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20-HETE inotropic effects involve the activation of a nonselective cationic current in airway smooth muscle

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

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 
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 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).

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 
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 C_max 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 H_n is the Hill coefficient. Patch-clamp analysis was performed with homemade software.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 
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).

View larger version (15K): [in this window] [in a new window]   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.

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.

View larger version (19K): [in this window] [in a new window]   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.

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).

View larger version (20K): [in this window] [in a new window]   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.

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.

View larger version (16K): [in this window] [in a new window]   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.

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.

View larger version (16K): [in this window] [in a new window]   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.

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; I_b-a (20-HETE) = I_b (total) - I_a (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).

View larger version (24K): [in this window] [in a new window]   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.

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).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 
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

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 
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 12^th Ave. N., Sherbrooke, Quebec, Canada J1H 5N4 (E-mail: Eric.Rousseau{at}USherbrooke.ca).

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	REFERENCES

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 

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