Le Bilarium, Department of Physiology and Biophysics, Faculty of Medicine, University of Sherbrooke, Sherbrooke, Quebec, Canada J1H 5N4
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ABSTRACT |
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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
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INTRODUCTION |
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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.
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MATERIALS AND METHODS |
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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 M) 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].
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RESULTS |
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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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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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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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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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DISCUSSION |
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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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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.
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