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Serotonin-induced activation of TRPV4-like current in rat intrapulmonary arterial smooth muscle cells
Cell Calcium 43 (2008) 315–323
Serotonin-induced activation of TRPV4-like current in rat intrapulmonary arterial smooth muscle cells Thomas Ducret a,b,∗ , Christelle Guibert a,b , Roger Marthan a,b , Jean-Pierre Savineau a,b a
Universit´e Bordeaux 2, Laboratoire de Physiologie Cellulaire Respiratoire, 146 rue L´eo-Saignat, F-33076 Bordeaux, France b INSERM, U885, 146 rue L´ eo-Saignat, F-33076 Bordeaux, France Received 22 January 2007; received in revised form 20 April 2007; accepted 30 May 2007 Available online 31 July 2007
Abstract In the present study, we investigated the implication of transient receptor potential vanilloid (TRPV)-related channels in the 5hydroxytryptamine (5-HT)-induced both intracellular calcium response and mitogenic effect in rat pulmonary arterial smooth muscle cells (PASMC). Using microspectrofluorimetry (indo-1 as Ca2+ fluorescent probe) and the patch-clamp technique (in whole-cell configuration), we found that 5-HT (10 M) induced a transient intracellular calcium mobilization followed by a sustained calcium entry. This latter was partly blocked by an inhibitor of cytochrome P450 epoxygenase (17-ODYA) and insensitive to cyclo-oxygenase and lipoxygenase inhibitors (indomethacin and CDC), suggesting the involvement of arachidonic acid metabolization by cytochrome P450 epoxygenase. This calcium influx was also sensitive to Ni2+ and to ruthenium red, a TRPV channel blocker, and mimicked by 4␣-phorbol-12,13-didecanoate (4␣-PDD), a TRPV4 channel agonist. In patched PASMC, 5-HT and 4␣-PDD-activated TRPV4-like ruthenium red sensitive currents with typical characteristics. Furthermore, 5-HT induced a ruthenium red sensitive increase in BrdU incorporation levels in PASMC. The present study provides evidence that 5-HT activates a TRPV4-like current, potentially involved in PASMC proliferation. The signalling pathway between proliferation and ion channel activation remains to be determined and may represent a molecular target for the treatment of vascular diseases such as pulmonary hypertension. © 2007 Elsevier Ltd. All rights reserved. Keywords: Arachidonic acid metabolites; Calcium influx; Serotonin; Smooth muscle cells proliferation; TRPV channels; Vascular smooth muscle
1. Introduction In pulmonary arteries, 5-hydroxytryptamine (5-HT) exerts vasoconstrictor and mitogenic effects [1,2] both phenomena being dependent on an increase in cytosolic calcium concentration ([Ca2+ ]i ) [3]. Pulmonary arterial hypertension (PAH) is characterized by high circulating 5-HT concentration [4], 5-HT-induced hyperreactivity and SMC proliferation [2,5,6] suggesting a major role for 5-HT in both vascular wall remodelling and elevated vascular resistances that characterize PAH. Thus, a better understanding of the calcium-related signalling pathway activated by 5-HT is of major importance in the pulmonary vascular bed. ∗
Corresponding author. Tel.: +33 5 57 57 16 94; fax: +33 5 57 57 16 95. E-mail address: [email protected] (T. Ducret).
0143-4160/$ – see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.ceca.2007.05.018
However, calcium sources involved in 5-HT-induced calcium response are still unresolved. In rat isolated small intrapulmonary arteries (IPA) and primary cultured pulmonary arterial smooth muscle cells (PASMC), we recently demonstrated that activation of 5-HT2A receptors by 5-HT led to a biphasic increase in [Ca2+ ]i , namely a transient phase due to the mobilization of Ca2+ stored in intracellular compartments, followed by a sustained phase due to Ca2+ entry from the extracellular medium [5,7]. We have previously found that the sustained phase was a non-capacitative Ca2+ entry corresponding at least, in part, to a voltage-independent Ca2+ influx sensitive to arachidonic acid production [7]. In a variety of tissues, transient receptor potential (TRP) proteins have been proposed as good candidates for the molecular structure underlying voltage-independent Ca2+ influx [8].
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TRP channels are non-selective cationic channels expressed in a large variety of tissues including smooth muscles. They are implicated in diverse cellular functions such as calcium homeostasis and cell proliferation [9,10]. TRP channels are characterized by six transmembranespanning domains, which form tetramers [11]. Based on structural homology, TRP superfamily can be subdivided into seven main subfamilies: TRPC (‘Canonical’), TRPV (‘Vanilloid’), TRPM (‘Melastatin’), TRPP (‘Polycystin’), TRPML (‘Mucolipin’), TRPA (‘Ankyrin’) and TRPN (‘NOMPC’). In PASMC, TRPC, TRPV and TRPM are expressed and functional [12,13] and among the TRPV subfamily, TRPV4 was the most abundantly expressed [13]. TRPV4 is a Ca2+ permeable non-selective cationic channel, activated by a wide variety of stimuli including physical, thermal and chemical stimuli such as the synthetic agonist 4␣-phorbol-12,13didecanoate (4␣-PDD), the endocannabinoid anandamine and the arachidonic acid metabolite epoxyeicosatrienoic acid (EET) [14] in particular. Moreover, 4␣-PDD induced an increase in [Ca2+ ]i in rat PASMC [13]. TRPV4 thus appears as a good candidate for mediating the Ca2+ response to 5-HT in PASMC. In the present study, we have examined the calcium influx activated by 5-HT in rat intrapulmonary arteries. We focused our study on the arachidonic acid sensitive TRPV channel such as the TRPV4-like channel by recording [Ca2+ ]i (microspectrofluorimetric assay) and plasma membrane currents (patch-clamp in a “whole-cell” mode). Since intracellular Ca2+ and 5-HT interfere with SMC proliferation, we also explored the role of the TRPV-like channels in 5-HT-induced PASMC proliferation (BrdU incorporation).
2. Materials and methods 2.1. PASMC isolation and culture As previously described [15], intrapulmonary arteries were dissected from lungs of male Wistar rats (250–350 g body weight), according to the animal care and use of our local ethic committee (Comit´e d’´ethique r´egional d’Aquitaine, AP 2/11/2005). Briefly, the entire heart-lung preparation was rapidly removed en bloc and rinsed in culture medium (DMEM-HEPES supplemented with 1% penicillin–streptomycin, 1% Na-pyruvate and 1% nonessential amino acids). Intrapulmonary arteries of the first and second orders from the left lung were dissected free from surrounding connective tissues under binocular control and sterile conditions. Endothelium was removed by rubbing the luminal surface. The arteries were then cut in several pieces (1 mm × 1 mm) and seeded on round glass coverslips (30 mm diameter) for microspectrofluorimetry experiments and Petri dishes for electrophysiological experiments. Explants were cultured in culture medium enriched with 10% foetal calf serum (FCS). They were maintained at 37 ◦ C in a humidified atmosphere gassed with 5% CO2 . Fifty percent of the medium
was changed everyday until cells go out of the explants (3–7 days after seeding). Before the experiments, PASMC were growth-arrested during 48 h by using serum-free culture medium supplemented with 1% insulin-transferrin-selenium (ITS). Smooth muscle characteristics (elongated fusiform shape and formation of “ hills and valleys”) of isolated cells were confirmed by positive immunostaining with an anti-␣ smooth muscle actin antibody (Sigma). 2.2. Microspectrofluorimetric assay of cytosolic calcium The Ca2+ -sensitive fluorescent probe indo-1 was used to record changes in [Ca2+ ]i . The cells plated on glass coverslips were incubated with 5 M indo-1 penta-acetoxymethyl ester (indo-1/AM) in Krebs-HEPES solution (see composition below) at room temperature for 40 min, then washed and maintained at room temperature in the same saline solution before the fluorescence measurements. For single cell measurements, the dual emission microspectrofluorimeter was constructed from a Nikon Diaphot inverted microscope fitted with epifluorescence (40× oil immersion fluorescence objective; numerical aperture, 1.3). For excitation of indo-1, a collimated light beam from a 100-W mercury arc lamp (Nikon) was filtered at 355 nm and reflected from a dichroic mirror (380 nm). The emitted fluorescence signal was passed through a pinhole diaphragm slightly larger than the selected cell and directed onto another dichroic mirror (455 nm). Transmitted light was filtered at 480 nm, reflected light was filtered at 405 nm and the intensities were recorded by separate photometers (P100, Nikon). Under these experimental conditions, the fluorescence ratio (F405 /F480 ) was calculated and recorded on-line as a voltage signal. [Ca2+ ]i was estimated from the F405 /F480 using the formula derived by Grynkiewicz et al. [16] after Ca2+ calibration for indo-1 determined within cells as previously described [17]. The sustained [Ca2+ ]i value was estimated as the amplitude of the plateau phase measured 30 s after the peak was reached. 2.3. Electrophysiological recordings The whole-cell recording mode of the patch-clamp technique was used [18]. The electrodes were pulled on a PC-10 (Narishige) puller in two stages from borosilicate glass capillaries (1.5 mm o.d., 1.16 mm i.d., Harvard Apparatus). The pipettes had a mean resistance of 4–6 M when measured in standard recording conditions. Cells were viewed under phase contrast with a Nikon Diaphot inverted microscope. A RK-400 patch-clamp amplifier (Biologic) was used for whole-cell recordings. Stimulus control, data acquisition, and processing were carried out on a PC computer fitted with a Digidata 1200 interface, using pCLAMP 6.0.2 software (interface and software, Axon Instruments). Current records were filtered with a Bessel filter at 2 kHz and digitized at 10 kHz for storage and analysis. For all experiments, currents were normalized to cell capacitance.
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2.4. Recording solutions and application of substances
2.6. Reagents
For whole-cell voltage-clamp and microspectrofluorimetry studies, the standard Krebs-HEPES extracellular solution contained (in mM): 118.4 NaCl, 4.7 KCl, 2 CaCl2 , 1.2 MgSO4 , 4 NaHCO3 , 1.2 KH2 PO4 , 6 d-glucose and 10 N-2hydroxyethylpiperazine-N -2-ethanesulfonic acid (HEPES). The osmolality (measured with a cryoosmometer type 15 L¨oser) of the external salt solution was adjusted to 300 mosm/kg with sucrose, and pH adjusted to 7.4 with NaOH. Solutions nominally free of Ca2+ were prepared by omitting CaCl2 and adding 0.4 mM EGTA. For steady-state whole-cell studies, the recording pipette was filled with an artificial intracellular saline containing (in mM): 130 CsCl, 2 MgCl2 , 0.2 EGTA, 3 Na2 ATP and 10 HEPES (pH 7.2; osmolality: 290 mosm/kg). To eliminate K+ currents, TEA (10 mM) was added to the extracellular solution. For voltage ramp whole-cell studies, the recording pipette was filled with (in mM): 100 Na-glutamate, 20 NaCl, 1 MgCl2 , 1 EGTA, 4 Na2 ATP and 10 HEPES (pH 7.2; osmolality: 290 mosm/kg). For electrophysiological and microspectrofluorimetric studies, 5-HT, arachidonic acid and 4␣-PDD were applied to the recorded cell by pressure ejection from a glass pipette located close to the cell for the period indicated on the records. It was verified, in control experiments, that no change in [Ca2+ ]i was observed during test ejections of Krebs-HEPES.
General salts were from VWR. All other chemicals were purchased from Sigma, except DMEM-HEPES, FCS and penicillin–streptomycin which were from Gibco.
2.5. Cell proliferation assessment Quantitative determination of DNA synthesis using the Cell Proliferation ELISA, BrdU colorimetric method (Roche Applied Science), was used to assess PASMC proliferation. Briefly, cells were seeded at a density of 5 × 103 cells per well in a standard 96-well tissue culture plate in a volume of 100 l medium/well and cultured in complete culture medium containing 10% FCS in a humidified incubator at 37 ◦ C. After 48 h, the medium was removed and replaced by a serum-free medium containing 1% ITS. After an additional period of 48 h at 37 ◦ C, the medium was changed to complete culture medium containing 0.2% serum, supplemented with 5-HT (10 M), or 5-HT (10 M) plus ruthenium red (10 M), or 4␣-PDD (5 M), or 4␣-PDD (5 M) plus ruthenium red (10 M). To avoid 5-HT degradation, iproniazid (100 M, a monoamine oxidase inhibitor) and ascorbic acid (600 M, an antioxidant) were added in the 5-HT containing well [19]. Reagents-free media (0.2% and 10% FCS) were used as control conditions. Each condition was tested in triplicate well, and experiments were performed with three rats. Following 24 h incubation under these conditions, the media were removed and replaced with medium containing BrdU (10 M), and cells were incubated for an additional 2 h at 37 ◦ C. DNA synthesis was then assayed using colorimetric method, according to the manufacturer’s instructions. Newly synthesized BrdU-DNA was determined using an EL808 ultra microplate reader (Bio-Tek Instruments) at a wavelength of 380 nm with a reference wavelength of 490 nm.
2.7. Data and statistical analysis Results are expressed as mean ± S.E.M. Each experiment was repeated several times (n indicates the number of cells). Student’s t-test was used for statistical comparison among means and differences with P < 0.05 were considered significant.
3. Results 3.1. Role of extracellular and intracellular calcium in 5-HT response All the cells analyzed exhibited a stable resting [Ca2+ ]i (155 ± 8 nM, n = 19). In 73% of tested cells (n = 26), application of 5-HT (10 M) for 1 min elicited a fast increase in [Ca2+ ]i (Fig. 1Aa). This 5-HT-induced response was characterized by a biphasic increase in [Ca2+ ]i , consisting in a transient phase (named “calcium peak”) followed by a sustained phase (named “calcium plateau”). In the absence of extracellular Ca2+ , resting [Ca2+ ]i was only slightly reduced (106 ± 10 nM, n = 11, P < 0.05). In this condition, the “calcium plateau” of the 5-HT-induced [Ca2+ ]i response disappeared (Fig. 1Ab). Indeed, the amplitude of the “calcium plateau” was 10 ± 4 nM (n = 11) and 70 ± 9 nM (n = 19) in the absence and presence of external Ca2+ , respectively (Fig. 1Bb). On the other hand, the amplitude of the “calcium peak” was not significantly different in the absence or presence of external Ca2+ (119 ± 15 nM, n = 11 and 151 ± 14 nM, n = 19, respectively, Fig. 1Ba), confirming that the calcium peak was related to intracellular Ca2+ release, as already described [20]. 3.2. Role of arachidonic acid sensitive signalling pathway in the 5-HT-induced sustained calcium response We have previously shown that 5-HT induces an arachidonic acid-sensitive calcium influx in PASMC [7]. In the following set of experiments, we focused on 5-HT-induced sustained Ca2+ influx and we investigated whether this 5-HT calcium influx requires the arachidonic acid metabolization. First, we checked that arachidonic acid (10 M) induces a [Ca2+ ]i -response which was devoid of a transient phase (Fig. 2Aa). Indeed, the amplitude of the plateau reached 111 ± 16 nM (n = 11). In addition, ETYA (5,8,11,13eicosatetraynoic acid, 10 M), a non-metabolizable analogue of arachidonic acid, did not increase [Ca2+ ]i (n = 19, Fig. 2Aa), suggesting that 5-HT activates a Ca2+ influx via an arachidonic acid metabolite. PASMC were then bathed in
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Fig. 1. Role of extracellular calcium in the serotonin-induced calcium response in PASMC. (A) [Ca2+ ]i determination was carried out on single cell using indo-1 as Ca2+ probe. Cells were bathed in recording medium containing 2 mM Ca2+ (a) or in Ca2+ -free solution (b). Typical recordings when 5-HT (10 M) was continuously applied for the period indicated by the horizontal bar are shown in (a) and (b). (B) Amplitude of the transient (a) and sustained (b) components of the Ca2+ response to 5-HT in the presence (grey bar) or absence (white bar) of extracellular Ca2+ . Data are mean value ± S.E.M. The number of cells is indicated in the brackets. Significant difference is indicated by two asterisks when P < 0.01, ns indicates a non-significant difference.
Krebs-HEPES solution containing inhibitors of the different aracidonic acid metabolism pathways (Fig. 2Ab). Whatever the inhibitor used, the 5-HT-induced transient phase was unaffected (Fig. 2B). ETYA (10 M), a non-specific blocker of all arachidonic acid-metabolizing enzymes, significantly inhibited the sustained Ca2+ response to 5-HT by 46% (n = 10, Fig. 2Ba and C). Indomethacin (50 M) (Fig. 2Bb), a cyclo-oxygenase inhibitor or CDC (cinnamyl3,4-dihydroxy-cyanocinnamic acid, 10 M) (Fig. 2Bc), a lipoxygenase inhibitor, did not significantly modified the 5HT response (n = 12, Fig. 2C). Inversely, in the presence of 17-ODYA (17-octadecynoic acid, 5 M), an inhibitor of the cytochrome P450 epoxygenase (Fig. 2Bd), the sustained Ca2+ response was reduced by 48% (n = 13, Fig. 2C). These data point out, for the first time, the involvement of the cytochrome P450 epoxygenase pathway in the 5-HTinduced [Ca2+ ]i response in PASMC. 3.3. Implication of TRPV in the 5-HT-induced sustained calcium response Previous studies have shown that the arachidonic acid metabolite epoxyeicosatrienoic acid (EET) activates TRPV4 [21,22] and that TRPV4 is the most abundantly expressed TRPV in PASMC [13]. We thus focused on the involvement of TRPV4 channels in the serotonin-induced sustained calcium response. In the presence of 300 M Ni2+ , a non-selective inorganic Ca2+ channel blocker, or 10 M ruthenium red, a TRPV channels blocker [23], the peak of the 5-HT-induced Ca2+ response was not affected whereas the calcium plateau was significantly decreased (Fig. 3Aa and Ab), the plateau response was reduced by 58 and 57%, respectively (n = 10 and 12, Fig. 3Ac).
In addition, the non-PKC activating phorbol derivative 4␣-PDD (5 M), a specific TRPV4 agonist [23], induced a [Ca2+ ]i response in PASMC (Fig. 3B) the amplitude of which was 114 ± 15 nM (n = 6). Interestingly, it should be noted that typically, the 4␣-PDD-induced [Ca2+ ]i response was deprived of an early transient phase. 3.4. Activation of a TRPV4-like current by 5-HT We then examined whether the 5-HT-induced sustained Ca2+ influx could be mediated by a cationic current through TRPV channels. The effect of 5-HT on basal ionic conductance was investigated using whole-cell patch-clamp recording. PASMC were clamped close to resting potential and 20 mV hyperpolarizing steps were periodically (every 30 s) injected to monitor membrane conductance. In these conditions, 10 M 5-HT induced a steady-state inward current (−67 ± 25 pA/pF, n = 4) associated with an increase in membrane conductance, as shown by the variation in current pulses resulting from constant hyperpolarizing pulses (Fig. 4A). The current–voltage relations were monitored by application of 500 ms voltage ramps from −120 to +80 mV, from a holding potential of 0 mV. In the presence of 5-HT (10 M), an inward and outward current was activated with the following characteristics: a moderately outward rectification, a current density of 11 ± 2 pA/pF (n = 6) at +80 mV and a reversal potential value of −19 ± 4 mV (n = 6, Fig. 4B). After pretreatment of PASMC with ruthenium red (10 M), the 5-HT-activated currents were markedly inhibited (Fig. 4B). Indeed, the current densities at −100 and +80 mV were decreased by 78% and 90%, respectively (Fig. 4C, n = 6). In addition, stimulation of the cells with 4␣-PDD (5 M) activated an outwardly rectifying current (Fig. 5A) with sim-
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Fig. 2. Effects of the blockers of arachidonic acid metabolism on the serotonin-induced calcium response in PASMC. (A) (a) [Ca2+ ]i determination was carried out on single cell using indo-1 as Ca2+ probe. Cells were bathed in recording medium containing 2 mM Ca2+ . Typical recordings when arachidonic acid (AA, 10 M, black trace) or 5,8,11,13-eicosatetraynoic acid (ETYA, 10 M, grey trace) were continuously applied for the period indicated by the horizontal bar. (b) Schematic overview of the arachidonic acid metabolization, including the metabolizing enzymes and corresponding inhibitors. 17-ODYA, 17-octadecynoic acid; CDC, cinnamyl-3,4-dihydroxy-cyanocinnamic acid; COX, cyclo-oxygenase; CYP450 , cytochrome P450 epoxygenase; EET, epoxyeicosatrienoic acids; ETYA, 5,8,11,13-eicosatetraynoic acid; HETE, hydroxyeicosatetraenoic acids; HPTE, hydroperoxyeicosatetraenoic acids; LOX, lipoxygenase; PG, prostaglandin; TX, thromboxane. (B) [Ca2+ ]i determination was carried out on single cell, bathed in recording medium containing 10 M ETYA (a), 50 M indomethacin (b), 10 M CDC (c) or 5 M 17-ODYA (d). Typical recordings when 5-HT (10 M) was continuously applied for the period indicated by the horizontal bar are shown in (a)–(d). (C) Amplitude of the sustained Ca2+ response to 5-HT in the presence of the mentioned inhibitors. Data are mean value ± S.E.M. The number of cells is indicated in the brackets. Significant difference is indicated by one asterisk when P < 0.05, ns indicates a non-significant difference.
ilar characteristics to the 5-HT-activated current (Fig. 4B). 4␣-PDD-activated current was also markedly inhibited by ruthenium red (10 M) (Fig. 5A). Indeed, the current densities at −100 and +80 mV were decreased by 97% and 85%, respectively (Fig. 5B, n = 4). Thus, taken together, our data are consistent with the contribution of a TRPV4-like current to the serotonin-induced sustained Ca2+ influx.
3.5. Implication of TRPV4-like current in 5-HT-induced proliferation of PASMC In another set of experiments, we investigated the potential implication of this TRPV4-like current in 5-HT mediated PASMC proliferation [2,24,25]. As shown in Fig. 6, when cells were incubated with 10 M 5-HT for 24 h in DMEM (0.2% FCS), 5-HT induced a significant stimulation of prolif-
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Fig. 3. Pharmacological characterization of the serotonin-induced calcium response in PASMC. (A) [Ca2+ ]i determination was carried out on single cell using indo-1 as Ca2+ probe. Cells were bathed in recording medium containing 300 M Ni2+ (a) or 10 M ruthenium red (b). Typical recordings when 5-HT (10 M) was continuously applied for the period indicated by the horizontal bar are shown in (a) and (b). (c) Amplitude of the sustained component of the Ca2+ response in the presence of the mentioned inhibitors. Data are mean value ± S.E.M. The number of cells is indicated in the brackets. Significant difference is indicated by one asterisk when P < 0.05. (B) Typical recording of [Ca2+ ]i determination, carried out on single cell, bathed in recording medium containing 2 mM Ca2+ , after stimulation with 4␣PDD (5 M) continuously applied for the period indicated by the horizontal bar.
eration (3.2-fold increase), close to that induced by DMEM (10% FCS). This 5-HT proliferative effect was inhibited by addition of ruthenium red (10 M). On the other hand, stimulation of cells with 4␣-PDD (5 M) did not significantly increase cell proliferation.
Fig. 4. Effects of serotonin on global basal ionic conductance and outwardly rectifying currents in PASMC. (A) Typical recordings obtained under voltage-clamp conditions. Cells were clamped close to resting potential. Hyperpolarizing steps (−20 mV) were periodically injected (every 30 s) to monitor membrane conductance. 5-HT (10 M) was continuously applied for the period indicated by the horizontal bar. (B) Whole-cell patch-clamp recording showing the representative trace of 10 M 5-HT-activated difference current elicited by 500 ms voltage ramps from −120 to +80 mV (see the inset) in control condition (2 mM Ca2+ ) and after prior administration of ruthenium red (10 M). The holding potential was 0 mV. (C) Summary data for 5-HT-induced mean difference current densities at −100 and +80 mV recorded under control conditions (2 mM Ca2+ ) or in the presence of ruthenium red (n = 6). Data are mean value ± S.E.M. Asterisk: Significantly different (P < 0.05) from control.
4. Discussion In this study, we have examined the effects of 5-HT on [Ca2+ ]i regulation and proliferation of PASMC. Our results indicate that both phenomena, which are key cellular elements in the pathogenesis of human pulmonary arterial hypertension [26], implicate the activation of a TRPV-like current. In accordance with a previous report [7], we have demonstrated that 5-HT increases [Ca2+ ]i as the result of a transient intracellular calcium mobilization followed by a sustained calcium entry. This latter has already been described as a voltage-independent, non-capacitative and arachidonic acid production-dependent calcium entry [7]. In the present paper, we first investigated whether arachidonic
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Fig. 5. Effects of 4␣-phorbol-12,13-didecanoate on outwardly rectifying currents in PASMC. (A) Whole-cell patch-clamp recording showing the representative trace of 5 M 4␣-PDD-activated difference current elicited by 500 ms voltage ramps from −120 to +80 mV (see the inset) in control condition (2 mM Ca2+ ) and after prior administration of ruthenium red (10 M). The holding potential was 0 mV. (B) Summary data for 4␣-PDD-induced mean difference current densities at −100 and +80 mV recorded under control conditions (2 mM Ca2+ ) or in the presence of ruthenium red (n = 4). Data are mean value ± S.E.M. Asterisk: Significantly different (P < 0.05) from control.
acid directly stimulates a Ca2+ -permeable channel or requires its metabolization. Using corresponding inhibitors of the different arachidonic acid-metabolizing enzymes, we demonstrated that the 5-HT-induced [Ca2+ ]i elevation implicates the cytochrome P450 epoxygenase and not the cyclo-oxygenase and lipoxygenase. Whereas the effect of cyclo-oxygenase and lipoxygenase products of arachidonic acid metabolism within the lung has been well characterized on vascular smooth
Fig. 6. Effects of ruthenium red on PASMC proliferation. Cell proliferation was assessed by quantitative determination of DNA synthesis using the Cell Proliferation ELISA BrdU colorimetric method. PASMC were incubated for 24 h in DMEM containing 0.2% FCS supplemented with 5-HT (10 M), or 5-HT (10 M) plus ruthenium red (10 M), or 4␣-PDD (5 M), or 4␣-PDD (5 M) plus ruthenium red (10 M) or DMEM containing 10% FCS before BrdU incorporation. Data represent the mean ± S.E.M. obtained in triplicate well, performed in three rats. RR: Ruthenium red. Asterisk: Significantly different (P < 0.01) from control (DMEM—0.2% FCS). ##: Significantly different (P < 0.01) from 5-HT. ns indicates a non-significant difference from control (DMEM—0.2% FCS).
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muscle, much less is known about the cytochrome P450 epoxygenase metabolites. On the one hand, the enzyme has been identified (mRNA and protein expression) in the lungs and in the pulmonary vascular smooth muscle cells in particular [27]. On the other hand, using gas chromatography/mass spectrometry [27,28], endogenous epoxyeicosatrienoic acids have also been detected, suggesting that these metabolites could modulate pulmonary vascular tone. However, experiments in isolated perfused lungs [29,30], isolated pressurized pulmonary arteries and pulmonary arterial rings [28] have generated conflicting results, depending on factors such as species, characteristics of the vessels (size, arteries versus veins), concentration of EET, potential influence of surrounding tissues, etc. Interestingly, Zhu et al. [28] have observed that, in rabbit pulmonary arteries, the vasopressor response to EET was concentration dependent and that the greatest constriction was observed in arteries stimulated with 5,6-EET which is also known to stimulate TRPV4 [22]. Moreover, a recent publication [31] showed that cytochrome P450 epoxygenase and its metabolites elicited pulmonary vasoconstriction in mice exposed to hypoxia. Indeed, cytochrome P450 epoxygenase protein expression and EET production were significantly increased after exposure to hypoxia. A selective epoxygenase inhibition attenuated acute hypoxic pulmonary vasoconstriction and chronic hypoxia-induced pulmonary vascular remodelling, whereas inhibitor of the soluble epoxide hydrolase enhanced the vasoconstrictor response. These findings might be related to the high circulating serotonin [4], hyperreactivity to 5-HT [6] and mitogenic effect of 5-HT [2] reported in various forms of pulmonary arterial hypertension. Our results indicate that the 5-HT-induced sustained Ca2+ influx could be mediated by Ca2+ influx through TRPV4 channels. Indeed, using microspectrofluorimetry and patchclamp in whole-cell configuration, we showed that serotonin induces a calcium influx whose properties are consistent with those previously described of TRPV4 current in vascular smooth muscle cells [21], i.e., a moderately outwardly rectifying current, a similar current density and reversal potential, and finally a similar sensitivity to pharmacological agents such as Ni2+ and ruthenium red [13,21]. Moreover, both 5-HT-induced sustained [Ca2+ ]i increase and current were mimicked by the selective TRPV4 agonist 4␣-PDD. Recently, in cerebral arterial smooth muscle cells, TRPV4 was shown to form a Ca2+ signalling complex with ryanodine receptors and BKCa channels [21]. Its activation by 11,12-EET induced a Ca2+ influx, which then activated ryanodine receptors to generate Ca2+ sparks activating, in turn, the closely coupled KCa channels resulting in spontaneous transient outward currents, to elicite membrane hyperpolarization and vasodilatation. However, in PASMC, activation of TRPVlike current by 5-HT would probably result in contraction because 11,12-EET was reported to elicit vasoconstriction in rabbit pulmonary arteries [28] and Ca2+ sparks cause membrane depolarization instead of hyperpolarization [32]. Furthermore, our results suggest the implication of TRPV4-like current in potentialization of 5-HT-induced
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PASMC proliferation since 5-HT-induced BrdU incorporation levels was inhibited by ruthenium red. If the role of 5-HT in cell proliferation is well established, the mechanism by which 5-HT intervened is still a matter of debate. In PASMC, 5-HT receptor activation increases [Ca2+ ]i . This latter may promote PASMC proliferation by induction of proliferation-associated immediate early genes, including cfos and c-jun, and activating cytoplasmic signal transduction proteins such as Ca2+ /cAMP response element binding protein (CREB) and mitogen-activated protein kinase (MAPK) [33]. In most cases, cell proliferation requires millimolar external calcium concentrations and is inhibited when cells are cultured in a Ca2+ -deprived or Ca2+ channel blockerssupplemented culture medium. The role of ion channels in PASMC proliferation is currently under investigation since the link between proliferation and ion channel activation may represent a molecular target for the treatment of pulmonary hypertension. Participation of TRPV channels in mitosis has already been demonstrated in different cell models. Indeed, TRPV6 was shown to potentiate Ca2+ -dependent HEK-293 cell proliferation [34]. In addition, TRPV6 may play an important role in the progression of prostate cancer: the appearance of TRPV6 transcripts significantly correlates with the prostate cancer stages [35]. Another publication [36] demonstrated the role of TRPV2 in the mitosis neuronal precursor and neuroendocrine cells. However, in our present paper, PASMC stimulation with 4␣-PDD did not significantly increase cell proliferation, showing that the Ca2+ influx through TRPV4 channels per se is not efficient to induce PASMC proliferation and that this former only potentiates the 5-HT proliferative effect. In conclusion, this study demonstrates for the first time, the implication of TRPV4-like current in 5-HT-induced Ca2+ response in PASMC. We propose that TRPV4 channels are responsible for the sustained Ca2+ elevation contributing to both PASMC contraction and proliferation as the result of arachidonic acid-derived EET stimulation. The signalling pathway between proliferation and ion channel activation remains to be determined and may represent a molecular target for the treatment of vascular diseases such as pulmonary hypertension. Acknowledgements We are grateful to Pr. P. Gailly for helpful discussions. This work was supported by the Conseil R´egional d’Aquitaine (200220301301A), the Agence National de la Recherche (ANR06-physio-015-01) and the Fondation de France (2006005603). References [1] M.R. MacLean, P. Herve, S. Eddahibi, S. Adnot, 5-Hydroxytryptamine and the pulmonary circulation: receptors, transporters and relevance to pulmonary arterial hypertension, Br. J. Pharmacol. 131 (2000) 161–168.
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Serotonin-induced activation of TRPV4-like current in rat intrapulmonary arterial smooth muscle cells Thomas Ducret a,b,∗ , Christelle Guibert a,b , Roger Marthan a,b , Jean-Pierre Savineau a,b a
Universit´e Bordeaux 2, Laboratoire de Physiologie Cellulaire Respiratoire, 146 rue L´eo-Saignat, F-33076 Bordeaux, France b INSERM, U885, 146 rue L´ eo-Saignat, F-33076 Bordeaux, France Received 22 January 2007; received in revised form 20 April 2007; accepted 30 May 2007 Available online 31 July 2007
Abstract In the present study, we investigated the implication of transient receptor potential vanilloid (TRPV)-related channels in the 5hydroxytryptamine (5-HT)-induced both intracellular calcium response and mitogenic effect in rat pulmonary arterial smooth muscle cells (PASMC). Using microspectrofluorimetry (indo-1 as Ca2+ fluorescent probe) and the patch-clamp technique (in whole-cell configuration), we found that 5-HT (10 M) induced a transient intracellular calcium mobilization followed by a sustained calcium entry. This latter was partly blocked by an inhibitor of cytochrome P450 epoxygenase (17-ODYA) and insensitive to cyclo-oxygenase and lipoxygenase inhibitors (indomethacin and CDC), suggesting the involvement of arachidonic acid metabolization by cytochrome P450 epoxygenase. This calcium influx was also sensitive to Ni2+ and to ruthenium red, a TRPV channel blocker, and mimicked by 4␣-phorbol-12,13-didecanoate (4␣-PDD), a TRPV4 channel agonist. In patched PASMC, 5-HT and 4␣-PDD-activated TRPV4-like ruthenium red sensitive currents with typical characteristics. Furthermore, 5-HT induced a ruthenium red sensitive increase in BrdU incorporation levels in PASMC. The present study provides evidence that 5-HT activates a TRPV4-like current, potentially involved in PASMC proliferation. The signalling pathway between proliferation and ion channel activation remains to be determined and may represent a molecular target for the treatment of vascular diseases such as pulmonary hypertension. © 2007 Elsevier Ltd. All rights reserved. Keywords: Arachidonic acid metabolites; Calcium influx; Serotonin; Smooth muscle cells proliferation; TRPV channels; Vascular smooth muscle
1. Introduction In pulmonary arteries, 5-hydroxytryptamine (5-HT) exerts vasoconstrictor and mitogenic effects [1,2] both phenomena being dependent on an increase in cytosolic calcium concentration ([Ca2+ ]i ) [3]. Pulmonary arterial hypertension (PAH) is characterized by high circulating 5-HT concentration [4], 5-HT-induced hyperreactivity and SMC proliferation [2,5,6] suggesting a major role for 5-HT in both vascular wall remodelling and elevated vascular resistances that characterize PAH. Thus, a better understanding of the calcium-related signalling pathway activated by 5-HT is of major importance in the pulmonary vascular bed. ∗
Corresponding author. Tel.: +33 5 57 57 16 94; fax: +33 5 57 57 16 95. E-mail address: [email protected] (T. Ducret).
0143-4160/$ – see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.ceca.2007.05.018
However, calcium sources involved in 5-HT-induced calcium response are still unresolved. In rat isolated small intrapulmonary arteries (IPA) and primary cultured pulmonary arterial smooth muscle cells (PASMC), we recently demonstrated that activation of 5-HT2A receptors by 5-HT led to a biphasic increase in [Ca2+ ]i , namely a transient phase due to the mobilization of Ca2+ stored in intracellular compartments, followed by a sustained phase due to Ca2+ entry from the extracellular medium [5,7]. We have previously found that the sustained phase was a non-capacitative Ca2+ entry corresponding at least, in part, to a voltage-independent Ca2+ influx sensitive to arachidonic acid production [7]. In a variety of tissues, transient receptor potential (TRP) proteins have been proposed as good candidates for the molecular structure underlying voltage-independent Ca2+ influx [8].
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TRP channels are non-selective cationic channels expressed in a large variety of tissues including smooth muscles. They are implicated in diverse cellular functions such as calcium homeostasis and cell proliferation [9,10]. TRP channels are characterized by six transmembranespanning domains, which form tetramers [11]. Based on structural homology, TRP superfamily can be subdivided into seven main subfamilies: TRPC (‘Canonical’), TRPV (‘Vanilloid’), TRPM (‘Melastatin’), TRPP (‘Polycystin’), TRPML (‘Mucolipin’), TRPA (‘Ankyrin’) and TRPN (‘NOMPC’). In PASMC, TRPC, TRPV and TRPM are expressed and functional [12,13] and among the TRPV subfamily, TRPV4 was the most abundantly expressed [13]. TRPV4 is a Ca2+ permeable non-selective cationic channel, activated by a wide variety of stimuli including physical, thermal and chemical stimuli such as the synthetic agonist 4␣-phorbol-12,13didecanoate (4␣-PDD), the endocannabinoid anandamine and the arachidonic acid metabolite epoxyeicosatrienoic acid (EET) [14] in particular. Moreover, 4␣-PDD induced an increase in [Ca2+ ]i in rat PASMC [13]. TRPV4 thus appears as a good candidate for mediating the Ca2+ response to 5-HT in PASMC. In the present study, we have examined the calcium influx activated by 5-HT in rat intrapulmonary arteries. We focused our study on the arachidonic acid sensitive TRPV channel such as the TRPV4-like channel by recording [Ca2+ ]i (microspectrofluorimetric assay) and plasma membrane currents (patch-clamp in a “whole-cell” mode). Since intracellular Ca2+ and 5-HT interfere with SMC proliferation, we also explored the role of the TRPV-like channels in 5-HT-induced PASMC proliferation (BrdU incorporation).
2. Materials and methods 2.1. PASMC isolation and culture As previously described [15], intrapulmonary arteries were dissected from lungs of male Wistar rats (250–350 g body weight), according to the animal care and use of our local ethic committee (Comit´e d’´ethique r´egional d’Aquitaine, AP 2/11/2005). Briefly, the entire heart-lung preparation was rapidly removed en bloc and rinsed in culture medium (DMEM-HEPES supplemented with 1% penicillin–streptomycin, 1% Na-pyruvate and 1% nonessential amino acids). Intrapulmonary arteries of the first and second orders from the left lung were dissected free from surrounding connective tissues under binocular control and sterile conditions. Endothelium was removed by rubbing the luminal surface. The arteries were then cut in several pieces (1 mm × 1 mm) and seeded on round glass coverslips (30 mm diameter) for microspectrofluorimetry experiments and Petri dishes for electrophysiological experiments. Explants were cultured in culture medium enriched with 10% foetal calf serum (FCS). They were maintained at 37 ◦ C in a humidified atmosphere gassed with 5% CO2 . Fifty percent of the medium
was changed everyday until cells go out of the explants (3–7 days after seeding). Before the experiments, PASMC were growth-arrested during 48 h by using serum-free culture medium supplemented with 1% insulin-transferrin-selenium (ITS). Smooth muscle characteristics (elongated fusiform shape and formation of “ hills and valleys”) of isolated cells were confirmed by positive immunostaining with an anti-␣ smooth muscle actin antibody (Sigma). 2.2. Microspectrofluorimetric assay of cytosolic calcium The Ca2+ -sensitive fluorescent probe indo-1 was used to record changes in [Ca2+ ]i . The cells plated on glass coverslips were incubated with 5 M indo-1 penta-acetoxymethyl ester (indo-1/AM) in Krebs-HEPES solution (see composition below) at room temperature for 40 min, then washed and maintained at room temperature in the same saline solution before the fluorescence measurements. For single cell measurements, the dual emission microspectrofluorimeter was constructed from a Nikon Diaphot inverted microscope fitted with epifluorescence (40× oil immersion fluorescence objective; numerical aperture, 1.3). For excitation of indo-1, a collimated light beam from a 100-W mercury arc lamp (Nikon) was filtered at 355 nm and reflected from a dichroic mirror (380 nm). The emitted fluorescence signal was passed through a pinhole diaphragm slightly larger than the selected cell and directed onto another dichroic mirror (455 nm). Transmitted light was filtered at 480 nm, reflected light was filtered at 405 nm and the intensities were recorded by separate photometers (P100, Nikon). Under these experimental conditions, the fluorescence ratio (F405 /F480 ) was calculated and recorded on-line as a voltage signal. [Ca2+ ]i was estimated from the F405 /F480 using the formula derived by Grynkiewicz et al. [16] after Ca2+ calibration for indo-1 determined within cells as previously described [17]. The sustained [Ca2+ ]i value was estimated as the amplitude of the plateau phase measured 30 s after the peak was reached. 2.3. Electrophysiological recordings The whole-cell recording mode of the patch-clamp technique was used [18]. The electrodes were pulled on a PC-10 (Narishige) puller in two stages from borosilicate glass capillaries (1.5 mm o.d., 1.16 mm i.d., Harvard Apparatus). The pipettes had a mean resistance of 4–6 M when measured in standard recording conditions. Cells were viewed under phase contrast with a Nikon Diaphot inverted microscope. A RK-400 patch-clamp amplifier (Biologic) was used for whole-cell recordings. Stimulus control, data acquisition, and processing were carried out on a PC computer fitted with a Digidata 1200 interface, using pCLAMP 6.0.2 software (interface and software, Axon Instruments). Current records were filtered with a Bessel filter at 2 kHz and digitized at 10 kHz for storage and analysis. For all experiments, currents were normalized to cell capacitance.
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2.4. Recording solutions and application of substances
2.6. Reagents
For whole-cell voltage-clamp and microspectrofluorimetry studies, the standard Krebs-HEPES extracellular solution contained (in mM): 118.4 NaCl, 4.7 KCl, 2 CaCl2 , 1.2 MgSO4 , 4 NaHCO3 , 1.2 KH2 PO4 , 6 d-glucose and 10 N-2hydroxyethylpiperazine-N -2-ethanesulfonic acid (HEPES). The osmolality (measured with a cryoosmometer type 15 L¨oser) of the external salt solution was adjusted to 300 mosm/kg with sucrose, and pH adjusted to 7.4 with NaOH. Solutions nominally free of Ca2+ were prepared by omitting CaCl2 and adding 0.4 mM EGTA. For steady-state whole-cell studies, the recording pipette was filled with an artificial intracellular saline containing (in mM): 130 CsCl, 2 MgCl2 , 0.2 EGTA, 3 Na2 ATP and 10 HEPES (pH 7.2; osmolality: 290 mosm/kg). To eliminate K+ currents, TEA (10 mM) was added to the extracellular solution. For voltage ramp whole-cell studies, the recording pipette was filled with (in mM): 100 Na-glutamate, 20 NaCl, 1 MgCl2 , 1 EGTA, 4 Na2 ATP and 10 HEPES (pH 7.2; osmolality: 290 mosm/kg). For electrophysiological and microspectrofluorimetric studies, 5-HT, arachidonic acid and 4␣-PDD were applied to the recorded cell by pressure ejection from a glass pipette located close to the cell for the period indicated on the records. It was verified, in control experiments, that no change in [Ca2+ ]i was observed during test ejections of Krebs-HEPES.
General salts were from VWR. All other chemicals were purchased from Sigma, except DMEM-HEPES, FCS and penicillin–streptomycin which were from Gibco.
2.5. Cell proliferation assessment Quantitative determination of DNA synthesis using the Cell Proliferation ELISA, BrdU colorimetric method (Roche Applied Science), was used to assess PASMC proliferation. Briefly, cells were seeded at a density of 5 × 103 cells per well in a standard 96-well tissue culture plate in a volume of 100 l medium/well and cultured in complete culture medium containing 10% FCS in a humidified incubator at 37 ◦ C. After 48 h, the medium was removed and replaced by a serum-free medium containing 1% ITS. After an additional period of 48 h at 37 ◦ C, the medium was changed to complete culture medium containing 0.2% serum, supplemented with 5-HT (10 M), or 5-HT (10 M) plus ruthenium red (10 M), or 4␣-PDD (5 M), or 4␣-PDD (5 M) plus ruthenium red (10 M). To avoid 5-HT degradation, iproniazid (100 M, a monoamine oxidase inhibitor) and ascorbic acid (600 M, an antioxidant) were added in the 5-HT containing well [19]. Reagents-free media (0.2% and 10% FCS) were used as control conditions. Each condition was tested in triplicate well, and experiments were performed with three rats. Following 24 h incubation under these conditions, the media were removed and replaced with medium containing BrdU (10 M), and cells were incubated for an additional 2 h at 37 ◦ C. DNA synthesis was then assayed using colorimetric method, according to the manufacturer’s instructions. Newly synthesized BrdU-DNA was determined using an EL808 ultra microplate reader (Bio-Tek Instruments) at a wavelength of 380 nm with a reference wavelength of 490 nm.
2.7. Data and statistical analysis Results are expressed as mean ± S.E.M. Each experiment was repeated several times (n indicates the number of cells). Student’s t-test was used for statistical comparison among means and differences with P < 0.05 were considered significant.
3. Results 3.1. Role of extracellular and intracellular calcium in 5-HT response All the cells analyzed exhibited a stable resting [Ca2+ ]i (155 ± 8 nM, n = 19). In 73% of tested cells (n = 26), application of 5-HT (10 M) for 1 min elicited a fast increase in [Ca2+ ]i (Fig. 1Aa). This 5-HT-induced response was characterized by a biphasic increase in [Ca2+ ]i , consisting in a transient phase (named “calcium peak”) followed by a sustained phase (named “calcium plateau”). In the absence of extracellular Ca2+ , resting [Ca2+ ]i was only slightly reduced (106 ± 10 nM, n = 11, P < 0.05). In this condition, the “calcium plateau” of the 5-HT-induced [Ca2+ ]i response disappeared (Fig. 1Ab). Indeed, the amplitude of the “calcium plateau” was 10 ± 4 nM (n = 11) and 70 ± 9 nM (n = 19) in the absence and presence of external Ca2+ , respectively (Fig. 1Bb). On the other hand, the amplitude of the “calcium peak” was not significantly different in the absence or presence of external Ca2+ (119 ± 15 nM, n = 11 and 151 ± 14 nM, n = 19, respectively, Fig. 1Ba), confirming that the calcium peak was related to intracellular Ca2+ release, as already described [20]. 3.2. Role of arachidonic acid sensitive signalling pathway in the 5-HT-induced sustained calcium response We have previously shown that 5-HT induces an arachidonic acid-sensitive calcium influx in PASMC [7]. In the following set of experiments, we focused on 5-HT-induced sustained Ca2+ influx and we investigated whether this 5-HT calcium influx requires the arachidonic acid metabolization. First, we checked that arachidonic acid (10 M) induces a [Ca2+ ]i -response which was devoid of a transient phase (Fig. 2Aa). Indeed, the amplitude of the plateau reached 111 ± 16 nM (n = 11). In addition, ETYA (5,8,11,13eicosatetraynoic acid, 10 M), a non-metabolizable analogue of arachidonic acid, did not increase [Ca2+ ]i (n = 19, Fig. 2Aa), suggesting that 5-HT activates a Ca2+ influx via an arachidonic acid metabolite. PASMC were then bathed in
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Fig. 1. Role of extracellular calcium in the serotonin-induced calcium response in PASMC. (A) [Ca2+ ]i determination was carried out on single cell using indo-1 as Ca2+ probe. Cells were bathed in recording medium containing 2 mM Ca2+ (a) or in Ca2+ -free solution (b). Typical recordings when 5-HT (10 M) was continuously applied for the period indicated by the horizontal bar are shown in (a) and (b). (B) Amplitude of the transient (a) and sustained (b) components of the Ca2+ response to 5-HT in the presence (grey bar) or absence (white bar) of extracellular Ca2+ . Data are mean value ± S.E.M. The number of cells is indicated in the brackets. Significant difference is indicated by two asterisks when P < 0.01, ns indicates a non-significant difference.
Krebs-HEPES solution containing inhibitors of the different aracidonic acid metabolism pathways (Fig. 2Ab). Whatever the inhibitor used, the 5-HT-induced transient phase was unaffected (Fig. 2B). ETYA (10 M), a non-specific blocker of all arachidonic acid-metabolizing enzymes, significantly inhibited the sustained Ca2+ response to 5-HT by 46% (n = 10, Fig. 2Ba and C). Indomethacin (50 M) (Fig. 2Bb), a cyclo-oxygenase inhibitor or CDC (cinnamyl3,4-dihydroxy-cyanocinnamic acid, 10 M) (Fig. 2Bc), a lipoxygenase inhibitor, did not significantly modified the 5HT response (n = 12, Fig. 2C). Inversely, in the presence of 17-ODYA (17-octadecynoic acid, 5 M), an inhibitor of the cytochrome P450 epoxygenase (Fig. 2Bd), the sustained Ca2+ response was reduced by 48% (n = 13, Fig. 2C). These data point out, for the first time, the involvement of the cytochrome P450 epoxygenase pathway in the 5-HTinduced [Ca2+ ]i response in PASMC. 3.3. Implication of TRPV in the 5-HT-induced sustained calcium response Previous studies have shown that the arachidonic acid metabolite epoxyeicosatrienoic acid (EET) activates TRPV4 [21,22] and that TRPV4 is the most abundantly expressed TRPV in PASMC [13]. We thus focused on the involvement of TRPV4 channels in the serotonin-induced sustained calcium response. In the presence of 300 M Ni2+ , a non-selective inorganic Ca2+ channel blocker, or 10 M ruthenium red, a TRPV channels blocker [23], the peak of the 5-HT-induced Ca2+ response was not affected whereas the calcium plateau was significantly decreased (Fig. 3Aa and Ab), the plateau response was reduced by 58 and 57%, respectively (n = 10 and 12, Fig. 3Ac).
In addition, the non-PKC activating phorbol derivative 4␣-PDD (5 M), a specific TRPV4 agonist [23], induced a [Ca2+ ]i response in PASMC (Fig. 3B) the amplitude of which was 114 ± 15 nM (n = 6). Interestingly, it should be noted that typically, the 4␣-PDD-induced [Ca2+ ]i response was deprived of an early transient phase. 3.4. Activation of a TRPV4-like current by 5-HT We then examined whether the 5-HT-induced sustained Ca2+ influx could be mediated by a cationic current through TRPV channels. The effect of 5-HT on basal ionic conductance was investigated using whole-cell patch-clamp recording. PASMC were clamped close to resting potential and 20 mV hyperpolarizing steps were periodically (every 30 s) injected to monitor membrane conductance. In these conditions, 10 M 5-HT induced a steady-state inward current (−67 ± 25 pA/pF, n = 4) associated with an increase in membrane conductance, as shown by the variation in current pulses resulting from constant hyperpolarizing pulses (Fig. 4A). The current–voltage relations were monitored by application of 500 ms voltage ramps from −120 to +80 mV, from a holding potential of 0 mV. In the presence of 5-HT (10 M), an inward and outward current was activated with the following characteristics: a moderately outward rectification, a current density of 11 ± 2 pA/pF (n = 6) at +80 mV and a reversal potential value of −19 ± 4 mV (n = 6, Fig. 4B). After pretreatment of PASMC with ruthenium red (10 M), the 5-HT-activated currents were markedly inhibited (Fig. 4B). Indeed, the current densities at −100 and +80 mV were decreased by 78% and 90%, respectively (Fig. 4C, n = 6). In addition, stimulation of the cells with 4␣-PDD (5 M) activated an outwardly rectifying current (Fig. 5A) with sim-
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Fig. 2. Effects of the blockers of arachidonic acid metabolism on the serotonin-induced calcium response in PASMC. (A) (a) [Ca2+ ]i determination was carried out on single cell using indo-1 as Ca2+ probe. Cells were bathed in recording medium containing 2 mM Ca2+ . Typical recordings when arachidonic acid (AA, 10 M, black trace) or 5,8,11,13-eicosatetraynoic acid (ETYA, 10 M, grey trace) were continuously applied for the period indicated by the horizontal bar. (b) Schematic overview of the arachidonic acid metabolization, including the metabolizing enzymes and corresponding inhibitors. 17-ODYA, 17-octadecynoic acid; CDC, cinnamyl-3,4-dihydroxy-cyanocinnamic acid; COX, cyclo-oxygenase; CYP450 , cytochrome P450 epoxygenase; EET, epoxyeicosatrienoic acids; ETYA, 5,8,11,13-eicosatetraynoic acid; HETE, hydroxyeicosatetraenoic acids; HPTE, hydroperoxyeicosatetraenoic acids; LOX, lipoxygenase; PG, prostaglandin; TX, thromboxane. (B) [Ca2+ ]i determination was carried out on single cell, bathed in recording medium containing 10 M ETYA (a), 50 M indomethacin (b), 10 M CDC (c) or 5 M 17-ODYA (d). Typical recordings when 5-HT (10 M) was continuously applied for the period indicated by the horizontal bar are shown in (a)–(d). (C) Amplitude of the sustained Ca2+ response to 5-HT in the presence of the mentioned inhibitors. Data are mean value ± S.E.M. The number of cells is indicated in the brackets. Significant difference is indicated by one asterisk when P < 0.05, ns indicates a non-significant difference.
ilar characteristics to the 5-HT-activated current (Fig. 4B). 4␣-PDD-activated current was also markedly inhibited by ruthenium red (10 M) (Fig. 5A). Indeed, the current densities at −100 and +80 mV were decreased by 97% and 85%, respectively (Fig. 5B, n = 4). Thus, taken together, our data are consistent with the contribution of a TRPV4-like current to the serotonin-induced sustained Ca2+ influx.
3.5. Implication of TRPV4-like current in 5-HT-induced proliferation of PASMC In another set of experiments, we investigated the potential implication of this TRPV4-like current in 5-HT mediated PASMC proliferation [2,24,25]. As shown in Fig. 6, when cells were incubated with 10 M 5-HT for 24 h in DMEM (0.2% FCS), 5-HT induced a significant stimulation of prolif-
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Fig. 3. Pharmacological characterization of the serotonin-induced calcium response in PASMC. (A) [Ca2+ ]i determination was carried out on single cell using indo-1 as Ca2+ probe. Cells were bathed in recording medium containing 300 M Ni2+ (a) or 10 M ruthenium red (b). Typical recordings when 5-HT (10 M) was continuously applied for the period indicated by the horizontal bar are shown in (a) and (b). (c) Amplitude of the sustained component of the Ca2+ response in the presence of the mentioned inhibitors. Data are mean value ± S.E.M. The number of cells is indicated in the brackets. Significant difference is indicated by one asterisk when P < 0.05. (B) Typical recording of [Ca2+ ]i determination, carried out on single cell, bathed in recording medium containing 2 mM Ca2+ , after stimulation with 4␣PDD (5 M) continuously applied for the period indicated by the horizontal bar.
eration (3.2-fold increase), close to that induced by DMEM (10% FCS). This 5-HT proliferative effect was inhibited by addition of ruthenium red (10 M). On the other hand, stimulation of cells with 4␣-PDD (5 M) did not significantly increase cell proliferation.
Fig. 4. Effects of serotonin on global basal ionic conductance and outwardly rectifying currents in PASMC. (A) Typical recordings obtained under voltage-clamp conditions. Cells were clamped close to resting potential. Hyperpolarizing steps (−20 mV) were periodically injected (every 30 s) to monitor membrane conductance. 5-HT (10 M) was continuously applied for the period indicated by the horizontal bar. (B) Whole-cell patch-clamp recording showing the representative trace of 10 M 5-HT-activated difference current elicited by 500 ms voltage ramps from −120 to +80 mV (see the inset) in control condition (2 mM Ca2+ ) and after prior administration of ruthenium red (10 M). The holding potential was 0 mV. (C) Summary data for 5-HT-induced mean difference current densities at −100 and +80 mV recorded under control conditions (2 mM Ca2+ ) or in the presence of ruthenium red (n = 6). Data are mean value ± S.E.M. Asterisk: Significantly different (P < 0.05) from control.
4. Discussion In this study, we have examined the effects of 5-HT on [Ca2+ ]i regulation and proliferation of PASMC. Our results indicate that both phenomena, which are key cellular elements in the pathogenesis of human pulmonary arterial hypertension [26], implicate the activation of a TRPV-like current. In accordance with a previous report [7], we have demonstrated that 5-HT increases [Ca2+ ]i as the result of a transient intracellular calcium mobilization followed by a sustained calcium entry. This latter has already been described as a voltage-independent, non-capacitative and arachidonic acid production-dependent calcium entry [7]. In the present paper, we first investigated whether arachidonic
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Fig. 5. Effects of 4␣-phorbol-12,13-didecanoate on outwardly rectifying currents in PASMC. (A) Whole-cell patch-clamp recording showing the representative trace of 5 M 4␣-PDD-activated difference current elicited by 500 ms voltage ramps from −120 to +80 mV (see the inset) in control condition (2 mM Ca2+ ) and after prior administration of ruthenium red (10 M). The holding potential was 0 mV. (B) Summary data for 4␣-PDD-induced mean difference current densities at −100 and +80 mV recorded under control conditions (2 mM Ca2+ ) or in the presence of ruthenium red (n = 4). Data are mean value ± S.E.M. Asterisk: Significantly different (P < 0.05) from control.
acid directly stimulates a Ca2+ -permeable channel or requires its metabolization. Using corresponding inhibitors of the different arachidonic acid-metabolizing enzymes, we demonstrated that the 5-HT-induced [Ca2+ ]i elevation implicates the cytochrome P450 epoxygenase and not the cyclo-oxygenase and lipoxygenase. Whereas the effect of cyclo-oxygenase and lipoxygenase products of arachidonic acid metabolism within the lung has been well characterized on vascular smooth
Fig. 6. Effects of ruthenium red on PASMC proliferation. Cell proliferation was assessed by quantitative determination of DNA synthesis using the Cell Proliferation ELISA BrdU colorimetric method. PASMC were incubated for 24 h in DMEM containing 0.2% FCS supplemented with 5-HT (10 M), or 5-HT (10 M) plus ruthenium red (10 M), or 4␣-PDD (5 M), or 4␣-PDD (5 M) plus ruthenium red (10 M) or DMEM containing 10% FCS before BrdU incorporation. Data represent the mean ± S.E.M. obtained in triplicate well, performed in three rats. RR: Ruthenium red. Asterisk: Significantly different (P < 0.01) from control (DMEM—0.2% FCS). ##: Significantly different (P < 0.01) from 5-HT. ns indicates a non-significant difference from control (DMEM—0.2% FCS).
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muscle, much less is known about the cytochrome P450 epoxygenase metabolites. On the one hand, the enzyme has been identified (mRNA and protein expression) in the lungs and in the pulmonary vascular smooth muscle cells in particular [27]. On the other hand, using gas chromatography/mass spectrometry [27,28], endogenous epoxyeicosatrienoic acids have also been detected, suggesting that these metabolites could modulate pulmonary vascular tone. However, experiments in isolated perfused lungs [29,30], isolated pressurized pulmonary arteries and pulmonary arterial rings [28] have generated conflicting results, depending on factors such as species, characteristics of the vessels (size, arteries versus veins), concentration of EET, potential influence of surrounding tissues, etc. Interestingly, Zhu et al. [28] have observed that, in rabbit pulmonary arteries, the vasopressor response to EET was concentration dependent and that the greatest constriction was observed in arteries stimulated with 5,6-EET which is also known to stimulate TRPV4 [22]. Moreover, a recent publication [31] showed that cytochrome P450 epoxygenase and its metabolites elicited pulmonary vasoconstriction in mice exposed to hypoxia. Indeed, cytochrome P450 epoxygenase protein expression and EET production were significantly increased after exposure to hypoxia. A selective epoxygenase inhibition attenuated acute hypoxic pulmonary vasoconstriction and chronic hypoxia-induced pulmonary vascular remodelling, whereas inhibitor of the soluble epoxide hydrolase enhanced the vasoconstrictor response. These findings might be related to the high circulating serotonin [4], hyperreactivity to 5-HT [6] and mitogenic effect of 5-HT [2] reported in various forms of pulmonary arterial hypertension. Our results indicate that the 5-HT-induced sustained Ca2+ influx could be mediated by Ca2+ influx through TRPV4 channels. Indeed, using microspectrofluorimetry and patchclamp in whole-cell configuration, we showed that serotonin induces a calcium influx whose properties are consistent with those previously described of TRPV4 current in vascular smooth muscle cells [21], i.e., a moderately outwardly rectifying current, a similar current density and reversal potential, and finally a similar sensitivity to pharmacological agents such as Ni2+ and ruthenium red [13,21]. Moreover, both 5-HT-induced sustained [Ca2+ ]i increase and current were mimicked by the selective TRPV4 agonist 4␣-PDD. Recently, in cerebral arterial smooth muscle cells, TRPV4 was shown to form a Ca2+ signalling complex with ryanodine receptors and BKCa channels [21]. Its activation by 11,12-EET induced a Ca2+ influx, which then activated ryanodine receptors to generate Ca2+ sparks activating, in turn, the closely coupled KCa channels resulting in spontaneous transient outward currents, to elicite membrane hyperpolarization and vasodilatation. However, in PASMC, activation of TRPVlike current by 5-HT would probably result in contraction because 11,12-EET was reported to elicit vasoconstriction in rabbit pulmonary arteries [28] and Ca2+ sparks cause membrane depolarization instead of hyperpolarization [32]. Furthermore, our results suggest the implication of TRPV4-like current in potentialization of 5-HT-induced
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PASMC proliferation since 5-HT-induced BrdU incorporation levels was inhibited by ruthenium red. If the role of 5-HT in cell proliferation is well established, the mechanism by which 5-HT intervened is still a matter of debate. In PASMC, 5-HT receptor activation increases [Ca2+ ]i . This latter may promote PASMC proliferation by induction of proliferation-associated immediate early genes, including cfos and c-jun, and activating cytoplasmic signal transduction proteins such as Ca2+ /cAMP response element binding protein (CREB) and mitogen-activated protein kinase (MAPK) [33]. In most cases, cell proliferation requires millimolar external calcium concentrations and is inhibited when cells are cultured in a Ca2+ -deprived or Ca2+ channel blockerssupplemented culture medium. The role of ion channels in PASMC proliferation is currently under investigation since the link between proliferation and ion channel activation may represent a molecular target for the treatment of pulmonary hypertension. Participation of TRPV channels in mitosis has already been demonstrated in different cell models. Indeed, TRPV6 was shown to potentiate Ca2+ -dependent HEK-293 cell proliferation [34]. In addition, TRPV6 may play an important role in the progression of prostate cancer: the appearance of TRPV6 transcripts significantly correlates with the prostate cancer stages [35]. Another publication [36] demonstrated the role of TRPV2 in the mitosis neuronal precursor and neuroendocrine cells. However, in our present paper, PASMC stimulation with 4␣-PDD did not significantly increase cell proliferation, showing that the Ca2+ influx through TRPV4 channels per se is not efficient to induce PASMC proliferation and that this former only potentiates the 5-HT proliferative effect. In conclusion, this study demonstrates for the first time, the implication of TRPV4-like current in 5-HT-induced Ca2+ response in PASMC. We propose that TRPV4 channels are responsible for the sustained Ca2+ elevation contributing to both PASMC contraction and proliferation as the result of arachidonic acid-derived EET stimulation. The signalling pathway between proliferation and ion channel activation remains to be determined and may represent a molecular target for the treatment of vascular diseases such as pulmonary hypertension. Acknowledgements We are grateful to Pr. P. Gailly for helpful discussions. This work was supported by the Conseil R´egional d’Aquitaine (200220301301A), the Agence National de la Recherche (ANR06-physio-015-01) and the Fondation de France (2006005603). References [1] M.R. MacLean, P. Herve, S. Eddahibi, S. Adnot, 5-Hydroxytryptamine and the pulmonary circulation: receptors, transporters and relevance to pulmonary arterial hypertension, Br. J. Pharmacol. 131 (2000) 161–168.
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