20-HETE inotropic effects involve the activation of a nonselective cationic current in airway smooth muscle
Martin Cloutier,
Shirley Campbell,
Nuria Basora,
Sonia Proteau,
Marcel D. Payet, and
Eric Rousseau
Faculty of Medicine, Department of Physiology and Biophysics, University
of Sherbrooke, Sherbrooke, Quebec, Canada J1H 5N4
Submitted 8 November 2002
; accepted in final form 5 May 2003
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ABSTRACT
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20-Hydroxyeicosatetraenoic acid (20-HETE) controls several mechanisms such
as vasoactivity, mitogenicity, and ion transport in various tissues. Our goal
was to quantify the effects of 20-HETE on the electrophysiological properties
of airway smooth muscle (ASM). Isometric tension measurements, performed on
guinea pig ASM, showed that 20-HETE induced a dose-dependent inotropic effect
with an EC50 value of 1.5 µM. This inotropic response was
insensitive to GF-109203X, a PKC inhibitor. The sustained contraction,
requiring Ca2+ entry, was partially blocked by either
100 µM Gd3+ or 1 µM nifedipine, revealing the
involvement of noncapacitative Ca2+ entry and L-type
Ca2+ channels, respectively. Microelectrode measurements
showed that 3 µM 20-HETE depolarized the membrane potential in guinea pig
ASM by 13 ± 2mV(n = 7), as did 30 µM
1-oleoyl-2-acetyl-sn-glycerol. Depolarizing effects were also
observed in the absence of epithelium. Patch-clamp recordings demonstrated
that 1 µM 20-HETE activated a nonselective cationic inward current that may
be supported by the activation of transient receptor potential channels. The
presence of canonical transient receptor potential mRNA was confirmed by
RT-PCR in guinea pig ASM cells.
20-hydroxyeicosatetraenoic acid; calcium; isometric tension; membrane potential; transient receptor potential; nonselective cationic current
THE AIRWAY SMOOTH MUSCLES (ASM) contract in response to various
agonist stimuli, involving the activation of G protein-coupled receptors
(16). These inotropic
responses are modulated by membrane depolarization, which relies on ion
channel activation and conductance changes
(19). These are mediated by
various means, including lipidic activation by arachidonic acid (AA)
metabolites. 20-Hydroxyeicosatetraenoic acid (20-HETE) is an AA metabolite
produced by cytochrome P-450 (CYP-450)
-hydroxylases. The
CYP-450 enzymes are predominantly detected in liver
(26), heart
(38), vasculature,
gastrointestinal tract, kidney
(32), and lung
(40). In guinea pig, 20-HETE
induces an increase of ASM basal tone
(39) and may have additive
effects on sustained contraction of ASM preconstricted using carbachol (CCh).
In contrast, 20-HETE relaxes rabbit
(18) and human
(41) bronchi preconstricted
with histamine or KCl. It has also been reported that 20-HETE causes
contraction of vascular smooth muscle (VSM) in various species, including cat,
dog, and rat, although it causes relaxation of human and rabbit VSM in
pulmonary circulation (28).
These relaxant effects are inhibited by indomethacin, a cyclooxygenase (Cox)
inhibitor, or by removal of the endothelium lining
(18). However, little is known
about the mechanisms that lead to contraction in guinea pig ASM. Experiments
involving exogenous addition of 20-HETE might help to discriminate its mode of
action on electrophysiological parameters. In the sustained portion of a
contraction of bovine tracheal smooth muscle strips induced by muscarinic
stimulation, the majority of Ca2+ entry from the
extracellular medium is not mediated via voltage-operated
Ca2+ channels
(34) but possibly via a
nonspecific cation channel, such as the transient receptor potential (TRP)
channels (4,
8). In VSM, it has been shown
that the mechanism that leads to activation of canonical transient receptor
potential (TRPC) 6 is associated with Gq-coupled receptors, such as
the
1-adrenoceptor, which activate phospholipase C (PLC; see
Ref. 6). The PLC produces
diacylglycerol (DAG) and inositol trisphosphate from phosphatidylinositol
4,5-bisphosphate. DAG, via a protein kinase C (PKC)-independent mechanism,
plays a central role in the activation of TRPC6
(17,
21,
22). It has been shown that
AA, produced by DAG lipase from DAG, activates a noncapacitative
Ca2+ entry in A7r5 smooth muscle cells stimulated with
low concentrations of vasopressin
(6). Moreover, Welsh et al.
(37) show that suppressing the
protein expression of TRPC6 using specific antisense oligonucleotides reduced
the current density of a major cation current in resistance artery smooth
muscle, which suggests that a nonselective cationic current might be important
in smooth muscle cell electrophysiology
(30).
The aim of the present study was to test whether or not 20-HETE, an AA
metabolite, can induce ASM contraction and modulate membrane potential in
relation to ionic conductance activation. We assessed the mechanical and
electrophysiological effects of 20-HETE on ASM at the tissue and cellular
levels, respectively. We used the following three complementary experimental
approaches: 1) isometric tension measurements on guinea pig ASM
induced by 20-HETE, 2) membrane potential measurements using the
classical microelectrode technique and quantification of the pharmacological
effects of 20-HETE and 1-oleoyl-2-acetyl-sn-glycerol (OAG), and
3) patch clamp to assess the effects of 20-HETE on macroscopic
currents. Our results show that 20-HETE induced concentration-dependent
contractions of ASM, depolarized membrane potential, and activated a
nonselective cationic current across the surface membrane of ASM cells. Part
of this work has been communicated elsewhere in abstract form
(9).
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MATERIALS AND METHODS
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Isometric tension measurements. The mechanical effects of 20-HETE
were measured on helically cut trachea and main bronchi taken from albino
guinea pigs (weighing 250-300 g; Hartley), as previously reported
(5,
7,
10). A Krebs solution,
containing (in mM) 118.1 NaCl, 4.7 KCl, 1.2 MgSO4 ·
7H2O, 1.2 KH2PO4, 25 NaHCO3, 2.5
CaCl2, and 11.1 glucose, pH 7.4, was used as physiological medium.
The effects of 20-HETE on basal tone were measured using a Grass polygraph.
The inotropic effects of 20-HETE were quantified and normalized to those
induced by 0.1 µM CCh. All procedures involving animal tissues were
performed according to current Canadian Council on Animal Care guidelines.
Electrophysiological recording. Albino guinea pigs were
anesthetized with pentobarbital sodium (35 mg/kg). The trachea was removed
rapidly and placed in oxygenated (95% O2-5% CO2) Krebs
solution at room temperature. A longitudinal section was made to expose the
luminal face of the trachea. The epithelium was removed mechanically by
delicate rubbing of the surface with an applicator, when required. Tissue was
cut into strips 10-12 mm long. 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 (5,
29). The tissues were
superfused at a constant flow rate of 2 ml/min with standard Krebs solution
and allowed to equilibrate for 20 min followed by another 20 min with 5 µM
wortmannin to prevent spontaneous smooth muscle contractures at the time of
impalements. Unused tissues were kept at 4°C in oxygenated Krebs solution
for several hours. Membrane potential was measured using conventional
intracellular borosilicate microelectrodes filled with 3 M KCl and resistance
ranging from 30 to 50 M
. The microelectrodes were connected via an
Ag/AgCl2 pellet to the headstage of an amplifier mounted on a
No13004 micromanipulator from Narishige (Tokyo, Japan). Measurements were
performed with a KS-700 amplifier from World Precision Instruments (Sarasota,
FL). Electrical signals were monitored continuously on a TDS 310 oscilloscope
(Tektroniks, Beaverton, OR). The membrane potential was digitized and recorded
using a Digidata 1200B interface and Axoscope 7.0 software from Axon
Intruments (Union City, CA). Data were stored on disk for further analysis.
Electrophysiological measurements were also performed on albino rabbits
(1.5-2.5 kg) and mongrel dog tracheas. In these experiments, a Tyrode solution
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, was used.
Patch-clamp recording. Whole cell currents were measured at room
temperature from guinea pig ASM cells
(15) using fire-polished patch
pipettes (3-6 M
) with uncompensated series resistance. Currents were
recorded with an Axo-Patch amplifier (Axon Instruments), controlled by
homemade software. The standard holding potential was -40 mV, and membrane
currents were filtered at 500 Hz and acquired at 1,000 Hz. The standard
intracellular solution contained (in mM) 140 CsAsp, 1 CaCl2, 11
EGTA, 2 MgCl2, 18 NaCl, 10 HEPES, 0.3 ATP, and 0.03 GTP, pH 7.2
(calculated free internal Ca2+: 100 nM). The standard
bath solution contained (in mM) 140 NaCl, 1.8 CaCl2, 1.2
MgCl2, 15 HEPES, and 10 glucose, pH 7.4. The final concentration of
1 µM 20-HETE was added to the perfusion solution containing (in mM) 145
NaCl, 2.5 EGTA, and 5 HEPES, pH 7.4, by a local perfusion system (Perfusion
fast-step; Harvard Apparatus, Holliston, MA). Depolarizing voltage ramps were
applied at a rate of 100 mV/s, from -100 to +60 mV.
Cell culture. Male or female albino guinea pigs (weighing 350-450
g; Hartley) were anesthetized by a lethal dose of pentobarbital sodium (50
mg/kg ip) and killed by abdominal exsanguinations. The trachea was excised
aseptically and placed immediately on ice in sterile Krebs solution (see
composition above). Under sterile conditions, and on ice, the trachea was cut
free of excess tissue and cut longitudinally on the opposite side of the
smooth muscle. The epithelial cells were removed mechanically with a sterile
cotton swab. The smooth muscle tissue was minced, washed in MEM containing 200
µM free Ca2+, and centrifuged at 80 g for 1
min. The pellet was resuspended and dissociated in 200 µM
Ca2+ MEM with 640 U/ml collagenase (type IV), 10 U/ml
elastase (type IV), and 20 µg/ml DNase (type I), all from Sigma-Aldrich
(Oakville, ON, Canada). The tissue was digested in a cell incubator at
37°C for 3 x 20 min with agitation at each step. The cell suspension
was then filtered through a 100-µm Nylon Cell Strainer, and the filtrate
was washed with 900 µMCa2+ MEM. The cells were
centrifuged at 80 g for 10 min, and the pellet was resuspended in 1
ml Opti-MEM supplemented with 2% FBS and 1% penicillin-streptomycin. The cells
were plated in 35 mm-dishes with
103 cells for each dish, and,
after 30 min incubation at 37°C, the dishes were completed with 2 ml
Opti-MEM.
Molecular biology. Guinea pig ASM cells were isolated and cultured
on plastic dishes as described above. Total RNA was extracted using the
RNaqueous method according to the manufacturer (Ambion, Austin, TX). RNA (5
µg) was used for each preparation and was reverse transcribed into
first-strand cDNA, oligo(dT) (5 units), dNTP (10 mM) from Amersham-Pharmacia
Biotech (Piscataway, NJ), DTT (0.1 M), Moloney murine leukemia virus, and
RNasin (all from Promega, Madison, WI). cDNA was amplified for each PCR
reaction by using specific primers based on published sequences for TRPC1, -3,
-4, and -5 (13) or primers
designed based on the GenBank sequence for TRPC6. The sequences of the primer
were as follows: 1) mouse (m) TRPC1: sense
5'-CAAGATTTTGGGAAATTTCTGG-3' and antisense
5'-TTTATCCTCAT-GATTTGCTAT-3'; 2) human (h) TRPC3: sense
5'-TGACT-TCCGTTGTGCTCAAATATG-3' and antisense
5'-CCTTCTGAAGCCTTCTCCTTCTGC-3'; 3) mTRPC4: sense
5'-TCTGCAGATATCTCTGGGAAGGATGC-3' and antisense
5'-AAGCT-TTGTTCGAGCAAATTTCCATTC-3'; 4) mTRPC5: sense
5'-ATCTACTGC-CTAGTACTACTGGCT-3' and antisense
5'-CAGCATGATCGGCAATGAGCTG-3'; and 5) rat TRPC6: sense
5'-AACAAAAGCATGACTCCTTCAG-3' and antisense
5'-AAGGAGCA-CACCAGTATATGAGA-3'. GAPDH was used as a control of RNA
integrity. Amplification was performed using Taq polymerase on a
Perkin-Elmer amplification system for 34 cycles consisting of 30 s at
94°C, 60 s at 58°C, and 2 min at 72°C for extension for all
samples. Products were loaded on a 2% agarose gel in Tris-acetate-EDTA buffer
with 0.1 µg/ml ethidium bromide. After electrophoresis, the gel was scanned
by a Fluorimager (Alpha Innotech, San Leandro, CA).
Drugs and chemical reagents. 20-HETE from Cayman Chemical (Ann
Arbor, MI) was dissolved in 100% ethanol and stored as 1 mM stock solutions.
The vehicle was tested separately at the maximal concentration used in the
presence of active compound. CCh, nifedipine, and iberiotoxin were purchased
from Sigma (St. Louis, MO). Gadolinium chloride was purchased from ICN
Biomedicals (Cleveland, OH), and OAG was from Calbiochem (San Diego, CA). FBS,
penicillin-streptomycin, and all cell media were purchased from GIBCO
Invitrogen (Burlington, ON, Canada).
Data analysis and statistics. Results were expressed as means
± SE; n indicates the number of experiments. Statistical
analyses were performed using either paired or unpaired Student's
t-tests, as well as ANOVA. Values of P < 0.05 were
considered significant. Data curve fittings were performed using Sigma Plot
8.0 (SPSS-Science, Chicago, IL). The concentration-response curve was fitted
to the equation
 | (1) |
where C and Cmax are the amplitude of contraction, X is
the concentration of 20-HETE, EC50 is the concentration of 20-HETE
that produces half-maximal amplitude of contraction, and
Hn is the Hill coefficient. Patch-clamp analysis was
performed with homemade software.
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RESULTS
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Tension measurements. The addition of cumulative concentrations of
20-HETE produced sustained contractions that were reversed by washing with
fresh, oxygenated (95% O2-5% CO2) Krebs solution
(Fig. 1A). In guinea
pig preparations, 20-HETE induced reversible, concentration-dependent positive
inotropic responses. Vehicle alone, ethanol, was tested, and the various
quantities used were shown to have no effects on the resting tone of guinea
pig ASM (Fig. 1B). To
account for biological variations, the data were normalized using the
amplitude of contraction induced by 0.1 µM CCh obtained for each
preparation at the beginning of all experiments. 20-HETE inotropic effects
were expressed as a percentage (%) of the contraction induced by 0.1 µM
CCh, a concentration that was previously determined as the EC30 on
guinea pig ASM (3).
Figure 1C shows the
dose-response curve for 20-HETE. This eicosanoid-induced tonic
concentration-dependent response was undetected below 0.03 µM and
saturating above 10 µM. An EC50 value of 1.5 µM and a Hill
coefficient of 0.77 were obtained by fitting Eq. 1 to the
experimental data. These results suggest the presence of a receptor for
20-HETE that remains to be characterized
(11).

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Fig. 1. Dose-dependent effects of 20-hydroxyeicosatetraenoic acid (20-HETE) on
guinea pig airway smooth muscle (ASM) with intact epithelium. A:
representative trace showing the dose-dependent inotropic effect of 20-HETE.
Cumulative concentrations of 0.5, 1.0, and 1.5 µM 20-HETE have been used
and directly applied on 1 g basal tone. B: effect of vehicle (100%
ethanol) on tone at the corresponding concentrations as those used for the
active compound delivery. Arrows indicate the times of sequential addition of
20-HETE, vehicle, and washout (W). C: concentration-response curve of
20-HETE on guinea pig ASM with intact epithelium (n = 3-8). The
continuous curve was obtained by fitting Eq. 1 to the experimental
data points, with EC50 and Hill coefficient
(Hn) values of 1.5 µM and 0.77, respectively. The
positive inotropic effects of 20-HETE were calculated as a percentage of the
tension induced by 0.1 µM carbachol (CCh) on the same tissue.
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Effects of Ca2+ release and Ca2+ entry on the
inotropic effect of 20-HETE. Figure
2A shows the sustained contractions induced by 20-HETE in
the presence of Krebs solution containing 2.5 mM Ca2+.
In contrast, 20-HETE induced only a transient contraction in
Ca2+-free Krebs solution
(Fig. 2B). When
tension returned to basal levels, addition of 2.5 mM CaCl2 induced
a large tension increase. This effect was fully reversible upon washout of
20-HETE (Fig. 2B). To
test the relative contribution of nonselective cation channels and
voltage-dependent L-type Ca2+ channels,
Gd3+, which had no effect on the resting tone (data not
shown), and nifedipine were used sequentially. Gd3+ (100
µM), a nonspecific cation channel blocker, has been reported to block
activities of the TRPC6 implicated in the control of myogenic tone in VSM
(21,
37).
Gd3+ (100 µM) induced relaxation of guinea pig ASM
precontracted with 1 µM 20-HETE (Fig. 2,
C and D), whereas 1 µM nifedipine, used to
block voltage-dependent L-type Ca2+ channels, relaxed
the remaining tension. Table 1
summarizes the results of two series of complementary and comparative
experiments where the relaxing effects of 1 µM nifedipine were measured on
20-HETE (n = 12)- and KCl (n = 4)-induced tension in guinea
pig ASM. Nifedipine was much more effective in inhibiting KCl-induced
responses than those induced by 20-HETE. However, 100 µM
Gd3+ had no effect on high KCl-induced tension (data not
shown). In contrast, the remaining tension, after nifedipine-induced
relaxation on the 20-HETE responses, was abolished by the addition of 100
µM Gd3+ (n = 12). Together, these results
suggest that both L-type Ca2+ channels and nonselective
cation channels are involved in the pharmacomechanical coupling induced by
20-HETE.

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Fig. 2. Roles of intracellular Ca2+ release and
Ca2+ entry in guinea pig ASM upon 20-HETE stimulation on
intact epithelium. A: sustained tension induced by 20-HETE in normal
Krebs solution. B: transient effect of 20-HETE in
Ca2+-free Krebs solution, followed by tonic tension
increase upon addition of 2.5 mM CaCl2 in the experimental chamber.
C: relaxant effect of 100 µM Gd3+ after
precontraction with 1 µM 20-HETE. D: relaxing effects of 100 µM
Gd3+ and 1 µM nifedipine after precontraction with 1
µM 20-HETE. All of these effects were reversible after washout.
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Table 1. Average relaxing effects of nifedipine on 20-HETE and KCI-induced
tensions in guinea pig airway smooth muscle
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Effects of PKC inhibitor on 20-HETE inotropic response. It has
been reported that, in cerebral arteries, nonselective cation channels
involved in myogenic tone are controlled by PKC activation
(30). Thus it was of interest
to test whether or not a membrane-permeable PKC inhibitor, such as GF-109203X,
might affect the tonic responses to 20-HETE in the airways.
Figure 3, A and
B, demonstrates that a 15-min preincubation period with 1
µM GF-109203X does not modify the amplitude nor the time course of the
inotropic responses induced by 1 µM 20-HETE in guinea pig ASM. In fact, the
PKC inhibitor displays a very slight relaxing effect while the average
response to 20-HETE, in the presence of GF-109203X, was not statistically
different (Fig.
3B).

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Fig. 3. Protein kinase C (PKC) is not involved in the 20-HETE inotropic response.
A: paired recordings of 1 µM 20-HETE challenges in the absence
(control) and after GF-103209X preincubation on guinea pig ASM. B:
quantitative analysis of the average responses for paired challenges. Reported
values are means ± SE (n = 12). Paired t-test shows
that the difference was not statistically significant.
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Effect of 20-HETE on ASM membrane potential. The effects of
20-HETE on the membrane potential of guinea pig ASM cells were assessed after
microelectrode impalement and continuous recordings. The muscle strip was
superfused with a physiological solution for several minutes, and then
micromolar concentrations of 20-HETE were applied. After a stable membrane
potential of -60 mV was obtained in this sample where the epithelium had been
kept intact, 3 µM 20-HETE was superfused, and depolarization was recorded
after a short delay, as shown in Fig.
4A. 20-HETE (3 µM) depolarized the membrane potential
of ASM by 13 ± 2 mV (n = 7), an effect that was fully
reversible within a few minutes. The mean electrophysiological effect of
20-HETE on guinea pig ASM and on tissue recovery are shown in
Fig. 4B.

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Fig. 4. Effect of 20-HETE on guinea pig ASM membrane potential. A:
representative trace of a continuous recording after impalement of a guinea
pig ASM cell (microelectrode resistance: 45 M ). The change in membrane
potential was induced by addition of 3 µM 20-HETE to the Krebs solution. At
a flow rate of 2 ml/min, this effect was fully reversible after 5 min.
B: quantification of the effect of 3 µM 20-HETE during continuous
recordings of the membrane potential from guinea pig ASM cells
(*P < 0.05, n = 7). Recovery was measured 7
min after washout with the Krebs solution. All data were obtained on
epithelium-intact tracheas.
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Since it was reported that the effects of 20-HETE could be different from
one species to the next, complementary experiments were performed on rabbit
and canine tissues. Because it is quite difficult to maintain microelectrode
impalements for long periods, we used a multi-impalement method during
sequential changes in experimental conditions. For instance, in rabbit ASM, 1
µM 20-HETE induced an average depolarization of 15 ± 5 mV
(n = 9, data not shown). This result is consistent with the data
obtained in guinea pig preparations. We also tested the effects of 20-HETE on
epithelium-denuded ASM to verify the putative contribution of epithelial cells
in the 20-HETE-induced depolarization. In the absence of epithelium, the mean
resting membrane potential value was -50 ± 0.6 mV (n = 23), as
previously reported by our group
(29). On the same tissues,
superfusion of 0.3 µM 20-HETE induced a depolarization of 3.4 ± 0.5
mV (data not shown). The depolarization induced by 20-HETE under these
conditions is fully reversible after the recovery period.
Effect of OAG on ASM membrane potential. OAG, a permeable analog
of DAG described as a PKC activator, has recently been reported to be a TRPC
channel activator (12,
27). Using the
multi-impalement method, we tested its putative depolarizing effect on ASM
membrane potential. Figure
5A shows that application of 30 µM OAG depolarized
canine ASM cells. The data also show that the basal membrane potential was
recovered upon washout of OAG with a physiological solution. The mean
depolarization induced by 30 µM OAG was 5.7 ± 1 mV (n =
14), and this effect was shown to be statistically significant
(Fig. 5B). Tissue
recovery was obtained and quantified 10 min after OAG removal
(Fig. 5B). The mean
value was not statistically significant when compared with control.

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Fig. 5. Effect of 1-oleoyl-2-acetyl-sn-glycerol (OAG) on ASM membrane
potential with epithelium, as assessed by the multi-impalement method.
A: time-dependent effect of OAG on the membrane potential of ASM
cells and reversibility. OAG (30 µM) depolarizes canine ASM cells.
B: quantitative analysis of the effect of 30 µM OAG on the
membrane potential of ASM (*P < 0.05, n = 14)
from 3 independent experiments. Recovery, after a 10-min washout with
physiological solution.
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Effect of 20-HETE on macroscopic nonselective currents in guinea pig
ASM cells. Patch-clamp experiments were performed on primary cultured ASM
cells from guinea pig, as described in MATERIALS AND METHODS.
20-HETE (1 µM) activates a nonselective cationic current under experimental
conditions used to measure the current supported by TRPC channel proteins
(Fig. 6A). The current
(I) activated by 20-HETE during a voltage ramp (-100 to +60 mV) was
visualized by data subtraction [Fig.
6B; Ib-a
(20-HETE) = Ib (total) - Ia
(control)], as reported in Fig.
6A. Hence, the increase in current density generated by
20-HETE was calculated and reported in Fig.
6C. This inward current activated by 20-HETE was likely
generated by TRPC channel openings. Furthermore, it has been reported recently
by our group that the current supported by the TRPC6 channel proteins
overexpressed in HEK293 cells was increased by 20-HETE and OAG and inhibited
by either Gd3+ or
N-methyl-D-glucamine
(1).

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Fig. 6. 20-HETE activates a nonselective inward current in guinea pig ASM cells.
A: whole cell recording of a nonselective cationic current at -50 mV
as a function of time, before and after addition of 1 µM 20-HETE in the
perfusion solution. Voltage ramps (-100 to +60 mV) were applied in control (a)
and upon 20-HETE superfusion (b). HP, holding potential. B: current
(I)-voltage (V) curves taken at control (a) and upon
superfusion of 1 µM 20-HETE (b) as indicated in A. The current
activated by 20-HETE was obtained by subtraction (b-a). The net inward current
displays a reversal potential close to 0 mV. These results are representative
of 5 independent experiments (n = 5). C: average increase in
current density induced by 1 µM 20-HETE on isolated guinea pig ASM cells.
Data represent mean values ± SE (n = 5; *P
< 0.05). D: RT-PCR analyses were carried out on total mRNA of
isolated guinea pig ASM cells using specific primers for canonical transient
receptor potential channel (TRPC)-1, -3, -4, -5, and -6, and GAPDH was used as
a loading control. B, brain; T, trachea. Amplified cDNA was analyzed on a 2%
agarose gel containing 0.1 µg/ml ethidium bromide.
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Expression of TRPCs in guinea pig ASM. RT-PCR was used to identify
the types of TRPC channels present in guinea pig ASM cells. TRPC1, -3, -4, -5,
and -6 were amplified using specific primers (see Molecular biology)
to visualize which forms are present in this tissue. Our results showed that
TRPC3, -4, -5, and -6 were expressed in guinea pig ASM cells, but not TRPC1,
although the signal was present in brain tissues used as a control
(Fig. 6D).
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DISCUSSION
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This work provides the first direct evidence that the mode of action of
20-HETE on ASM involves the activation of a nonselective cationic conductance,
in addition to L-type Ca2+ channels. Our results also
show that 20-HETE induces concentration-dependent tension increases in guinea
pig ASM. Regardless of the Ca2+ released from
intracellular stores during the contraction, 20-HETE also induced
Ca2+ entry during the development of the tonic response.
Moreover, 20-HETE depolarized the membrane of guinea pig ASM cells in a
similar manner as OAG in canine ASM cells. Hence, OAG has been shown to
activate nonselective currents in other tissues
(35), and, based on the
similar effects of OAG and 20-HETE on membrane potential, it was suggested
that the mechanism of action of 20-HETE could also involve nonselective
cationic currents generated by TRPC channels. This working hypothesis has now
been tested in native ASM cells. Our results correlate well with other data
recently reported in the literature on VSM
(6,
30,
37) and further support the
physiological role of nonselective cationic currents in the tonic responses
triggered by eicosanoids.
Pharmacological responses to 20-HETE. Because of the biological
variability of the tonic responses after eicosanoid challenge on airway
tissues, we measured the positive inotropic effects of 20-HETE on guinea pig
ASM and normalized the mechanical responses as a percentage of the response
induced by 0.1 µM CCh on the same tissue. Concentrations of 20-HETE
>0.03 µM induced tonic concentration-dependent responses, saturating
above 10 µM. In contrast, 20-HETE has been reported to relax rabbit bronchi
preconstricted with histamine or KCl
(18) and human bronchi
preconstricted with histamine
(41). The relaxant effect of
20-HETE on these ASM tissues was blocked by indomethacin or after epithelium
removal. These results indicate that the effects of 20-HETE are species
dependent and could be related to differential expression profiles of Cox
isozymes and metabolite production. The concentration-response curve performed
on guinea pig ASM revealed an EC50 value of 1.5 µM for 20-HETE.
In contrast, other related eicosanoids, such as the epoxyeicosatrienoic acid
regioisomers, which are produced by the CYP-450 epoxygenase, were shown to
trigger relaxing and hyperpolarizing responses in guinea pig ASM preparations
(5,
10).
The positive inotropic effects of 20-HETE mobilize
Ca2+ from intracellular stores, as attested by the
transient responses observed in the absence of extracellular
Ca2+. However, Ca2+ entry was
necessary to trigger and maintain the sustained inotropic responses that
occurred upon addition of 2.5 mM extracellular Ca2+
concentration (Fig.
2B). It was already reported that, in bronchia,
Ca2+ entry could be the result of activation of the
capacitative Ca2+ entry after depletion of intracellular
Ca2+ stores upon ACh stimulation
(33). The tonic response
induced by 20-HETE was partially relaxed by 100 µM
Gd3+, a concentration known to block noncapacitative
Ca2+ entry
(25), suggesting a putative
role for this molecular process. Taking into account that nifedipine alone
partially relaxes the tonic responses induced by 20-HETE
(Table 1), it was suggested
that L-type Ca2+ channels play a complementary role in
ASM contraction (20). Indeed,
pharmacological maneuvers and electrophysiological experiments in animal and
human ASM have demonstrated the existence of dihydropyridine-sensitive
Ca2+ currents in these samples
(24). On the other hand, it
has been reported that 20-HETE induced an increase in intracellular free
Ca2+ concentration with a concomitant activation of
L-type Ca2+ channels in VSM
(14). Thus 20-HETE challenges
could be involved in the activation of Ca2+ selective
channels and a nonselective cationic pathway. In ASM cells, this process would
be independent of PKC activation, since the inotropic effect of 20-HETE was
not modified in the presence of PKC inhibitor according to the results
reported in Fig. 3, A and
B.
Electrophysiological effects of 20-HETE. Our results show that
20-HETE depolarized the membrane of guinea pig ASM cells and that this effect
was fully reversible. This behavior was also observed in rabbit ASM in the
presence and in the absence of the epithelial layer, suggesting that 20-HETE
interacted directly with membrane components of ASM cells. Similar results
were observed in canine renal arteries, where 20-HETE is associated with a
10-mV depolarization of the membrane potential
(23). OAG is a stable and
membrane-permeable analog of DAG that has been reported to activate the
TRPC3/6/7 channels in Jurkat cells and human peripheral blood T lymphocytes
(17,
27). Our results indicate as
well that 30 µM OAG depolarizes ASM cells and that this effect was
reversible (Fig. 5B).
It was previously shown that OAG also depolarizes the membrane of T
lymphocytes in Ca2+-free media
(12).
The depolarizing effects of 20-HETE and OAG on ASM membrane potential could
be explained by the activation of an inward cationic current. Hence, the
electrophysiological effects of 20-HETE were sensitive to
Gd3+, a blocker of nonselective conductances. Thus we
tested the effects of 20-HETE on the macroscopic inward currents, which under
our experimental conditions may be generated by activation of TRPC channels in
smooth muscle, as previously reported by other laboratories working on various
biological structures (6,
21,
30). Addition of exogenous
20-HETE consistently activated nonselective cationic currents. Such currents
had already been reported to be activated by DAG
(2,
36) and OAG
(17,
27). The activation of an
inward cationic current might partially explain the depolarization induced by
20-HETE and OAG on ASM. These observations do not rule out the putative
contribution of other pharmacological (via eicosanoid receptors or lipid-gated
channels) and biochemical (via the activation of intracellular cascades)
pathways. However, they provide evidence that this hydrophobic eicosanoid,
generated in vivo by CYP-450
-hydroxylases upon AA release after
phospholipase A2 activation, might regulate surface membrane
conductances, which are likely involved in the control of the basal ASM tone
(30). A role of TRPC channels
in controlling the VSM myogenic tone has already been demonstrated by two
independent groups (6,
37). However, to date, the
role of TRP channels and more specifically the TRPC6 isoform has not been
precisely forecast in ASM, except by Snetkov et al.
(31), who had envisioned their
implication on bronchoactive leukotreine D4 stimulation. Recently,
in a set of key experiments, our group has demonstrated that exogenous 20-HETE
and OAG, in the micromolar concentration range, activate nonselective cationic
currents in HEK293 cells stably overexpressing TRPC6
(1). The direct implication of
TRPC6 in the mode of action of 20-HETE in ASM cells is plausible since the
addition of 100 µMGd3+, which has been reported to
block the noncapacitative entry of Ca2+
(25) and more directly the
TRPC6 channel isoform (21),
partially relaxes its positive inotropic effect as shown and discussed above.
Although we have not tested the effects of 20-HETE on TRPC activity in an
overexpression system, our present results reveal the presence of the TRPC3,
-4, -5, and -6 mRNA in guinea pig ASM cells
(9).
In summary, 20-HETE induces concentration-dependent positive tonic
responses in guinea pig ASM. This tonic response is triggered by intracellular
Ca2+ release and is maintained by
Ca2+ entry, the latter being related to a depolarization
of the membrane potential as shown on the same preparation. The following two
currents are likely to be involved in this process: voltage-dependent L-type
Ca2+ currents and nonselective cationic currents.
Although this hypothesis remains to be assessed, our results suggest that TRPC
channels are likely to support some of the latter currents, even if
lipid-gated channels related to the vanilloid receptor family cannot be
disregarded (4).
 |
DISCLOSURES
|
---|
This work was supported by a Canadian Institutes of Health Research Grant
MOP-57677. E. Rousseau is a National Scholar and a member of the Health
Respiratory Network of the Fonds de la Recherche en Santé du
Quebec.
 |
ACKNOWLEDGMENTS
|
---|
We thank Solange Cloutier and Frederic Mercier for technical assistance
with the pharmacological and electrophysiological measurements, Dr. Alain
Cadieux for advice and scientific comments, and Dr. Dany Salvail for reading
and discussing the manuscript.
 |
FOOTNOTES
|
---|
Address for reprint requests and other correspondence: E. Rousseau, Le
Bilarium, Faculty of Medicine, Dept. of Physiology and Biophysics, Univ. of
Sherbrooke, 3001 12th Ave. N., Sherbrooke, Quebec, Canada J1H 5N4
(E-mail:
Eric.Rousseau{at}USherbrooke.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.
 |
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