(CCh), which resulted in a significant inhibition of the EET-induced
relaxation. More specifically, the relaxations induced by 3 µM 8,9-, 11,12-, and 14,15-EET were reduced by 45, 45, and 47%, respectively
(Fig. 2B). Note that IbTX had partial (17%) inhibitory
effects on the relaxation induced by 3 µM 5,6-EET. The IbTX
sensitivity of the relaxations induced by the other EET regioisomers
appeared very similar, which strongly supports the role of
BKCa channels in the control of the relaxation process
induced by EETs. Thus the present results confirm and complement the
observations reported previously on the same preparation (12).
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Effect of EETs on ASM membrane potential.
The effects of 8,9- and 14,15-EETs were tested on membrane potential.
Impaled microelectrodes were used with tracheal epithelium-denuded rabbit ASM. Because of tissue vibrations, cell depolarization, and/or
rupture on impalement, continuous recordings were very difficult to
obtain and a multi-impalement protocol consisting of repetitive
penetrations in single ASM cells was designed. In control conditions,
the experimental chamber was continuously perfused with normal Tyrode
solution (37°C), and the mean membrane potential of
epithelium-denuded cells was measured at 50 ± 1.7 mV. The
addition of 8,9-EET consistently induced a hyperpolarization of the
membrane potential (Fig. 3A).
Concentration-response curves (Fig. 3B) were obtained on
cumulative addition of EETs (0.01-5 µM); 8,9- and 14,15-EET had
maximal hyperpolarizing effects of
12 ± 3.5 and
16 ± 3.0 mV, respectively. Corresponding EC50 values of 0.13 and
0.14 µM were derived from the sigmoidal response curves obtained.
These EET-induced hyperpolarizing effects were fully reversible on
washout of the exogenous EETs with normal Tyrode solution for 20 min.
The vehicle (ethanol) had basically no effect on the membrane potential
when tested at the same concentration used in the presence of the EETs
except at the higher concentration tested (Fig. 3B, solid
squares). Overall, ASM membrane potential was much more sensitive
to the actions of EETs than tissue isometric tension.
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IbTX sensitivity of hyperpolarizations induced by EETs.
To test for the involvement of BKCa channels on the robust
hyperpolarizations induced by both EETs tested, two protocols were used: IbTX pretreatment before EET challenges and IbTX reversal of
EET-induced hyperpolarizations. IbTX alone had no significant effect on
the membrane potential (49.9 ± 2 mV) as illustrated in Fig.
4A. The data reveal that 10 nM
IbTX completely prevented the hyperpolarizations normally induced by
the two EET isomers; subsequent addition of the EC50 doses
of 8,9- and 14,15-EET to the perfusion chamber did not cause any
hyperpolarization of the membrane potential. On the other hand, 8,9- and 14,15-EET (3 µM) induced membrane potential hyperpolarizations
that were completely reversed by subsequent addition of 10 nM IbTX to
the perfusion medium (Fig. 4B). Both IbTX effects attest to
the role of BKCa channels in the EET-induced
hyperpolarizations. However, IbTX effects on ASM relaxation by the EETs
demonstrate that BKCa activation by the latter only
accounted for one-half of the relaxation, suggesting the involvement of
other pathways in the control of the relaxation process elicited by
EETs.
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Effect of EETs on activation of reconstituted BKCa
channels in PLB.
To assess the effects of 8,9- and 14,15-EET on channel activity,
BKCa channels were reconstituted in a symmetrical (250/250 mM) KCl buffer system containing, initially, 10 µM free
Ca2+ concentration ([Ca2+]).
[Ca2+] was reduced on addition of EGTA to the trans
chamber (cytoplasmic side of the channel), and channel open
probability (Po) decreased due to its
Ca2+ sensitivity. Figure
5A illustrates representative
single BKCa channel recordings in control conditions (+30
mV, 0.6 µM free [Ca2+ ]) and following
sequential additions of increasing concentrations of 8,9-EET in the
cis chamber. These current recordings show that increasing
concentrations of 8,9-EET enhanced BKCa activity (Fig. 5A). Similar observations were made with 14,15-EET (Fig.
6A), which demonstrated the
capacity of this eicosanoid to also activate BKCa channels
in a concentration-dependent manner but with a lower effectiveness. The
lowest concentration displaying a significant effect was 1.5 µM in
each case, which nearly doubled the Po of the
channel (Fig. 6B). At the maximal concentration tested (10 µM), 8,9- and 14,15-EET caused an increase in
Po from nearly zero to 0.70 ± 0.09 and
0.09 ± 0.03, respectively. The activating effects of 8,9- and
14,15-EET were not voltage dependent over the range of potentials
tested (60 to 60 mV; data not shown). They were observed at negative
potentials compatible with the resting membrane potential of ASM under
physiological conditions. For instance, at
50 mV, in control
conditions (low free [Ca2+]), the
Po value increased from 0.07 to 0.32 in the
presence of 6.0 µM 8,9-EET (data not illustrated). Current-voltage
curves obtained in control conditions and in the presence of increasing concentrations of 8,9- or 14,15-EET (Figs. 5C and
6C) reveal that EETs had no effect on current amplitudes and
unitary conductances.
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IbTX sensitivity of EET activation of the BKCa channel.
Figure 7A shows single-channel
recordings with a low Po (0.02) in the presence
of 0.5 µM free [Ca2+]. Subsequent addition of 3 µM
8,9-EET enhanced the activity of this channel, an effect that was
completely blocked on addition of 10 nM IbTX on the extracellular side
of the channel. Similar results were obtained in the presence of 6 µM
14,15-EET and 10 nM IbTX (Fig. 7B). Thus both EETs activate
the BKCa channel at low free [Ca2+], an
effect completely blocked by IbTX.
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Effects of EETs in the presence of NOS and COX inhibitors.
Figure 8 shows the effects of 100 µM
NG-nitro-L-arginine methyl ester
(L-NAME; a NOS inhibitor) and 1 µM indomethacin
(a COX inhibitor) used alone and combined. There is basically no
significant effect of these inhibitors on the membrane potential of
epithelium-denuded ASM cells. In the presence of both inhibitors, the
addition of either 8,9- or 14,15-EET (3 µM) induced a
hyperpolarization of 5 and
8 mV, respectively. These results
demonstrate that, in the presence of NOS and COX inhibitors, exogenous
EETs have electrophysiological effects with the hallmark of
epithelium-derived hyperpolarizing factor (EpDHF). However, we
must consider that the effects of both isomers were smaller than their
effects measured at the same concentration (3 µM) in the absence of
inhibitors (Fig. 3B). Together these results suggest that
the EETs might also influence other intracellular enzymatic systems.
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DISCUSSION |
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In this work, we investigated the ability of four EET regioisomers
to relax tissues precontracted by muscarinic stimulation. This
relaxation was associated with a hyperpolarization of ASM cells via an
IbTX-sensitive mechanism. Using complementary experimental procedures,
we have shown that 8,9- and 14,15-EET induced cellular hyperpolarizations, which in turn resulted from BKCa
channel activation and were completely blocked or reversed by the
application of 10 nM IbTX. These results obtained on ASM are consistent
with those from other reports on VSM in which the EETs were considered as "EDHFs" [see Hecker (18) for a recent review].
Moreover, it has been shown that 5,6- and 11,12-EET were able to
directly activate BKCa channels (12) and
inhibit Cl channels (33) from bovine ASM.
Although the mechanisms by which EETs produce ASM relaxation remain to
be clarified, we have shown that the eicosanoids, produced in vivo by
CYP450 epoxygenase, induce a consistent hyperpolarization involving
BKCa channel activation. More precisely, this work focuses
on the mechanism of action of the two enantioisomers, 8,9- and
14,15-EET, and proposes that these molecules can play the role of EpDHF
in the respiratory tract.
Isometric tension measurements of epithelium-denuded precontracted ASM
reveal that the potency order of the four EETs is 14,15- > 8,9- > 11,12- 5,6-EET, with 14,15-EET being the most potent EET
regioisomer. This result is different from that obtained in VSM, in
which 11,12-EET is recognized as the most potent isomer (6). Despite the fact that a direct action of EETs has
been reported in the literature, such as the direct inhibition of
cardiac L-type Ca2+ channels (10), the
presence of a membrane receptor remains to be verified. Some reports
have highlighted the endogenous production of EETs by airway epithelium
and ASM cells (19, 23, 42). In this study, we assessed the
effects of exogenous EETs on ASM and therefore used epithelium-denuded
tracheae and bronchia to prevent the production of other or additional
endogenous modulators by the epithelial cells. Thus no epoxygenase
inhibitor was used in the present study. Previous work had revealed
that exogenous application of 5,6- and 11,12-EETs had no significant
relaxing effects on ASM in the presence of intact epithelium
(12). In vivo, agonist stimulation of the epithelium could
result in endogenous EET release and therefore in the modulation of ASM
tone. The partial inhibition by IbTX of the EET relaxation reveals the
implication of K+ conductances in the control of this relaxation.
The intracellular microelectrode technique, using a multiple-impalement
protocol, revealed that both 8,9- and 14,15-EET induced significant
concentration-dependent hyperpolarizations of ASM cells, with
EC50 values of 0.14 and 0.13 µM, respectively. The present results can also be compared with the previous membrane potential measurements performed with 5,6- and 11,12-EET (0.75 µM),
which induced 5- to
3-mV hyperpolarizations, respectively (33). Thus the 8,9- and 14,15-EET isomers were slightly
more potent than the two regioisomers previously tested
(12). Because IbTX prevented or completely abolished the
hyperpolarizing effects induced by both EETs, BKCa channel
activation appears to be a key determinant in the hyperpolarization
process. However, the difference in IbTX sensitivity between
hyperpolarization and relaxation measurements is consistent with the
involvement of other mechanisms in the regulation of muscle tone. In
VSM, charybdotoxin (a less selective BKCa channel blocker)
has been shown to inhibit hyperpolarizations induced by bradykinin,
which corresponded to an EDHF effect, since it was observed in the
presence of L-NAME and indomethacin, specific inhibitors of
the NOS and COX pathways, respectively (2). We are now
reporting that, in ASM cells, EETs have hyperpolarizing effects (Fig.
8) in the presence of NOS and COX inhibitors. In human coronary
arterioles, the EDHF effect was insensitive to 1 µM glibenclamide (an
ATP-sensitive K+ channel blocker) and to 0.1 µM apamin (a
small-conductance KCa channel blocker) (27).
These observations support the fact that BKCa channels are
major effectors of EDHF and therefore probably of EpDHF
(27).
On the other hand, membrane reconstitution of BKCa channels
allowed the evaluation of direct effects of EETs on the
BKCa single-channel properties. 8,9-EET and 14,15-EET were
added to the extracellular side of the channel to mimic their
physiological release by epithelial cells and/or other lung cells
(4). Moreover, the sidedness of the effects of EETs on
BKCa channels, reconstituted in PLB, was previously
assessed by our group and revealed that EETs had no effect on the
channel when added to the trans side (intracellular face) of
the channel (12). Both EETs induced a
concentration-dependent activation of BKCa channels,
without affecting current amplitudes and unitary channel conductances.
This partial activation of the reconstituted channels proves that the
bioactive molecules tested herein had a direct effect on
BKCa channels. One has to consider that the
trans chamber (intracellular face) did not contain GTP, ATP,
cGMP, cAMP, or other metabolites that could induce or be involved in
indirect channel activation. Thus the direct effects of EETs on the
channel could be induced by interaction of these eicosanoids with
either the - or
-subunits of the BKCa channel with
neighboring proteins or membrane phospholipids. For instance, it has
recently been demonstrated in an heterologous expression system that
estradiol binding to the
-subunit of hSlo induced an acute
activation of BKCa channel (38). Other reports
revealed that EETs induce BKCa channel activation by
stimulation of a Gs protein that modulates channel gating
(25). However, these EET-induced BKCa channel
activations by intracellular mechanisms remain controversial; a recent
study in VSM showed that EET could modulate the channel activity via a
cAMP-dependent protein kinase (PKA)-dependent signaling pathway
(20). In ASM it has been shown that BKCa
channels could be activated by either PKA-dependent or cGMP-dependent
protein kinase-dependent phosphorylations (1, 24,
34). However, to date, our results do not demonstrate the
existence of a specific intracellular mechanism explaining
BKCa channel activation by EETs. Such a possibility could
yield a plausible explanation for the differences in EET sensitivity at
the tissular and molecular levels; the results reported here suggest
the existence of specific binding sites on cell membrane proteins
and/or interactions with membrane lipid components. The existence of
specific binding sites for 14(R),15(S)-EET have
been reported in mononuclear cells (41) and for 11,12-EET
in cell membrane of cerebral vascular beds (26). Further
investigations will be required to verify the existence of an EET
receptor on ASM cells. For practical purposes, we used three different
animal species; there is no evidence that the role of EET in modulating
ASM tone is significantly different in these mammalian species. In
fact, the evidence gathered from various reports has clearly shown that
EETs have similar effects in several species (12, 43).
Aside from the mechanisms by which the EETs induce ASM relaxation, another aim of this study was to assess whether or not the EETs could act as an EpDHF. Based on the accepted definition of an EDHF (18), a candidate molecule must be able to modulate smooth muscle tone via a hyperpolarization of the membrane potential through BKCa channel activation, which should be independent from NO and prostaglandin production. The results obtained in the present work reveal that EET isomers induce ASM relaxation mainly via membrane hyperpolarizations induced by direct BKCa activation. Thus the mechanistic characteristics of EET relaxation, hyperpolarization, and BKCa activation are consistent with a role of the EETs as EpDHF in the airways.
In summary, both EET isomers induced concentration-dependent relaxing effects on ASM, which were related to hyperpolarizations and were likely due to BKCa channel activation. These eicosanoids were able to directly activate reconstituted channels, although the quantitative analysis, derived from the partial IbTX-inhibitory effects on tension measurements, suggests that a second intracellular mechanism could also be operable. Altogether, our data support the view and bring new evidence that, in the airway, EET regioisomers may behave as EpDHF-like agents.
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ACKNOWLEDGEMENTS |
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We are indebted to Sophie B. Gaudreault and Dr. Alain Cadieux for assistance with the pharmacological measurements.
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FOOTNOTES |
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This study was supported by a Medical Research Council of Canada Grant MT-15173.
D. Salvail is the recipient of a student fellowship from the Fonds de la Recherche en Santé du Québec (FRSQ)-Fonds pour la Formation de Chercheurs et d'Aide à la Recherche. E. Rousseau is a National Scholar and member of the Health Respiratory Network of the FRSQ.
Address for reprint requests and other correspondence: E. Rousseau, Dept. of Physiology and Biophysics, Faculty of Medicine, Univ. of Sherbrooke, 3001, 12th Ave. North, Sherbrooke, Quebec, Canada J1H 5N4 (E-mail: erouss01{at}courrier.usherb.ca).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 2 May 2000; accepted in final form 8 November 2000.
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